pPerturb: A Server for Predicting Long-Distance Energetic Couplings and Mutation-Induced Stability Changes in Proteins via PerturbationsClick to copy article linkArticle link copied!
- Soundhararajan GopiSoundhararajan GopiDepartment of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras (IITM), Chennai 600036, IndiaMore by Soundhararajan Gopi
- Devanshu DevanshuDevanshu DevanshuDepartment of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras (IITM), Chennai 600036, IndiaMore by Devanshu Devanshu
- Nandakumar RajasekaranNandakumar RajasekaranDepartment of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras (IITM), Chennai 600036, IndiaDepartment of Biology, Johns Hopkins University, Baltimore, Maryland 21218, United StatesMore by Nandakumar Rajasekaran
- Sathvik AnantakrishnanSathvik AnantakrishnanDepartment of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras (IITM), Chennai 600036, IndiaMore by Sathvik Anantakrishnan
- Athi N. Naganathan*Athi N. Naganathan*E-mail: [email protected]Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras (IITM), Chennai 600036, IndiaMore by Athi N. Naganathan
Abstract
The strength of intraprotein interactions or contact network is one of the dominant factors determining the thermodynamic stabilities of proteins. The nature and the extent of connectivity of this network also play a role in allosteric signal propagation characteristics upon ligand binding to a protein domain. Here, we develop a server for rapid quantification of the strength of an interaction network by employing an experimentally consistent perturbation approach previously validated against a large data set of 375 mutations in 19 different proteins. The web server can be employed to predict the extent of destabilization of proteins arising from mutations in the protein interior in experimentally relevant units. Moreover, coupling distances—a measure of the extent of percolation on perturbation—and overall perturbation magnitudes are predicted in a residue-specific manner, enabling a first look at the distribution of energetic couplings in a protein or its changes upon ligand binding. We show specific examples of how the server can be employed to probe for the distribution of local stabilities in a protein, to examine changes in side chain orientations or packing before and after ligand binding, and to predict changes in stabilities of proteins upon mutations of buried residues. The web server is freely available at http://pbl.biotech.iitm.ac.in/pPerturb and supports recent versions of all major browsers.
Introduction
Computational Methods
Perturbation Protocol


Web Server Description
Figure 1
Figure 1. Flowchart depicting the organization of modules in the pPerturb web server. Once the protein structure is loaded into the server, perturbation profiles at the level of individual residues are generated for individual residues using eq 2, following which residue-specific parameters are provided for selected residues (perturbation profile) or all residues in the protein (interaction network profile). The residue-specific parameters are then colored on the protein structure to generate publication-quality images. Users can also request the prediction of changes in stability involving truncation mutations of uncharged residues wherein the mutational effects are introduced via eq 2. The model output can be downloaded as text files or high-resolution images.
Results and Discussion
Visualization of Local Stability or Interaction Network Profiles
Figure 2
Figure 2. Structural model of GPCR NTSR1 (PDB 6OS9) without (panel A) and with ∑ΔQ mapped on to the structure (panel B). The structure in panel B is colored in the spectral scale between the two extremes of well-packed residues (red) and weakly packed residues (blue). Note the stretch of dark blue in the TM helices 5 and 6 pointing to weak packing.
Structural Changes on Ligand Binding
Figure 3
Figure 3. Left column presents a superimposition of ligand/inhibitor-unbound and -bound structures of the proteins bACBP (PDB ids 2ABD/1ACA for ligand-unbound and -bound states), PDZ3 (1BFE/1BE9), and PFK (3PFK/6PFK for inhibitor-unbound and -bound states) in gray and light brown, respectively. The overall RMSD values (including Cα and side chain) between the bound (b) and unbound (u) forms are 2.3, 1.1, and 1.6 Å, respectively. The cartoons in the middle and right columns are colored in the spectral scale (red to blue as in the color bar provided) according to ∑ΔQb – ∑ΔQu. Spheres represent the Cα atoms of specific residues whose difference in ∑ΔQ fall in the extremes, with Z-score ≥ 1 in the middle column and Z-score ≤ −1 in the right column. Note that such vivid details in terms of packing differences (middle and right columns) cannot be extracted from structural superimposition alone (left column).
Predicting Stability Changes on Truncation Mutations
Figure 4
Figure 4. (A) Structure of ubiquitin (1UBQ) highlighting the position of I30 (cyan) together with first- and second-shell neighbors in blue and green, respectively. (B) Unfolding curves predicted by the WSME model for mutations at position 30 at pH 7.0 and 20 mM ionic strength by employing a 6 Å heavy-atom contact cutoff including the nearest neighbors.
Conclusions
Acknowledgments
A.N.N. is a Wellcome Trust/DBT India Alliance Intermediate Fellow. S.G. acknowledges the Initiative for Biological Systems Engineering, IIT Madras, India for the IBSE Ph.D. Studentship.
WSME | Wako–Saitô–Muñoz–Eaton |
NMR | nuclear magnetic resonance |
PDB | protein data bank |
GPCR | G-protein coupled receptor |
MD | molecular dynamics |
RMSD | root-mean-squared deviation |
vdW | van der Waals |
References
This article references 32 other publications.
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- 4Boyken, S. E.; Benhaim, M. A.; Busch, F.; Jia, M.; Bick, M. J.; Choi, H.; Klima, J. C.; Chen, Z.; Walkey, C.; Mileant, A.; Sahasrabuddhe, A.; Wei, K. Y.; Hodge, E. A.; Byron, S.; Quijano-Rubio, A.; Sankaran, B.; King, N. P.; Lippincott-Schwartz, J.; Wysocki, V. H.; Lee, K. K.; Baker, D. De novo design of tunable, pH-driven conformational changes. Science 2019, 364, 658– 664, DOI: 10.1126/science.aav7897Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXpvVChtbw%253D&md5=d58ec17696ffd269599466f1cf1c379aDe novo design of tunable, pH-driven conformational changesBoyken, Scott E.; Benhaim, Mark A.; Busch, Florian; Jia, Mengxuan; Bick, Matthew J.; Choi, Heejun; Klima, Jason C.; Chen, Zibo; Walkey, Carl; Mileant, Alexander; Sahasrabuddhe, Aniruddha; Wei, Kathy Y.; Hodge, Edgar A.; Byron, Sarah; Quijano-Rubio, Alfredo; Sankaran, Banumathi; King, Neil P.; Lippincott-Schwartz, Jennifer; Wysocki, Vicki H.; Lee, Kelly K.; Baker, DavidScience (Washington, DC, United States) (2019), 364 (6441), 658-664CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The ability of naturally occurring proteins to change conformation in response to environmental changes is crit. to biol. function. Although there have been advances in the de novo design of stable proteins with a single, deep free-energy min., the design of conformational switches remains challenging. We present a general strategy to design pH-responsive protein conformational changes by precisely preorganizing histidine residues in buried hydrogen-bond networks. We design homotrimers and heterodimers that are stable above pH 6.5 but undergo cooperative, large-scale conformational changes when the pH is lowered and electrostatic and steric repulsion builds up as the network histidine residues become protonated. The transition pH and cooperativity can be controlled through the no. of histidine-contg. networks and the strength of the surrounding hydrophobic interactions. Upon disassembly, the designed proteins disrupt lipid membranes both in vitro and after being endocytosed in mammalian cells. Our results demonstrate that environmentally triggered conformational changes can now be programmed by de novo protein design.
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- 9Naganathan, A. N. Modulation of allosteric coupling by mutations: from protein dynamics and packing to altered native ensembles and function. Curr. Opin. Struct. Biol. 2019, 54, 1– 9, DOI: 10.1016/j.sbi.2018.09.004Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslCju73I&md5=dfc261b3d6bd5d7b9764bb34d84a7bf5Modulation of allosteric coupling by mutations: from protein dynamics and packing to altered native ensembles and functionNaganathan, Athi N.Current Opinion in Structural Biology (2019), 54 (), 1-9CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. A large body of work has gone into understanding the effect of mutations on protein structure and function. Conventional treatments have involved quantifying the change in stability, activity and relaxation rates of the mutants with respect to the wild-type protein. However, it is now becoming increasingly apparent that mutational perturbations consistently modulate the packing and dynamics of a significant fraction of protein residues, even those that are located >10-15 Å from the mutated site. Such long-range modulation of protein features can distinctly tune protein stability and the native conformational ensemble contributing to allosteric modulation of function. In this review, I summarize a series of exptl. and computational observations that highlight the incredibly pliable nature of proteins and their response to mutational perturbations manifested via the intra-protein interaction network. I highlight how an intimate understanding of mutational effects could pave the way for integrating stability, folding, cooperativity and even allostery within a single phys. framework.
- 10Guarnera, E.; Berezovsky, I. N. On the perturbation nature of allostery: sites, mutations, and signal modulation. Curr. Opin. Struct. Biol. 2019, 56, 18– 27, DOI: 10.1016/j.sbi.2018.10.008Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitFSjs7vI&md5=2c6c003783e53393d435df408a0e9b35On the perturbation nature of allostery: sites, mutations, and signal modulationGuarnera, Enrico; Berezovsky, Igor N.Current Opinion in Structural Biology (2019), 56 (), 18-27CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. Regardless of the diversity of systems, allosteic signaling is found to be always caused by perturbations. This recurring trait of allostery serves as a foundation for developing different exptl. efforts and theor. models for the studies of allosteric mechanisms. Among computational approaches considered here particular emphasis is given to the structure-based statistical mech. model of allostery (SBSMMA), which allows one to study the causality and energetics of allosteric communication. We argue that the reverse allosteric signaling on the basis of SBSMMA can be used for predicting latent allosteric sites and inducing a tunable allosteric response. Per-residue allosteric effects of mutations can also be explored and 'latent drivers' expanding the cancer mutational landscape can be predicted using SBSMMA. Most recent and important implementations of computational models in web-resources along with a brief outlook on future research directions are also discussed.
- 11Ben-David, M.; Huang, H.; Sun, M. G. F.; Corbi-Verge, C.; Petsalaki, E.; Liu, K.; Gfeller, D.; Garg, P.; Tempel, W.; Sochirca, I.; Shifman, J. M.; Davidson, A.; Min, J.; Kim, P. M.; Sidhu, S. S. Allosteric Modulation of Binding Specificity by Alternative Packing of Protein Cores. J. Mol. Biol. 2019, 431, 336– 350, DOI: 10.1016/j.jmb.2018.11.018Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitlent7vJ&md5=514b0ebac210a850c301183bbf680382Allosteric Modulation of Binding Specificity by Alternative Packing of Protein CoresBen-David, Moshe; Huang, Haiming; Sun, Mark G. F.; Corbi-Verge, Carles; Petsalaki, Evangelia; Liu, Ke; Gfeller, David; Garg, Pankaj; Tempel, Wolfram; Sochirca, Irina; Shifman, Julia M.; Davidson, Alan; Min, Jinrong; Kim, Philip M.; Sidhu, Sachdev S.Journal of Molecular Biology (2019), 431 (2), 336-350CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)Hydrophobic cores are often viewed as tightly packed and rigid, but they do show some plasticity and could thus be attractive targets for protein design. Here we explored the role of different functional pressures on the core packing and ligand recognition of the SH3 domain from human Fyn tyrosine kinase. We randomized the hydrophobic core and used phage display to select variants that bound to each of three distinct ligands. The three evolved groups showed remarkable differences in core compn., illustrating the effect of different selective pressures on the core. Changes in the core did not significantly alter protein stability, but were linked closely to changes in binding affinity and specificity. Structural anal. and mol. dynamics simulations revealed the structural basis for altered specificity. The evolved domains had significantly reduced core vols., which in turn induced increased backbone flexibility. These motions were propagated from the core to the binding surface and induced significant conformational changes. These results show that alternative core packing and consequent allosteric modulation of binding interfaces could be used to engineer proteins with novel functions.
- 12Petrović, D.; Risso, V. A.; Kamerlin, S. C. L.; Sanchez-Ruiz, J. M. Conformational dynamics and enzyme evolution. J. R. Soc., Interface 2018, 15, 20180330 DOI: 10.1098/rsif.2018.0330Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXis1Cnsbg%253D&md5=aaf6752e8e06cd1d29a8668e6a241ad8Conformational dynamics and enzyme evolutionPetrovic, Dussan; Risso, Valeria A.; Kamerlin, Shina Caroline Lynn; Sanchez-Ruiz, Jose M.Journal of the Royal Society, Interface (2018), 15 (144), 20180330/1-20180330/18CODEN: JRSICU; ISSN:1742-5662. (Royal Society)Enzymes are dynamic entities, and their dynamic properties are clearly linked to their biol. function. It follows that dynamics ought to play an essential role in enzyme evolution. Indeed, a link between conformational diversity and the emergence of new enzyme functionalities has been recognized for many years. However, it is only recently that state-of-the-art computational and exptl. approaches are revealing the crucial mol. details of this link. Specifically, evolutionary trajectories leading to functional optimization for a given host environment or to the emergence of a new function typically involve enriching catalytically competent conformations and/or the freezing out of non-competent conformations of an enzyme. In some cases, these evolutionary changes are achieved through distant mutations that shift the protein ensemble towards productive conformations. Multifunctional intermediates in evolutionary trajectories are probably multi-conformational, i.e. able to switch between different overall conformations, each competent for a given function. Conformational diversity can assist the emergence of a completely new active site through a single mutation by facilitating transition-state binding. We propose that this mechanism may have played a role in the emergence of enzymes at the primordial, progenote stage, where it was plausibly promoted by high environmental temps. and the possibility of addnl. phenotypic mutations.
- 13Tokuriki, N.; Tawfik, D. S. Stability effects of mutations and protein evolvability. Curr. Opin. Struct. Biol. 2009, 19, 596– 604, DOI: 10.1016/j.sbi.2009.08.003Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXht1ylsLrP&md5=cd22e7bf4b26369fbb2dc517caa52e91Stability effects of mutations and protein evolvabilityTokuriki, Nobuhiko; Tawfik, Dan S.Current Opinion in Structural Biology (2009), 19 (5), 596-604CODEN: COSBEF; ISSN:0959-440X. (Elsevier B.V.)A review. The past several years have seen novel insights at the interface of protein biophysics and evolution. The accepted paradigm that proteins can tolerate nearly any amino acid substitution has been replaced by the view that the deleterious effects of mutations, and esp. their tendency to undermine the thermodn. and kinetic stability of protein, is a major constraint on protein evolvability, i.e, the ability of proteins to acquire changes in sequence and function. The authors summarize recent findings regarding how mutations affect protein stability, and how stability affects protein evolution. The authors describe ways of predicting and analyzing stability effects of mutations, and mechanisms that buffer or compensate for these destabilizing effects and thereby promote protein evolvability, in Nature and in the lab.
- 14Muñoz, V.; Campos, L. A.; Sadqi, M. Limited cooperativity in protein folding. Curr. Opin. Struct. Biol. 2016, 36, 58– 66, DOI: 10.1016/j.sbi.2015.12.001Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVGrtrfL&md5=20d2c592462d935d7b4040caa049204aLimited cooperativity in protein foldingMunoz, Victor; Campos, Luis A.; Sadqi, MouradCurrent Opinion in Structural Biology (2016), 36 (), 58-66CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. Theory and simulations predict that the structural concert of protein folding reactions is relatively low. Exptl., folding cooperativity has been difficult to study, but in recent years we have witnessed major advances. New anal. procedures in terms of conformational ensembles rather than discrete states, exptl. techniques with improved time, structural, or single-mol. resoln., and combined thermodn. and kinetic anal. of fast folding have contributed to demonstrate a general scenario of limited cooperativity in folding. Gradual structural disorder is already apparent on the unfolded and native states of slow, two-state folding proteins, and it greatly increases in magnitude for fast folding domains. These results demonstrate a direct link between how fast a single-domain protein folds and unfolds, and how cooperative (or structurally diverse) is its equil. unfolding process. Reducing cooperativity also destabilizes the native structure because it affects unfolding more than folding. We can thus define a continuous cooperativity scale that goes from the 'pliable' two-state character of slow folders to the gradual unfolding of one-state downhill, and eventually to intrinsically disordered proteins. The connection between gradual unfolding and intrinsic disorder is appealing because it suggests a conformational rheostat mechanism to explain the allosteric effects of folding coupled to binding.
- 15Chan, H. S.; Shimizu, S.; Kaya, H. Cooperativity principles in protein folding. Methods Enzymol. 2004, 380, 350– 379, DOI: 10.1016/S0076-6879(04)80016-8Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhs1Omurg%253D&md5=d28669191744b8cc3939b9d051a2f9e4Cooperativity principles in protein foldingChan, Hue Sun; Shimizu, Seishi; Kaya, HuseyinMethods in Enzymology (2004), 380 (Energetics of Biological Macromolecules, Part E), 350-379CODEN: MENZAU; ISSN:0076-6879. (Elsevier)A review. Extensive evaluations of coarse grained chain models suggest that the high degrees of thermodn. and kinetic cooperativity of small single-domain proteins may likely originate from many-body interactions in the form of a coupling between local conformational preferences and favorable nonlocal interactions. A discussion on the recent advances in these respects is presented.
- 16Stein, A.; Fowler, D. M.; Hartmann-Petersen, R.; Lindorff-Larsen, K. Biophysical and Mechanistic Models for Disease-Causing Protein Variants. Trends Biochem. Sci. 2019, 44, 575– 588, DOI: 10.1016/j.tibs.2019.01.003Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsVOqtbc%253D&md5=15b7d25b0dd21d04bc0bfcd873461fd7Biophysical and Mechanistic Models for Disease-Causing Protein VariantsStein, Amelie; Fowler, Douglas M.; Hartmann-Petersen, Rasmus; Lindorff-Larsen, KrestenTrends in Biochemical Sciences (2019), 44 (7), 575-588CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)A review. The rapid decrease in DNA sequencing cost is revolutionizing medicine and science. In medicine, genome sequencing has revealed millions of missense variants that change protein sequences, yet we only understand the mol. and phenotypic consequences of a small fraction. Within protein science, high-throughput deep mutational scanning expts. enable us to probe thousands of variants in a single, multiplexed expt. We review efforts that bring together these topics via exptl. and computational approaches to det. the consequences of missense variants in proteins. We focus on the role of changes in protein stability as a driver for disease, and how expts., biophys. models, and computation are providing a framework for understanding and predicting how changes in protein sequence affect cellular protein stability.
- 17Rajasekaran, N.; Suresh, S.; Gopi, S.; Raman, K.; Naganathan, A. N. A general mechanism for the propagation of mutational effects in proteins. Biochemistry 2017, 56, 294– 305, DOI: 10.1021/acs.biochem.6b00798Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitVKktLjF&md5=caf6021e1d6ba8325e2c2b66ae895dbfA General Mechanism for the Propagation of Mutational Effects in ProteinsRajasekaran, Nandakumar; Suresh, Swaathiratna; Gopi, Soundhararajan; Raman, Karthik; Naganathan, Athi N.Biochemistry (2017), 56 (1), 294-305CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Mutations in the hydrophobic interior of proteins are generally thought to weaken the interactions only in their immediate neighborhood. This forms the basis of protein-engineering based studies of folding mechanism and function. However, mutational work on diverse proteins has shown that distant residues are thermodynamically coupled, with the network of interactions within the protein acting as signal conduits, thus raising an intriguing paradox. Are mutational effects localized and if not, is there a general rule for the extent of percolation and on the functional form of this propagation. We explore these questions from multiple perspectives in the current work. Perturbation anal. of interaction networks within proteins and microsecond-long mol. dynamics simulations of several aliph. mutants of ubiquitin reveal strong evidence for distinct alteration of distal residue-residue communication networks. We find that mutational effects consistently propagate into the second shell of the altered site (even up to 15-20 Å) in proportion to the perturbation magnitude and dissipates exponentially with a decay distance-const. of ∼4-5 Å. We also report evidence for this phenomenon from published exptl. NMR data that strikingly resemble predictions from network theory and MD simulations. Reformulating these observations onto a statistical mech. model, we reproduce the stability changes of 375 mutations from 19 single-domain proteins. Our work thus reveals a robust energy dissipation-cum-signaling mechanism in the interaction network within proteins, quantifies the partitioning of destabilization energetics around the mutation neighborhood and presents a simple theor. framework for modeling the allosteric effects of point mutations.
- 18Rajasekaran, N.; Sekhar, A.; Naganathan, A. N. A Universal Pattern in the Percolation and Dissipation of Protein Structural Perturbations. J. Phys. Chem. Lett. 2017, 8, 4779– 4784, DOI: 10.1021/acs.jpclett.7b02021Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsV2js7rO&md5=3161d8efc35d5538417762478cd126a3A Universal Pattern in the Percolation and Dissipation of Protein Structural PerturbationsRajasekaran, Nandakumar; Sekhar, Ashok; Naganathan, Athi N.Journal of Physical Chemistry Letters (2017), 8 (19), 4779-4784CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)Understanding the extent to which information is transmitted through the intramol. interaction network of proteins upon a perturbation, i.e., an allosteric effect, has long remained an unsolved problem. Here, through an anal. of high-resoln. NMR data from the literature on 28 different proteins and 49 structural perturbations, we show that the extent of induced structural changes through mutations, and mol. events including protein-protein, protein-peptide, protein-ligand binding, and post-translational modifications exhibit a near-universal exponential functional form. The extent of percolation into the protein structures can be up to 20-25 Å despite no apparent change in the 3-dimensional structures. These observations were also consistent with theor. expectations, elementary graph theoretic anal. of protein structures, detailed mol. dynamics simulations, and exptl. double-mutant cycles. Our anal. highlighted that most mol. events would contribute to allosteric effects independent of protein structure, topol., or identity, and provides a simple avenue to test and potentially model their effects.
- 19Rajasekaran, N.; Naganathan, A. N. A self-consistent structural perturbation approach for determining the magnitude and extent of allosteric coupling in proteins. Biochem. J. 2017, 474, 2379– 2388, DOI: 10.1042/BCJ20170304Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFygsrjJ&md5=ab41998037500c5be60e1e7a8d8a1522A self-consistent structural perturbation approach for determining the magnitude and extent of allosteric coupling in proteinsRajasekaran, Nandakumar; Naganathan, Athi N.Biochemical Journal (2017), 474 (14), 2379-2388CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)Elucidating the extent of energetic coupling between residues in single-domain proteins, which is a fundamental determinant of allostery, information transfer and folding cooperativity, has remained a grand challenge. While several sequence- and structure-based approaches have been proposed, a self-consistent description that is simultaneously compatible with unfolding thermodn. is lacking. We recently developed a simple structural perturbation protocol that captures the changes in thermodn. stabilities induced by point mutations within the protein interior. Here, we show that a fundamental residue-specific component of this perturbation approach, the coupling distance, is uniquely sensitive to the environment of a residue in the protein to a distance of ∼15 A[n.778]. With just the protein contact map as an input, we reproduce the extent of percolation of perturbations within the structure as obsd. in network anal. of intra-protein interactions, mol. dynamics simulations and NMR-obsd. changes in chem. shifts. Using this rapid protocol that relies on a single structure, we explain the results of statistical coupling anal. (SCA) that requires hundreds of sequences to identify functionally crit. sectors, the propagation and dissipation of perturbations within proteins and the higher-order couplings deduced from detailed NMR expts. Our results thus shed light on the possible mechanistic origins of signaling through the interaction network within proteins, the likely distance dependence of perturbations induced by ligands and post-translational modifications and the origins of folding cooperativity through many-body interactions.
- 20Yu, M.; Chen, Y.; Wang, Z. L.; Liu, Z. Fluctuation correlations as major determinants of structure- and dynamics-driven allosteric effects. Phys. Chem. Chem. Phys. 2019, 21, 5200– 5214, DOI: 10.1039/C8CP07859AGoogle Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXislKksb0%253D&md5=70c29c723f859c99a4f52455850b7d85Fluctuation correlations as major determinants of structure- and dynamics-driven allosteric effectsYu, Miao; Chen, Yixin; Wang, Zi-Le; Liu, ZhirongPhysical Chemistry Chemical Physics (2019), 21 (9), 5200-5214CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)Allosteric control is essential for regulating biol. functions whereby stimuli such as ligand binding at one site on a protein cause a response at a distant functional site. Correlations between different sites in proteins have been used widely in identifying allosteric sites and pathways, and in designing allosteric drugs. However, the deterministic connection between correlations and allostery remains unsolved, esp. considering that there are various types of correlations. Here, we combine perturbation-theory anal. and numerical calcns. to study both structure- and dynamics-driven allosteric effects in an anisotropic network model (ANM). The results reveal that the allosteries are detd. by the correlation (covariance) of distance fluctuations, but are irrelevant to the usual displacement correlations or time-delayed correlations. Dynamics-driven allostery is weaker than structure-driven allostery by at least one to two orders of magnitude. The intrinsic allostery capacity decays with distance by an exponential law, with the resulting characteristic distance parameter lying in the range of 7-10 Å for structure-driven allostery and 4-5 Å for dynamics-driven allostery. The importance of the cutoff distance of the ANM is also addressed.
- 21Medina-Carmona, E.; Betancor-Fernández, I.; Santos, J.; Mesa-Torres, N.; Grottelli, S.; Batlle, C.; Naganathan, A. N.; Oppici, E.; Cellini, B.; Ventura, S.; Salido, E.; Pey, A. L. Insight into the specificity and severity of pathogenic mechanisms associated with missense mutations through experimental and structural perturbation analyses. Hum. Mol. Genet. 2019, 28, 1– 15, DOI: 10.1093/hmg/ddy323Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1CgsLrJ&md5=c32c562fe414bd244ac84bfdc2f0cd9aInsight into the specificity and severity of pathogenic mechanisms associated with missense mutations through experimental and structural perturbation analysesMedina-Carmona, Encarnacion; Betancor-Fernandez, Isabel; Santos, Jaime; Mesa-Torres, Noel; Grottelli, Silvia; Batlle, Cristina; Naganathan, Athi N.; Oppici, Elisa; Cellini, Barbara; Ventura, Salvador; Salido, Eduardo; Pey, Angel L.Human Molecular Genetics (2019), 28 (1), 1-15CODEN: HMGEE5; ISSN:1460-2083. (Oxford University Press)Most pathogenic missense mutations cause specific mol. phenotypes through protein destabilization. However, how protein destabilization is manifested as a given mol. phenotype is not well understood. We develop here a structural and energetic approach to describe mutational effects on specific traits such as function, regulation, stability, subcellular targeting or aggregation propensity. This approach is tested using large-scale exptl. and structural perturbation analyses in over thirty mutations in three different proteins (cancer-assocd. NQO1, transthyretin related with amyloidosis and AGT linked to primary hyperoxaluria type I) and comprising five very common pathogenic mechanisms (loss-of-function and gain-of-toxic function aggregation, enzyme inactivation, protein mistargeting and accelerated degrdn.). Our results revealed that the magnitude of destabilizing effects and, particularly, their propagation through the structure to promote disease-assocd. conformational states largely det. the severity and mol. mechanisms of disease-assocd. missense mutations. Modulation of the structural perturbation at a mutated site is also shown to cause switches between different mol. phenotypes. When very common disease-assocd. missense mutations were investigated, we also found that they were not among the most deleterious possible missense mutations at those sites, and required addnl. contributions from codon bias and effects of CpG sites to explain their high frequency in patients. Our work sheds light on the mol. basis of pathogenic mechanisms and genotype-phenotype relationships, with implications for discriminating between pathogenic and neutral changes within human genome variability from whole genome sequencing studies.
- 22Wako, H.; Saitô, N. Statistical Mechanical Theory of Protein Conformation.2. Folding Pathway for Protein. J. Phys. Soc. Jpn. 1978, 44, 1939– 1945, DOI: 10.1143/JPSJ.44.1939Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXkvVKjsLc%253D&md5=4cf3c7168e13ba4e51c78b6d1088b6e2Statistical mechanical theory of the protein conformation. II. Folding pathway for proteinWako, Hiroshi; Saito, NobuhikoJournal of the Physical Society of Japan (1978), 44 (6), 1939-45CODEN: JUPSAU; ISSN:0031-9015.The theory of a 1-dimensional lattice gas with long-range many-body interactions was applied to the folding pathways of proteins. This model presents the quant. informations about the order of inter-residue interactions, the location of nucleation, and so on. Three typical proteins are cited as examples and their folding pathways are traced according to the computation of some useful properties.
- 23Muñoz, V.; Eaton, W. A. A simple model for calculating the kinetics of protein folding from three-dimensional structures. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11311– 11316, DOI: 10.1073/pnas.96.20.11311Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXmvVCmsL8%253D&md5=1adf1ff76d654bab4842b90e0685e5acA simple model for calculating the kinetics of protein folding from three-dimensional structuresMunoz, Victor; Eaton, William A.Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (20), 11311-11316CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)An elementary statistical mech. model was used to calc. the folding rates for 22 proteins from their known three-dimensional structures. In this model, residues come into contact only after all of the intervening chain is in the native conformation. An addnl. simplifying assumption is that native structure grows from localized regions that then fuse to form the complete native mol. The free energy function for this model contains just two contributions-conformational entropy of the backbone and the energy of the inter-residue contacts. The matrix of interresidue interactions is obtained from the at. coordinates of the three-dimensional structure. For the 18 proteins that exhibit two-state equil. and kinetic behavior, profiles of the free energy vs. the no. of native peptide bonds show two deep min., corresponding to the native and denatured states. For four proteins known to exhibit intermediates in folding, the free energy profiles show addnl. deep min. The calcd. rates of folding the two-state proteins, obtained by solving a diffusion equation for motion on the free energy profiles, reproduce the exptl. detd. values surprisingly well. The success of these calcns. suggests that folding speed is largely detd. by the distribution and strength of contacts in the native structure. We also calcd. the effect of mutations on the folding kinetics of chymotrypsin inhibitor 2, the most intensively studied two-state protein, with some success.
- 24Naganathan, A. N. Predictions from an Ising-like Statistical Mechanical Model on the Dynamic and Thermodynamic Effects of Protein Surface Electrostatics. J. Chem. Theory Comput. 2012, 8, 4646– 4656, DOI: 10.1021/ct300676wGoogle Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsVOhs7bK&md5=61da68db7a56f01b51157ec9d42e3a89Predictions from an Ising-like Statistical Mechanical Model on the Dynamic and Thermodynamic Effects of Protein Surface ElectrostaticsNaganathan, Athi N.Journal of Chemical Theory and Computation (2012), 8 (11), 4646-4656CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)Charged residues on the surface of a protein are known hot-spots for post-translational modification, protein/ligand-binding, and tuning conformational stabilities. Recent exptl. evidence points to the fact that surface electrostatics can also modulate thermodn. barriers and hence folding mechanisms. To probe for this behavior across different proteins, we develop a novel version of the Wako-Sait̂o-Mu~noz-Eaton (WSME) model in which we include an electrostatic potential term in the energy function while simplifying the treatment of solvation free energy. Both of the energy terms are obtained by quant. fitting the model to differential scanning calorimetry (DSC) expts. that carry crit. information on the protein partition function. We characterize four sets of structural/functional homologs (HEWL/BLA, CspB, engrailed, α-spectrin) either by fitting the exptl. data of a single domain in the homologous set and predicting the conformational behavior of the rest with the same set of parameters or by performing semiblind predictions. The model with the added electrostatic term is able to successfully reproduce the order of thermodn. stabilities and relaxation rates of most of the homologs. In parallel, we predict diverse conformational features including a wide range of thermodn. barriers (∼9-40 kJ/mol), broad native ensembles in helical proteins, structured unfolded states and intermediates, rugged folding landscapes, and further provide an independent protein-specific est. of the folding speed limit at 298 K (1/(7-300 μs)). Our results are evidence that protein surface electrostatics can be tailored to not only engineer stabilities but also folding mechanisms and the ruggedness of the underlying landscape.
- 25Naganathan, A. N. A Rapid, Ensemble and Free Energy Based Method for Engineering Protein Stabilities. J. Phys. Chem. B 2013, 117, 4956– 4964, DOI: 10.1021/jp401588xGoogle Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXltVOmtbs%253D&md5=bb43890bae36523073911fda03e09062A Rapid, Ensemble and Free Energy Based Method for Engineering Protein StabilitiesNaganathan, Athi N.Journal of Physical Chemistry B (2013), 117 (17), 4956-4964CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)Engineering the conformational stabilities of proteins through mutations has immense potential in biotechnol. applications. It is, however, an inherently challenging problem given the weak noncovalent nature of the stabilizing interactions. In this regard, we present here a robust and fast strategy to engineer protein stabilities through mutations involving charged residues using a structure-based statistical mech. model that accounts for the ensemble nature of folding. We validate the method by predicting the abs. changes in stability for 138 exptl. mutations from 16 different proteins and enzymes with a correlation of 0.65 and importantly with a success rate of 81%. Multiple point mutants are predicted with a higher success rate (90%) that is validated further by comparing mesophile-thermophile protein pairs. In parallel, we devise a methodol. to rapidly engineer mutations in silico which we benchmark against exptl. mutations of ubiquitin (correlation of 0.95) and check for its feasibility on a larger therapeutic protein DNase I. We expect the method to be of importance as a first and rapid step to screen for protein mutants with specific stability in the biotechnol. industry, in the construction of stability maps at the residue level (i.e., hot spots), and as a robust tool to probe for mutations that enhance the stability of protein-based drugs.
- 26Gopi, S.; Devanshu, D.; Krishna, P.; Naganathan, A. N. pStab: prediction of stable mutants, unfolding curves, stability maps and protein electrostatic frustration. Bioinformatics 2018, 34, 875– 877, DOI: 10.1093/bioinformatics/btx697Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitlemtbzL&md5=bfc07fdf1c6bef731acf5b149201c4d6pStab: prediction of stable mutants, unfolding curves, stability maps and protein electrostatic frustrationGopi, Soundhararajan; Devanshu, Devanshu; Krishna, Praveen; Naganathan, Athi N.Bioinformatics (2018), 34 (5), 875-877CODEN: BOINFP; ISSN:1367-4811. (Oxford University Press)Summary: We present a web-server for rapid prediction of changes in protein stabilities over a range of temps. and exptl. conditions upon single- or multiple-point substitutions of charged residues. Potential mutants are identified by a charge-shuffling procedure while the stability changes (i.e. an unfolding curve) are predicted employing an ensemble-based statistical-mech. model. We expect this server to be a simple yet detailed tool for engineering stabilities, identifying electrostatically frustrated residues, generating local stability maps and in constructing fitness landscapes.
- 27Kato, H. E.; Zhang, Y.; Hu, H.; Suomivuori, C. M.; Kadji, F. M. N.; Aoki, J.; Krishna Kumar, K.; Fonseca, R.; Hilger, D.; Huang, W.; Latorraca, N. R.; Inoue, A.; Dror, R. O.; Kobilka, B. K.; Skiniotis, G. Conformational transitions of a neurotensin receptor 1-Gi1 complex. Nature 2019, 572, 80– 85, DOI: 10.1038/s41586-019-1337-6Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXht1yktL%252FL&md5=0ce265e5a9dc353e69d2c04032803399Conformational transitions of a neurotensin receptor 1-Gi1 complexKato, Hideaki E.; Zhang, Yan; Hu, Hongli; Suomivuori, Carl-Mikael; Kadji, Francois Marie Ngako; Aoki, Junken; Krishna Kumar, Kaavya; Fonseca, Rasmus; Hilger, Daniel; Huang, Weijiao; Latorraca, Naomi R.; Inoue, Asuka; Dror, Ron O.; Kobilka, Brian K.; Skiniotis, GeorgiosNature (London, United Kingdom) (2019), 572 (7767), 80-85CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Neurotensin receptor 1 (NTSR1) is a G-protein-coupled receptor (GPCR) that engages multiple subtypes of G protein, and is involved in the regulation of blood pressure, body temp., wt. and the response to pain. Here we present structures of human NTSR1 in complex with the agonist JMV449 and the heterotrimeric Gi1 protein, at a resoln. of 3 Å. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR-Gi/o complexes (in which the nucleotide-binding pocket adopts more flexible conformations that may facilitate nucleotide exchange), and a non-canonical state in which the G protein is rotated by about 45 degrees relative to the receptor and exhibits a more rigid nucleotide-binding pocket. In the non-canonical state, NTSR1 exhibits features of both active and inactive conformations, which suggests that the structure may represent an intermediate form along the activation pathway of G proteins. This structural information, complemented by mol. dynamics simulations and functional studies, provides insights into the complex process of G-protein activation.
- 28Kragelund, B. B.; Andersen, K. V.; Madsen, J. C.; Knudsen, J.; Poulsen, F. M. Three-dimensional structure of the complex between acyl-coenzyme A binding protein and palmitoyl-coenzyme A. J. Mol. Biol. 1993, 230, 1260– 1277, DOI: 10.1006/jmbi.1993.1240Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXkvV2gtrk%253D&md5=05aed1a07a17f17754d976b611755097Three-dimensional structure of the complex between acyl-coenzyme A binding protein and palmitoyl-coenzyme AKragelund, Birth B.; Andersen, Kim Vilbour; Madsen, Jens Chr.; Knudsen, Jens; Poulsen, Flemming M.Journal of Molecular Biology (1993), 230 (4), 1260-77CODEN: JMOBAK; ISSN:0022-2836.Multidimensional 1H, 13C, and 15N NMR spectroscopy was used to study the complex between palmitoyl-CoA and acyl-CoA binding protein (ACBP). The 1H and 15N spectra of holo-ACBP were almost completely assigned and so was most of the 1H spectrum of the CoA part of the protein-bound ligand. The palmitoyl part of the ligand was uniformly labeled with 13C and the NMR signals of the C atoms and their protons were assigned at the 2 ends of the hydrocarbon chain. A total of 1251 distance restraints from NOE and 131 dihedral angle restraints from 3-bond coupling consts. provided the basis for the structure calcn. A comparison of 20 structures calcd. from these data to the av. structure showed that they could be aligned with an at. root-mean-square deviation (r.m.s.d.) of 1.3 Å for all C, N, O, P and S atoms in protein and ligand. Apo-ACBP was a 4-helix protein and this structure was maintained in holo-ACBP. The 4 α-helixes were: Ac1 for residues 3-15; Ac2 for residues 20-36; Ac3 for residues 51-62; and Ac4 for residues 65-84. For the 4 α-helixes of the peptide backbone of holo-ACBP, the r.m.s.d. for the C, Cα, and N atoms was 0.42 Å. The binding site for the palmitoyl chain stretched between the N-terminal end of Ac3 where the carboxyl part binds, to the N-terminal of Ac3 where the ω-end of the palmitoyl part binds. The 3'-AMP was bound near residues of each of the 4 helixes in an arrangement where it could form salt bridges and/or H-bonds to either backbone or side-chain atoms of Ala-9, Tyr-28, Lys-32, Lys-54, and Tyr-73. The polar parts of the pantetheine and the pyrophosphate were structured in the bound ligand to form an interface with the solvent. Also, the ligand formed a set of nonpolar intramol. interactions where the adenine, the pantetheine, and the palmitoyl chain were assocd., so that the overall structure of the bound ligand appeared to be organized to protect the lipophilic palmitoyl part from the polar solvent.
- 29Schirmer, T.; Evans, P. R. Structural basis of the allosteric behaviour of phosphofructokinase. Nature 1990, 343, 140– 145, DOI: 10.1038/343140a0Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXpsVarsA%253D%253D&md5=373daba1e0eeedb16a98fd461eb5f694Structural basis of the allosteric behavior of phosphofructokinaseSchirmer, Tilman; Evans, Philip R.Nature (London, United Kingdom) (1990), 343 (6254), 140-5CODEN: NATUAS; ISSN:0028-0836.A review and discussion with 20 refs. A comparison between the crystal structures of low- and high-affinity forms of phosphofructokinase shows a close coupling between the change of quaternary structure and local changes triggered by binding of the allosteric effectors. These concerted changes link all the substrate and effector sites in the tetramer, and explain the change of affinity for the cooperative substrate.
- 30Doyle, D. A.; Lee, A.; Lewis, J.; Kim, E.; Sheng, M.; MacKinnon, R. Crystal structures of a complexed and peptide-free membrane protein-binding domain: molecular basis of peptide recognition by PDZ. Cell 1996, 85, 1067– 1076, DOI: 10.1016/S0092-8674(00)81307-0Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XjvF2iu7s%253D&md5=bcc54fe12efeda2bd672deb5487fb670Crystal structures of a complexed and peptide-free membrane protein-binding domain: molecular basis of peptide recognition by PDZDoyle, Declan A.; Lee, Alice; Lewis, John; Kim, Eunjoon; Sheng, Morgan; MacKinnon, RoderickCell (Cambridge, Massachusetts) (1996), 85 (7), 1067-1076CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Modular PDZ domains, found in many cell junction-assocd. proteins, mediate the clustering of membrane ion channels by binding to their C-terminus. The X-ray crystallog. structures of the third PDZ domain from the synaptic protein PSD-95 in complex with and in the absence of its peptide ligand were detd. at 1.8 Å and 2.3 Å resoln., resp. The structures reveal that a 4-residue C-terminal stretch (X-Thr/Ser-X-Val-COO-) engages the PDZ domain through antiparallel main chain interactions with a β sheet of the domain. Recognition of the terminal carboxylate group of the peptide is conferred by a cradle of main chain amides provided by a Gly-Leu-Gly-Phe loop as well as by an arginine side chain. Specific side chain interactions and a prominent hydrophobic pocket explain the selective recognition of the C-terminal consensus sequence.
- 31Hultqvist, G.; Haq, S. R.; Punekar, A. S.; Chi, C. N.; Engstrom, A.; Bach, A.; Stromgaard, K.; Selmer, M.; Gianni, S.; Jemth, P. Energetic pathway sampling in a protein interaction domain. Structure 2013, 21, 1193– 1202, DOI: 10.1016/j.str.2013.05.010Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtVehtr3J&md5=57ecfd7d086265edc7b4bd5d77f95a0bEnergetic Pathway Sampling in a Protein Interaction DomainHultqvist, Greta; Haq, S. Raza; Punekar, Avinash S.; Chi, Celestine N.; Engstroem, Aake; Bach, Anders; Stroemgaard, Kristian; Selmer, Maria; Gianni, Stefano; Jemth, PerStructure (Oxford, United Kingdom) (2013), 21 (7), 1193-1202CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)The affinity and specificity of protein-ligand interactions are influenced by energetic crosstalk within the protein domain. However, the mol. details of such intradomain allostery are still unclear. Here, we have exptl. detected and computationally predicted interaction pathways in the postsynaptic d. 95/disks large/zonula occludens 1 (PDZ)-peptide ligand model system using wild-type and circularly permuted PDZ proteins. The circular permutant introduced small perturbations in the tertiary structure and a concomitant rewiring of allosteric pathways, allowing us to describe how subtle changes may reshape energetic signaling. The results were analyzed in the context of other members of the PDZ family, which were found to contain distinct interaction pathways for different peptide ligands. The data reveal a fascinating scenario whereby several energetic pathways are sampled within one single domain and distinct pathways are activated by specific protein ligands.
- 32Petit, C. M.; Zhang, J.; Sapienza, P. J.; Fuentes, E. J.; Lee, A. L. Hidden dynamic allostery in a PDZ domain. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 18249– 18254, DOI: 10.1073/pnas.0904492106Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsVWrsrjO&md5=9ae15474e5320584dbe68dee381b2f5cHidden dynamic allostery in a PDZ domainPetit, Chad M.; Zhang, Jun; Sapienza, Paul J.; Fuentes, Ernesto J.; Lee, Andrew L.Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (43), 18249-18254CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Structure-function relationships in proteins are predicated on the spatial proximity of noncovalently interacting groups of atoms. Thus, structural elements located away from a protein's active site are typically presumed to serve a stabilizing or scaffolding role for the larger structure. Here we report a functional role for a distal structural element in a PDZ domain, even though it is not required to maintain PDZ structure. The third PDZ domain from PSD-95/SAP90 (PDZ3) has an unusual addnl. third alpha helix (α3) that packs in contiguous fashion against the globular domain. Although α3 lies outside the active site and does not make direct contact with C-terminal peptide ligand, removal of α3 reduces ligand affinity by 21-fold. Further investigation revealed that the difference in binding free energies between the full-length and truncated constructs is predominantly entropic in nature and that without α3, picosecond-nanosecond side-chain dynamics are enhanced throughout the domain, as detd. by 2H Me NMR relaxation. Thus, the distal modulation of binding function appears to occur via a delocalized conformational entropy mechanism. Without removal of α3 and characterization of side-chain dynamics, this dynamic allostery would have gone unnoticed. Moreover, what appeared at first to be an artificial modification of PDZ3 has been corroborated by exptl. verified phosphorylation of α3, revealing a tangible biol. mechanism for this novel regulatory scheme. This hidden dynamic allostery raises the possibility of as-yet unidentified or untapped allosteric regulation in this PDZ domain and is a very clear example of function arising from dynamics rather than from structure.
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Abstract
Figure 1
Figure 1. Flowchart depicting the organization of modules in the pPerturb web server. Once the protein structure is loaded into the server, perturbation profiles at the level of individual residues are generated for individual residues using eq 2, following which residue-specific parameters are provided for selected residues (perturbation profile) or all residues in the protein (interaction network profile). The residue-specific parameters are then colored on the protein structure to generate publication-quality images. Users can also request the prediction of changes in stability involving truncation mutations of uncharged residues wherein the mutational effects are introduced via eq 2. The model output can be downloaded as text files or high-resolution images.
Figure 2
Figure 2. Structural model of GPCR NTSR1 (PDB 6OS9) without (panel A) and with ∑ΔQ mapped on to the structure (panel B). The structure in panel B is colored in the spectral scale between the two extremes of well-packed residues (red) and weakly packed residues (blue). Note the stretch of dark blue in the TM helices 5 and 6 pointing to weak packing.
Figure 3
Figure 3. Left column presents a superimposition of ligand/inhibitor-unbound and -bound structures of the proteins bACBP (PDB ids 2ABD/1ACA for ligand-unbound and -bound states), PDZ3 (1BFE/1BE9), and PFK (3PFK/6PFK for inhibitor-unbound and -bound states) in gray and light brown, respectively. The overall RMSD values (including Cα and side chain) between the bound (b) and unbound (u) forms are 2.3, 1.1, and 1.6 Å, respectively. The cartoons in the middle and right columns are colored in the spectral scale (red to blue as in the color bar provided) according to ∑ΔQb – ∑ΔQu. Spheres represent the Cα atoms of specific residues whose difference in ∑ΔQ fall in the extremes, with Z-score ≥ 1 in the middle column and Z-score ≤ −1 in the right column. Note that such vivid details in terms of packing differences (middle and right columns) cannot be extracted from structural superimposition alone (left column).
Figure 4
Figure 4. (A) Structure of ubiquitin (1UBQ) highlighting the position of I30 (cyan) together with first- and second-shell neighbors in blue and green, respectively. (B) Unfolding curves predicted by the WSME model for mutations at position 30 at pH 7.0 and 20 mM ionic strength by employing a 6 Å heavy-atom contact cutoff including the nearest neighbors.
References
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- 1Baase, W. A.; Eriksson, A. E.; Zhang, X. J.; Heinz, D. W.; Sauer, U.; Blaber, M.; Baldwin, E. P.; Wozniak, J. A.; Matthews, B. W. Dissection of protein structure and folding by directed mutagenesis. Faraday Discuss. 1992, 93, 173– 181, DOI: 10.1039/fd99293001731https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXlsFChtbc%253D&md5=07e0fdbfd1cf1b007a7948044d8e048aDissection of protein structure and folding by directed mutagenesisBaase, Walter A.; Eriksson, A. Elisabeth; Zhang, Xue Jun; Heinz, Dirk W.; Sauer, Uwe; Blaber, Michael; Baldwin, Enoch P.; Wozniak, Joan A.; Matthews, Brian W.Faraday Discussions (1992), 93 (Structure and Activity of Enzymes), 173-81CODEN: FDISE6; ISSN:1359-6640.The lysozyme from bacteriophage T4 is being used as a model system to det. the roles of individual amino acids in the folding and stability of a typical globular protein. One general finding is that the protein is very adaptable, being able to accommodate many potentially destabilizing replacements. In order to det. the importance of 'α-helix propensity' in protein stability, different replacements have been made within α-helical segments of T4 lysozyme. Several such substitutions of the form Xaa → Ala increase the stability of the protein, supporting the idea that alanine is a strongly helix-favoring amino acid. It is possible to engineer a protein that has up to ten alanines in succession, yet still folds and has normal activity. This illustrates the redundancy that is present in the amino acid sequence. A no. of 'cavity-creating' mutants of the form Leu → Ala have been constructed to understand better the nature of hydrophobic stabilization. The structural consequences of these mutations differ from site to site. In some cases the protein structure hardly changes at all; in other cases removal of the wild-type side-chain allows surrounding atoms to move in and occupy the vacated space, although a cavity always remains. The destabilization of the protein assocd. with these cavity-creating mutations also varies from case to case. The results suggest how to reconcile recent conflicting reports concerning the strength of the hydrophobic effect in proteins.
- 2Eriksson, A. E.; Baase, W. A.; Zhang, X. J.; Heinz, D. W.; Blaber, M.; Baldwin, E. P.; Matthews, B. W. Response of a protein structure to cavity-creating mutations and its relation to the hydrophobic effect. Science 1992, 255, 178– 183, DOI: 10.1126/science.15535432https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XnvVynsg%253D%253D&md5=85479f782e001b1932949e2c1fd14ce9Response of a protein structure to cavity-creating mutations and its relation to the hydrophobic effectEriksson, A. E.; Baase, W. A.; Zhang, X. J.; Heinz, D. W.; Blaber, M.; Baldwin, E. P.; Matthews, B. W.Science (Washington, DC, United States) (1992), 255 (5041), 178-83CODEN: SCIEAS; ISSN:0036-8075.Six cavity-creating mutants, Leu46 → Ala (L46A), L99A, L118A, L121A, L133A, and Phe153 → Ala (F153A), were constructed within the hydrophobic core of phage T4 lysozyme. The substitutions decreased the stability of the protein at pH 3.0 by different amts., ranging from 2.7 kcal per mol (kcal mol-1) for L46A and L121A to 5.0 kcal mol-1 for L99A. The double mutant L99A/F153A was also constructed and decreased in stability by 8.3 kcal mol-1. The x-ray structures of all of the variants were detd. at high resoln. In every case, removal of the wild-type side chain allowed some of the surrounding atoms to move toward the vacated space but a cavity always remained, which ranged in vol. from 24 cubic angstroms (Å3) for L46A to 150 Å3 for L99A. No solvent mols. were obsd. in any of these cavities. The destabilization of the mutant Leu → Ala proteins relative to wild type can be approximated by a const. term (∼2.0 kcal mol-1) plus a term that increases in proportion to the size of the cavity. The const. term is approx. equal to the transfer free energy of leucine relative to alanine as detd. from partitioning between aq. and org. solvents. The energy term that increases with the size of the cavity can be expressed either in terms of the cavity vol. (24 to 33 cal mol1-1 Å-3) or in terms of the cavity surface area (20 cal mol-1 Å-2). The results suggest how to reconcile a no. of conflicting reports concerning the strength of the hydrophobic effect in proteins.
- 3Rocklin, G. J.; Chidyausiku, T. M.; Goreshnik, I.; Ford, A.; Houliston, S.; Lemak, A.; Carter, L.; Ravichandran, R.; Mulligan, V. K.; Chevalier, A.; Arrowsmith, C. H.; Baker, D. Global analysis of protein folding using massively parallel design, synthesis, and testing. Science 2017, 357, 168– 175, DOI: 10.1126/science.aan06933https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFOjs7rK&md5=0c089edbcc1309b72f412cfe72d149cfGlobal analysis of protein folding using massively parallel design, synthesis, and testingRocklin, Gabriel J.; Chidyausiku, Tamuka M.; Goreshnik, Inna; Ford, Alex; Houliston, Scott; Lemak, Alexander; Carter, Lauren; Ravichandran, Rashmi; Mulligan, Vikram K.; Chevalier, Aaron; Arrowsmith, Cheryl H.; Baker, DavidScience (Washington, DC, United States) (2017), 357 (6347), 168-175CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Proteins fold into unique native structures stabilized by thousands of weak interactions that collectively overcome the entropic cost of folding. Although these forces are "encoded" in the thousands of known protein structures, "decoding" them is challenging because of the complexity of natural proteins that have evolved for function, not stability. We combined computational protein design, next-generation gene synthesis, and a high-throughput protease susceptibility assay to measure folding and stability for more than 15,000 de novo designed miniproteins, 1000 natural proteins, 10,000 point mutants, and 30,000 neg. control sequences. This anal. identified more than 2500 stable designed proteins in four basic folds - a no. sufficient to enable us to systematically examine how sequence dets. folding and stability in uncharted protein space. Iteration between design and expt. increased the design success rate from 6% to 47%, produced stable proteins unlike those found in nature for topologies where design was initially unsuccessful, and revealed subtle contributions to stability as designs became increasingly optimized. Our approach achieves the long-standing goal of a tight feedback cycle between computation and expt. and has the potential to transform computational protein design into a data-driven science.
- 4Boyken, S. E.; Benhaim, M. A.; Busch, F.; Jia, M.; Bick, M. J.; Choi, H.; Klima, J. C.; Chen, Z.; Walkey, C.; Mileant, A.; Sahasrabuddhe, A.; Wei, K. Y.; Hodge, E. A.; Byron, S.; Quijano-Rubio, A.; Sankaran, B.; King, N. P.; Lippincott-Schwartz, J.; Wysocki, V. H.; Lee, K. K.; Baker, D. De novo design of tunable, pH-driven conformational changes. Science 2019, 364, 658– 664, DOI: 10.1126/science.aav78974https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXpvVChtbw%253D&md5=d58ec17696ffd269599466f1cf1c379aDe novo design of tunable, pH-driven conformational changesBoyken, Scott E.; Benhaim, Mark A.; Busch, Florian; Jia, Mengxuan; Bick, Matthew J.; Choi, Heejun; Klima, Jason C.; Chen, Zibo; Walkey, Carl; Mileant, Alexander; Sahasrabuddhe, Aniruddha; Wei, Kathy Y.; Hodge, Edgar A.; Byron, Sarah; Quijano-Rubio, Alfredo; Sankaran, Banumathi; King, Neil P.; Lippincott-Schwartz, Jennifer; Wysocki, Vicki H.; Lee, Kelly K.; Baker, DavidScience (Washington, DC, United States) (2019), 364 (6441), 658-664CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The ability of naturally occurring proteins to change conformation in response to environmental changes is crit. to biol. function. Although there have been advances in the de novo design of stable proteins with a single, deep free-energy min., the design of conformational switches remains challenging. We present a general strategy to design pH-responsive protein conformational changes by precisely preorganizing histidine residues in buried hydrogen-bond networks. We design homotrimers and heterodimers that are stable above pH 6.5 but undergo cooperative, large-scale conformational changes when the pH is lowered and electrostatic and steric repulsion builds up as the network histidine residues become protonated. The transition pH and cooperativity can be controlled through the no. of histidine-contg. networks and the strength of the surrounding hydrophobic interactions. Upon disassembly, the designed proteins disrupt lipid membranes both in vitro and after being endocytosed in mammalian cells. Our results demonstrate that environmentally triggered conformational changes can now be programmed by de novo protein design.
- 5Narayan, A.; Gopi, S.; Fushman, D.; Naganathan, A. N. A binding cooperativity switch driven by synergistic structural swelling of an osmo-regulatory protein pair. Nat. Commun. 2019, 10, 1995 DOI: 10.1038/s41467-019-10002-95https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3M7gvFeqsA%253D%253D&md5=326ce989246f23c2e4266107d34f78adA binding cooperativity switch driven by synergistic structural swelling of an osmo-regulatory protein pairNarayan Abhishek; Gopi Soundhararajan; Naganathan Athi N; Fushman DavidNature communications (2019), 10 (1), 1995 ISSN:.Uropathogenic E. coli experience a wide range of osmolarity conditions before and after successful infection. Stress-responsive regulatory proteins in bacteria, particularly proteins of the Hha family and H-NS, a transcription repressor, sense such osmolarity changes and regulate transcription through unknown mechanisms. Here we use an array of experimental probes complemented by molecular simulations to show that Cnu, a member of the Hha protein family, acts as an exquisite molecular sensor of solvent ionic strength. The osmosensory behavior of Cnu involves a fine-tuned modulation of disorder in the fourth helix and the three-dimensional structure in a graded manner. Order-disorder transitions in H-NS act synergistically with molecular swelling of Cnu contributing to a salt-driven switch in binding cooperativity. Thus, sensitivity to ambient conditions can be imprinted at the molecular level by tuning not just the degree of order in the protein conformational ensemble but also through population redistributions of higher-order molecular complexes.
- 6Luque, I.; Leavitt, S. A.; Freire, E. The linkage between protein folding and functional cooperativity: two sides of the same coin?. Annu. Rev. Biophys. Biomol. Struct. 2002, 31, 235– 256, DOI: 10.1146/annurev.biophys.31.082901.1342156https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XltVynurY%253D&md5=431f37939924e89292a6316fb538c09eThe linkage between protein folding and functional cooperativity: two sides of the same coin?Luque, Irene; Leavitt, Stephanie A.; Freire, ErnestoAnnual Review of Biophysics and Biomolecular Structure (2002), 31 (), 235-256CODEN: ABBSE4; ISSN:1056-8700. (Annual Reviews Inc.)A review with 81 refs. During the course of their biol. function, proteins undergo different types of structural rearrangements ranging from local to large-scale conformational changes. These changes are usually triggered by their interactions with small-mol.-wt. ligands or other macromols. Because binding interactions occur at specific sites and involve only a small no. of residues, a chain of cooperative interactions is necessary for the propagation of binding signals to distal locations within the protein structure. This process requires an uneven structural distribution of protein stability and cooperativity as revealed by NMR-detected 1H/2H exchange expts. under native conditions. The distribution of stabilizing interactions does not only provide the architectural foundation to the 3-dimensional structure of a protein, but it also provides the required framework for functional cooperativity. In this review, the statistical thermodn. linkage between protein stability, functional cooperativity, and ligand binding is discussed.
- 7Di Paola, L.; Giuliani, A. Protein contact network topology: a natural language for allostery. Curr. Opin. Struct. Biol. 2015, 31, 43– 48, DOI: 10.1016/j.sbi.2015.03.0017https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXktFOktr0%253D&md5=6436f436974d812914a3e2815d9b0166Protein contact network topology: a natural language for allosteryDi Paola, Luisa; Giuliani, AlessandroCurrent Opinion in Structural Biology (2015), 31 (), 43-48CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. Protein mols. work as a whole, so that any local perturbation may resonate on the entire structure; allostery deals with this general property of protein mols. It is worth noting that a perturbation does not necessarily involve a conformational change but, more generally, it travels across the structure as an 'energy signal'. The at. interactions within the network provide the structural support for this 'signaling highway'. Network descriptors, capturing network signaling efficiency, explain allostery in terms of signal transmission. Here, the authors survey the key applications of graph theory to protein allostery. The complex network approach introduces a new perspective in biochem.; as for applications, the development of new drugs relying on allosteric effects (allo-network drugs) represents a promising avenue of contact network formalism.
- 8Dokholyan, N. V. Controlling Allosteric Networks in Proteins. Chem. Rev. 2016, 116, 6463– 6487, DOI: 10.1021/acs.chemrev.5b005448https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XivVeltL0%253D&md5=9294593fdeb42a59d015bd84a10091b9Controlling Allosteric Networks in ProteinsDokholyan, Nikolay V.Chemical Reviews (Washington, DC, United States) (2016), 116 (11), 6463-6487CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review on the phys. and evolutionary origin of protein allostery defined as conformational changes induced by ligand binding, as well as its importance to protein regulation, drug discovery, and biol. processes in living systems; including exptl. and computational protein engineering approaches for control of protein function by modulation of allosteric sites.
- 9Naganathan, A. N. Modulation of allosteric coupling by mutations: from protein dynamics and packing to altered native ensembles and function. Curr. Opin. Struct. Biol. 2019, 54, 1– 9, DOI: 10.1016/j.sbi.2018.09.0049https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslCju73I&md5=dfc261b3d6bd5d7b9764bb34d84a7bf5Modulation of allosteric coupling by mutations: from protein dynamics and packing to altered native ensembles and functionNaganathan, Athi N.Current Opinion in Structural Biology (2019), 54 (), 1-9CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. A large body of work has gone into understanding the effect of mutations on protein structure and function. Conventional treatments have involved quantifying the change in stability, activity and relaxation rates of the mutants with respect to the wild-type protein. However, it is now becoming increasingly apparent that mutational perturbations consistently modulate the packing and dynamics of a significant fraction of protein residues, even those that are located >10-15 Å from the mutated site. Such long-range modulation of protein features can distinctly tune protein stability and the native conformational ensemble contributing to allosteric modulation of function. In this review, I summarize a series of exptl. and computational observations that highlight the incredibly pliable nature of proteins and their response to mutational perturbations manifested via the intra-protein interaction network. I highlight how an intimate understanding of mutational effects could pave the way for integrating stability, folding, cooperativity and even allostery within a single phys. framework.
- 10Guarnera, E.; Berezovsky, I. N. On the perturbation nature of allostery: sites, mutations, and signal modulation. Curr. Opin. Struct. Biol. 2019, 56, 18– 27, DOI: 10.1016/j.sbi.2018.10.00810https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitFSjs7vI&md5=2c6c003783e53393d435df408a0e9b35On the perturbation nature of allostery: sites, mutations, and signal modulationGuarnera, Enrico; Berezovsky, Igor N.Current Opinion in Structural Biology (2019), 56 (), 18-27CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. Regardless of the diversity of systems, allosteic signaling is found to be always caused by perturbations. This recurring trait of allostery serves as a foundation for developing different exptl. efforts and theor. models for the studies of allosteric mechanisms. Among computational approaches considered here particular emphasis is given to the structure-based statistical mech. model of allostery (SBSMMA), which allows one to study the causality and energetics of allosteric communication. We argue that the reverse allosteric signaling on the basis of SBSMMA can be used for predicting latent allosteric sites and inducing a tunable allosteric response. Per-residue allosteric effects of mutations can also be explored and 'latent drivers' expanding the cancer mutational landscape can be predicted using SBSMMA. Most recent and important implementations of computational models in web-resources along with a brief outlook on future research directions are also discussed.
- 11Ben-David, M.; Huang, H.; Sun, M. G. F.; Corbi-Verge, C.; Petsalaki, E.; Liu, K.; Gfeller, D.; Garg, P.; Tempel, W.; Sochirca, I.; Shifman, J. M.; Davidson, A.; Min, J.; Kim, P. M.; Sidhu, S. S. Allosteric Modulation of Binding Specificity by Alternative Packing of Protein Cores. J. Mol. Biol. 2019, 431, 336– 350, DOI: 10.1016/j.jmb.2018.11.01811https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitlent7vJ&md5=514b0ebac210a850c301183bbf680382Allosteric Modulation of Binding Specificity by Alternative Packing of Protein CoresBen-David, Moshe; Huang, Haiming; Sun, Mark G. F.; Corbi-Verge, Carles; Petsalaki, Evangelia; Liu, Ke; Gfeller, David; Garg, Pankaj; Tempel, Wolfram; Sochirca, Irina; Shifman, Julia M.; Davidson, Alan; Min, Jinrong; Kim, Philip M.; Sidhu, Sachdev S.Journal of Molecular Biology (2019), 431 (2), 336-350CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)Hydrophobic cores are often viewed as tightly packed and rigid, but they do show some plasticity and could thus be attractive targets for protein design. Here we explored the role of different functional pressures on the core packing and ligand recognition of the SH3 domain from human Fyn tyrosine kinase. We randomized the hydrophobic core and used phage display to select variants that bound to each of three distinct ligands. The three evolved groups showed remarkable differences in core compn., illustrating the effect of different selective pressures on the core. Changes in the core did not significantly alter protein stability, but were linked closely to changes in binding affinity and specificity. Structural anal. and mol. dynamics simulations revealed the structural basis for altered specificity. The evolved domains had significantly reduced core vols., which in turn induced increased backbone flexibility. These motions were propagated from the core to the binding surface and induced significant conformational changes. These results show that alternative core packing and consequent allosteric modulation of binding interfaces could be used to engineer proteins with novel functions.
- 12Petrović, D.; Risso, V. A.; Kamerlin, S. C. L.; Sanchez-Ruiz, J. M. Conformational dynamics and enzyme evolution. J. R. Soc., Interface 2018, 15, 20180330 DOI: 10.1098/rsif.2018.033012https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXis1Cnsbg%253D&md5=aaf6752e8e06cd1d29a8668e6a241ad8Conformational dynamics and enzyme evolutionPetrovic, Dussan; Risso, Valeria A.; Kamerlin, Shina Caroline Lynn; Sanchez-Ruiz, Jose M.Journal of the Royal Society, Interface (2018), 15 (144), 20180330/1-20180330/18CODEN: JRSICU; ISSN:1742-5662. (Royal Society)Enzymes are dynamic entities, and their dynamic properties are clearly linked to their biol. function. It follows that dynamics ought to play an essential role in enzyme evolution. Indeed, a link between conformational diversity and the emergence of new enzyme functionalities has been recognized for many years. However, it is only recently that state-of-the-art computational and exptl. approaches are revealing the crucial mol. details of this link. Specifically, evolutionary trajectories leading to functional optimization for a given host environment or to the emergence of a new function typically involve enriching catalytically competent conformations and/or the freezing out of non-competent conformations of an enzyme. In some cases, these evolutionary changes are achieved through distant mutations that shift the protein ensemble towards productive conformations. Multifunctional intermediates in evolutionary trajectories are probably multi-conformational, i.e. able to switch between different overall conformations, each competent for a given function. Conformational diversity can assist the emergence of a completely new active site through a single mutation by facilitating transition-state binding. We propose that this mechanism may have played a role in the emergence of enzymes at the primordial, progenote stage, where it was plausibly promoted by high environmental temps. and the possibility of addnl. phenotypic mutations.
- 13Tokuriki, N.; Tawfik, D. S. Stability effects of mutations and protein evolvability. Curr. Opin. Struct. Biol. 2009, 19, 596– 604, DOI: 10.1016/j.sbi.2009.08.00313https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXht1ylsLrP&md5=cd22e7bf4b26369fbb2dc517caa52e91Stability effects of mutations and protein evolvabilityTokuriki, Nobuhiko; Tawfik, Dan S.Current Opinion in Structural Biology (2009), 19 (5), 596-604CODEN: COSBEF; ISSN:0959-440X. (Elsevier B.V.)A review. The past several years have seen novel insights at the interface of protein biophysics and evolution. The accepted paradigm that proteins can tolerate nearly any amino acid substitution has been replaced by the view that the deleterious effects of mutations, and esp. their tendency to undermine the thermodn. and kinetic stability of protein, is a major constraint on protein evolvability, i.e, the ability of proteins to acquire changes in sequence and function. The authors summarize recent findings regarding how mutations affect protein stability, and how stability affects protein evolution. The authors describe ways of predicting and analyzing stability effects of mutations, and mechanisms that buffer or compensate for these destabilizing effects and thereby promote protein evolvability, in Nature and in the lab.
- 14Muñoz, V.; Campos, L. A.; Sadqi, M. Limited cooperativity in protein folding. Curr. Opin. Struct. Biol. 2016, 36, 58– 66, DOI: 10.1016/j.sbi.2015.12.00114https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVGrtrfL&md5=20d2c592462d935d7b4040caa049204aLimited cooperativity in protein foldingMunoz, Victor; Campos, Luis A.; Sadqi, MouradCurrent Opinion in Structural Biology (2016), 36 (), 58-66CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)A review. Theory and simulations predict that the structural concert of protein folding reactions is relatively low. Exptl., folding cooperativity has been difficult to study, but in recent years we have witnessed major advances. New anal. procedures in terms of conformational ensembles rather than discrete states, exptl. techniques with improved time, structural, or single-mol. resoln., and combined thermodn. and kinetic anal. of fast folding have contributed to demonstrate a general scenario of limited cooperativity in folding. Gradual structural disorder is already apparent on the unfolded and native states of slow, two-state folding proteins, and it greatly increases in magnitude for fast folding domains. These results demonstrate a direct link between how fast a single-domain protein folds and unfolds, and how cooperative (or structurally diverse) is its equil. unfolding process. Reducing cooperativity also destabilizes the native structure because it affects unfolding more than folding. We can thus define a continuous cooperativity scale that goes from the 'pliable' two-state character of slow folders to the gradual unfolding of one-state downhill, and eventually to intrinsically disordered proteins. The connection between gradual unfolding and intrinsic disorder is appealing because it suggests a conformational rheostat mechanism to explain the allosteric effects of folding coupled to binding.
- 15Chan, H. S.; Shimizu, S.; Kaya, H. Cooperativity principles in protein folding. Methods Enzymol. 2004, 380, 350– 379, DOI: 10.1016/S0076-6879(04)80016-815https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhs1Omurg%253D&md5=d28669191744b8cc3939b9d051a2f9e4Cooperativity principles in protein foldingChan, Hue Sun; Shimizu, Seishi; Kaya, HuseyinMethods in Enzymology (2004), 380 (Energetics of Biological Macromolecules, Part E), 350-379CODEN: MENZAU; ISSN:0076-6879. (Elsevier)A review. Extensive evaluations of coarse grained chain models suggest that the high degrees of thermodn. and kinetic cooperativity of small single-domain proteins may likely originate from many-body interactions in the form of a coupling between local conformational preferences and favorable nonlocal interactions. A discussion on the recent advances in these respects is presented.
- 16Stein, A.; Fowler, D. M.; Hartmann-Petersen, R.; Lindorff-Larsen, K. Biophysical and Mechanistic Models for Disease-Causing Protein Variants. Trends Biochem. Sci. 2019, 44, 575– 588, DOI: 10.1016/j.tibs.2019.01.00316https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsVOqtbc%253D&md5=15b7d25b0dd21d04bc0bfcd873461fd7Biophysical and Mechanistic Models for Disease-Causing Protein VariantsStein, Amelie; Fowler, Douglas M.; Hartmann-Petersen, Rasmus; Lindorff-Larsen, KrestenTrends in Biochemical Sciences (2019), 44 (7), 575-588CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)A review. The rapid decrease in DNA sequencing cost is revolutionizing medicine and science. In medicine, genome sequencing has revealed millions of missense variants that change protein sequences, yet we only understand the mol. and phenotypic consequences of a small fraction. Within protein science, high-throughput deep mutational scanning expts. enable us to probe thousands of variants in a single, multiplexed expt. We review efforts that bring together these topics via exptl. and computational approaches to det. the consequences of missense variants in proteins. We focus on the role of changes in protein stability as a driver for disease, and how expts., biophys. models, and computation are providing a framework for understanding and predicting how changes in protein sequence affect cellular protein stability.
- 17Rajasekaran, N.; Suresh, S.; Gopi, S.; Raman, K.; Naganathan, A. N. A general mechanism for the propagation of mutational effects in proteins. Biochemistry 2017, 56, 294– 305, DOI: 10.1021/acs.biochem.6b0079817https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XitVKktLjF&md5=caf6021e1d6ba8325e2c2b66ae895dbfA General Mechanism for the Propagation of Mutational Effects in ProteinsRajasekaran, Nandakumar; Suresh, Swaathiratna; Gopi, Soundhararajan; Raman, Karthik; Naganathan, Athi N.Biochemistry (2017), 56 (1), 294-305CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Mutations in the hydrophobic interior of proteins are generally thought to weaken the interactions only in their immediate neighborhood. This forms the basis of protein-engineering based studies of folding mechanism and function. However, mutational work on diverse proteins has shown that distant residues are thermodynamically coupled, with the network of interactions within the protein acting as signal conduits, thus raising an intriguing paradox. Are mutational effects localized and if not, is there a general rule for the extent of percolation and on the functional form of this propagation. We explore these questions from multiple perspectives in the current work. Perturbation anal. of interaction networks within proteins and microsecond-long mol. dynamics simulations of several aliph. mutants of ubiquitin reveal strong evidence for distinct alteration of distal residue-residue communication networks. We find that mutational effects consistently propagate into the second shell of the altered site (even up to 15-20 Å) in proportion to the perturbation magnitude and dissipates exponentially with a decay distance-const. of ∼4-5 Å. We also report evidence for this phenomenon from published exptl. NMR data that strikingly resemble predictions from network theory and MD simulations. Reformulating these observations onto a statistical mech. model, we reproduce the stability changes of 375 mutations from 19 single-domain proteins. Our work thus reveals a robust energy dissipation-cum-signaling mechanism in the interaction network within proteins, quantifies the partitioning of destabilization energetics around the mutation neighborhood and presents a simple theor. framework for modeling the allosteric effects of point mutations.
- 18Rajasekaran, N.; Sekhar, A.; Naganathan, A. N. A Universal Pattern in the Percolation and Dissipation of Protein Structural Perturbations. J. Phys. Chem. Lett. 2017, 8, 4779– 4784, DOI: 10.1021/acs.jpclett.7b0202118https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsV2js7rO&md5=3161d8efc35d5538417762478cd126a3A Universal Pattern in the Percolation and Dissipation of Protein Structural PerturbationsRajasekaran, Nandakumar; Sekhar, Ashok; Naganathan, Athi N.Journal of Physical Chemistry Letters (2017), 8 (19), 4779-4784CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)Understanding the extent to which information is transmitted through the intramol. interaction network of proteins upon a perturbation, i.e., an allosteric effect, has long remained an unsolved problem. Here, through an anal. of high-resoln. NMR data from the literature on 28 different proteins and 49 structural perturbations, we show that the extent of induced structural changes through mutations, and mol. events including protein-protein, protein-peptide, protein-ligand binding, and post-translational modifications exhibit a near-universal exponential functional form. The extent of percolation into the protein structures can be up to 20-25 Å despite no apparent change in the 3-dimensional structures. These observations were also consistent with theor. expectations, elementary graph theoretic anal. of protein structures, detailed mol. dynamics simulations, and exptl. double-mutant cycles. Our anal. highlighted that most mol. events would contribute to allosteric effects independent of protein structure, topol., or identity, and provides a simple avenue to test and potentially model their effects.
- 19Rajasekaran, N.; Naganathan, A. N. A self-consistent structural perturbation approach for determining the magnitude and extent of allosteric coupling in proteins. Biochem. J. 2017, 474, 2379– 2388, DOI: 10.1042/BCJ2017030419https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFygsrjJ&md5=ab41998037500c5be60e1e7a8d8a1522A self-consistent structural perturbation approach for determining the magnitude and extent of allosteric coupling in proteinsRajasekaran, Nandakumar; Naganathan, Athi N.Biochemical Journal (2017), 474 (14), 2379-2388CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)Elucidating the extent of energetic coupling between residues in single-domain proteins, which is a fundamental determinant of allostery, information transfer and folding cooperativity, has remained a grand challenge. While several sequence- and structure-based approaches have been proposed, a self-consistent description that is simultaneously compatible with unfolding thermodn. is lacking. We recently developed a simple structural perturbation protocol that captures the changes in thermodn. stabilities induced by point mutations within the protein interior. Here, we show that a fundamental residue-specific component of this perturbation approach, the coupling distance, is uniquely sensitive to the environment of a residue in the protein to a distance of ∼15 A[n.778]. With just the protein contact map as an input, we reproduce the extent of percolation of perturbations within the structure as obsd. in network anal. of intra-protein interactions, mol. dynamics simulations and NMR-obsd. changes in chem. shifts. Using this rapid protocol that relies on a single structure, we explain the results of statistical coupling anal. (SCA) that requires hundreds of sequences to identify functionally crit. sectors, the propagation and dissipation of perturbations within proteins and the higher-order couplings deduced from detailed NMR expts. Our results thus shed light on the possible mechanistic origins of signaling through the interaction network within proteins, the likely distance dependence of perturbations induced by ligands and post-translational modifications and the origins of folding cooperativity through many-body interactions.
- 20Yu, M.; Chen, Y.; Wang, Z. L.; Liu, Z. Fluctuation correlations as major determinants of structure- and dynamics-driven allosteric effects. Phys. Chem. Chem. Phys. 2019, 21, 5200– 5214, DOI: 10.1039/C8CP07859A20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXislKksb0%253D&md5=70c29c723f859c99a4f52455850b7d85Fluctuation correlations as major determinants of structure- and dynamics-driven allosteric effectsYu, Miao; Chen, Yixin; Wang, Zi-Le; Liu, ZhirongPhysical Chemistry Chemical Physics (2019), 21 (9), 5200-5214CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)Allosteric control is essential for regulating biol. functions whereby stimuli such as ligand binding at one site on a protein cause a response at a distant functional site. Correlations between different sites in proteins have been used widely in identifying allosteric sites and pathways, and in designing allosteric drugs. However, the deterministic connection between correlations and allostery remains unsolved, esp. considering that there are various types of correlations. Here, we combine perturbation-theory anal. and numerical calcns. to study both structure- and dynamics-driven allosteric effects in an anisotropic network model (ANM). The results reveal that the allosteries are detd. by the correlation (covariance) of distance fluctuations, but are irrelevant to the usual displacement correlations or time-delayed correlations. Dynamics-driven allostery is weaker than structure-driven allostery by at least one to two orders of magnitude. The intrinsic allostery capacity decays with distance by an exponential law, with the resulting characteristic distance parameter lying in the range of 7-10 Å for structure-driven allostery and 4-5 Å for dynamics-driven allostery. The importance of the cutoff distance of the ANM is also addressed.
- 21Medina-Carmona, E.; Betancor-Fernández, I.; Santos, J.; Mesa-Torres, N.; Grottelli, S.; Batlle, C.; Naganathan, A. N.; Oppici, E.; Cellini, B.; Ventura, S.; Salido, E.; Pey, A. L. Insight into the specificity and severity of pathogenic mechanisms associated with missense mutations through experimental and structural perturbation analyses. Hum. Mol. Genet. 2019, 28, 1– 15, DOI: 10.1093/hmg/ddy32321https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1CgsLrJ&md5=c32c562fe414bd244ac84bfdc2f0cd9aInsight into the specificity and severity of pathogenic mechanisms associated with missense mutations through experimental and structural perturbation analysesMedina-Carmona, Encarnacion; Betancor-Fernandez, Isabel; Santos, Jaime; Mesa-Torres, Noel; Grottelli, Silvia; Batlle, Cristina; Naganathan, Athi N.; Oppici, Elisa; Cellini, Barbara; Ventura, Salvador; Salido, Eduardo; Pey, Angel L.Human Molecular Genetics (2019), 28 (1), 1-15CODEN: HMGEE5; ISSN:1460-2083. (Oxford University Press)Most pathogenic missense mutations cause specific mol. phenotypes through protein destabilization. However, how protein destabilization is manifested as a given mol. phenotype is not well understood. We develop here a structural and energetic approach to describe mutational effects on specific traits such as function, regulation, stability, subcellular targeting or aggregation propensity. This approach is tested using large-scale exptl. and structural perturbation analyses in over thirty mutations in three different proteins (cancer-assocd. NQO1, transthyretin related with amyloidosis and AGT linked to primary hyperoxaluria type I) and comprising five very common pathogenic mechanisms (loss-of-function and gain-of-toxic function aggregation, enzyme inactivation, protein mistargeting and accelerated degrdn.). Our results revealed that the magnitude of destabilizing effects and, particularly, their propagation through the structure to promote disease-assocd. conformational states largely det. the severity and mol. mechanisms of disease-assocd. missense mutations. Modulation of the structural perturbation at a mutated site is also shown to cause switches between different mol. phenotypes. When very common disease-assocd. missense mutations were investigated, we also found that they were not among the most deleterious possible missense mutations at those sites, and required addnl. contributions from codon bias and effects of CpG sites to explain their high frequency in patients. Our work sheds light on the mol. basis of pathogenic mechanisms and genotype-phenotype relationships, with implications for discriminating between pathogenic and neutral changes within human genome variability from whole genome sequencing studies.
- 22Wako, H.; Saitô, N. Statistical Mechanical Theory of Protein Conformation.2. Folding Pathway for Protein. J. Phys. Soc. Jpn. 1978, 44, 1939– 1945, DOI: 10.1143/JPSJ.44.193922https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXkvVKjsLc%253D&md5=4cf3c7168e13ba4e51c78b6d1088b6e2Statistical mechanical theory of the protein conformation. II. Folding pathway for proteinWako, Hiroshi; Saito, NobuhikoJournal of the Physical Society of Japan (1978), 44 (6), 1939-45CODEN: JUPSAU; ISSN:0031-9015.The theory of a 1-dimensional lattice gas with long-range many-body interactions was applied to the folding pathways of proteins. This model presents the quant. informations about the order of inter-residue interactions, the location of nucleation, and so on. Three typical proteins are cited as examples and their folding pathways are traced according to the computation of some useful properties.
- 23Muñoz, V.; Eaton, W. A. A simple model for calculating the kinetics of protein folding from three-dimensional structures. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11311– 11316, DOI: 10.1073/pnas.96.20.1131123https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXmvVCmsL8%253D&md5=1adf1ff76d654bab4842b90e0685e5acA simple model for calculating the kinetics of protein folding from three-dimensional structuresMunoz, Victor; Eaton, William A.Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (20), 11311-11316CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)An elementary statistical mech. model was used to calc. the folding rates for 22 proteins from their known three-dimensional structures. In this model, residues come into contact only after all of the intervening chain is in the native conformation. An addnl. simplifying assumption is that native structure grows from localized regions that then fuse to form the complete native mol. The free energy function for this model contains just two contributions-conformational entropy of the backbone and the energy of the inter-residue contacts. The matrix of interresidue interactions is obtained from the at. coordinates of the three-dimensional structure. For the 18 proteins that exhibit two-state equil. and kinetic behavior, profiles of the free energy vs. the no. of native peptide bonds show two deep min., corresponding to the native and denatured states. For four proteins known to exhibit intermediates in folding, the free energy profiles show addnl. deep min. The calcd. rates of folding the two-state proteins, obtained by solving a diffusion equation for motion on the free energy profiles, reproduce the exptl. detd. values surprisingly well. The success of these calcns. suggests that folding speed is largely detd. by the distribution and strength of contacts in the native structure. We also calcd. the effect of mutations on the folding kinetics of chymotrypsin inhibitor 2, the most intensively studied two-state protein, with some success.
- 24Naganathan, A. N. Predictions from an Ising-like Statistical Mechanical Model on the Dynamic and Thermodynamic Effects of Protein Surface Electrostatics. J. Chem. Theory Comput. 2012, 8, 4646– 4656, DOI: 10.1021/ct300676w24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsVOhs7bK&md5=61da68db7a56f01b51157ec9d42e3a89Predictions from an Ising-like Statistical Mechanical Model on the Dynamic and Thermodynamic Effects of Protein Surface ElectrostaticsNaganathan, Athi N.Journal of Chemical Theory and Computation (2012), 8 (11), 4646-4656CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)Charged residues on the surface of a protein are known hot-spots for post-translational modification, protein/ligand-binding, and tuning conformational stabilities. Recent exptl. evidence points to the fact that surface electrostatics can also modulate thermodn. barriers and hence folding mechanisms. To probe for this behavior across different proteins, we develop a novel version of the Wako-Sait̂o-Mu~noz-Eaton (WSME) model in which we include an electrostatic potential term in the energy function while simplifying the treatment of solvation free energy. Both of the energy terms are obtained by quant. fitting the model to differential scanning calorimetry (DSC) expts. that carry crit. information on the protein partition function. We characterize four sets of structural/functional homologs (HEWL/BLA, CspB, engrailed, α-spectrin) either by fitting the exptl. data of a single domain in the homologous set and predicting the conformational behavior of the rest with the same set of parameters or by performing semiblind predictions. The model with the added electrostatic term is able to successfully reproduce the order of thermodn. stabilities and relaxation rates of most of the homologs. In parallel, we predict diverse conformational features including a wide range of thermodn. barriers (∼9-40 kJ/mol), broad native ensembles in helical proteins, structured unfolded states and intermediates, rugged folding landscapes, and further provide an independent protein-specific est. of the folding speed limit at 298 K (1/(7-300 μs)). Our results are evidence that protein surface electrostatics can be tailored to not only engineer stabilities but also folding mechanisms and the ruggedness of the underlying landscape.
- 25Naganathan, A. N. A Rapid, Ensemble and Free Energy Based Method for Engineering Protein Stabilities. J. Phys. Chem. B 2013, 117, 4956– 4964, DOI: 10.1021/jp401588x25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXltVOmtbs%253D&md5=bb43890bae36523073911fda03e09062A Rapid, Ensemble and Free Energy Based Method for Engineering Protein StabilitiesNaganathan, Athi N.Journal of Physical Chemistry B (2013), 117 (17), 4956-4964CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)Engineering the conformational stabilities of proteins through mutations has immense potential in biotechnol. applications. It is, however, an inherently challenging problem given the weak noncovalent nature of the stabilizing interactions. In this regard, we present here a robust and fast strategy to engineer protein stabilities through mutations involving charged residues using a structure-based statistical mech. model that accounts for the ensemble nature of folding. We validate the method by predicting the abs. changes in stability for 138 exptl. mutations from 16 different proteins and enzymes with a correlation of 0.65 and importantly with a success rate of 81%. Multiple point mutants are predicted with a higher success rate (90%) that is validated further by comparing mesophile-thermophile protein pairs. In parallel, we devise a methodol. to rapidly engineer mutations in silico which we benchmark against exptl. mutations of ubiquitin (correlation of 0.95) and check for its feasibility on a larger therapeutic protein DNase I. We expect the method to be of importance as a first and rapid step to screen for protein mutants with specific stability in the biotechnol. industry, in the construction of stability maps at the residue level (i.e., hot spots), and as a robust tool to probe for mutations that enhance the stability of protein-based drugs.
- 26Gopi, S.; Devanshu, D.; Krishna, P.; Naganathan, A. N. pStab: prediction of stable mutants, unfolding curves, stability maps and protein electrostatic frustration. Bioinformatics 2018, 34, 875– 877, DOI: 10.1093/bioinformatics/btx69726https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitlemtbzL&md5=bfc07fdf1c6bef731acf5b149201c4d6pStab: prediction of stable mutants, unfolding curves, stability maps and protein electrostatic frustrationGopi, Soundhararajan; Devanshu, Devanshu; Krishna, Praveen; Naganathan, Athi N.Bioinformatics (2018), 34 (5), 875-877CODEN: BOINFP; ISSN:1367-4811. (Oxford University Press)Summary: We present a web-server for rapid prediction of changes in protein stabilities over a range of temps. and exptl. conditions upon single- or multiple-point substitutions of charged residues. Potential mutants are identified by a charge-shuffling procedure while the stability changes (i.e. an unfolding curve) are predicted employing an ensemble-based statistical-mech. model. We expect this server to be a simple yet detailed tool for engineering stabilities, identifying electrostatically frustrated residues, generating local stability maps and in constructing fitness landscapes.
- 27Kato, H. E.; Zhang, Y.; Hu, H.; Suomivuori, C. M.; Kadji, F. M. N.; Aoki, J.; Krishna Kumar, K.; Fonseca, R.; Hilger, D.; Huang, W.; Latorraca, N. R.; Inoue, A.; Dror, R. O.; Kobilka, B. K.; Skiniotis, G. Conformational transitions of a neurotensin receptor 1-Gi1 complex. Nature 2019, 572, 80– 85, DOI: 10.1038/s41586-019-1337-627https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXht1yktL%252FL&md5=0ce265e5a9dc353e69d2c04032803399Conformational transitions of a neurotensin receptor 1-Gi1 complexKato, Hideaki E.; Zhang, Yan; Hu, Hongli; Suomivuori, Carl-Mikael; Kadji, Francois Marie Ngako; Aoki, Junken; Krishna Kumar, Kaavya; Fonseca, Rasmus; Hilger, Daniel; Huang, Weijiao; Latorraca, Naomi R.; Inoue, Asuka; Dror, Ron O.; Kobilka, Brian K.; Skiniotis, GeorgiosNature (London, United Kingdom) (2019), 572 (7767), 80-85CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Neurotensin receptor 1 (NTSR1) is a G-protein-coupled receptor (GPCR) that engages multiple subtypes of G protein, and is involved in the regulation of blood pressure, body temp., wt. and the response to pain. Here we present structures of human NTSR1 in complex with the agonist JMV449 and the heterotrimeric Gi1 protein, at a resoln. of 3 Å. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR-Gi/o complexes (in which the nucleotide-binding pocket adopts more flexible conformations that may facilitate nucleotide exchange), and a non-canonical state in which the G protein is rotated by about 45 degrees relative to the receptor and exhibits a more rigid nucleotide-binding pocket. In the non-canonical state, NTSR1 exhibits features of both active and inactive conformations, which suggests that the structure may represent an intermediate form along the activation pathway of G proteins. This structural information, complemented by mol. dynamics simulations and functional studies, provides insights into the complex process of G-protein activation.
- 28Kragelund, B. B.; Andersen, K. V.; Madsen, J. C.; Knudsen, J.; Poulsen, F. M. Three-dimensional structure of the complex between acyl-coenzyme A binding protein and palmitoyl-coenzyme A. J. Mol. Biol. 1993, 230, 1260– 1277, DOI: 10.1006/jmbi.1993.124028https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXkvV2gtrk%253D&md5=05aed1a07a17f17754d976b611755097Three-dimensional structure of the complex between acyl-coenzyme A binding protein and palmitoyl-coenzyme AKragelund, Birth B.; Andersen, Kim Vilbour; Madsen, Jens Chr.; Knudsen, Jens; Poulsen, Flemming M.Journal of Molecular Biology (1993), 230 (4), 1260-77CODEN: JMOBAK; ISSN:0022-2836.Multidimensional 1H, 13C, and 15N NMR spectroscopy was used to study the complex between palmitoyl-CoA and acyl-CoA binding protein (ACBP). The 1H and 15N spectra of holo-ACBP were almost completely assigned and so was most of the 1H spectrum of the CoA part of the protein-bound ligand. The palmitoyl part of the ligand was uniformly labeled with 13C and the NMR signals of the C atoms and their protons were assigned at the 2 ends of the hydrocarbon chain. A total of 1251 distance restraints from NOE and 131 dihedral angle restraints from 3-bond coupling consts. provided the basis for the structure calcn. A comparison of 20 structures calcd. from these data to the av. structure showed that they could be aligned with an at. root-mean-square deviation (r.m.s.d.) of 1.3 Å for all C, N, O, P and S atoms in protein and ligand. Apo-ACBP was a 4-helix protein and this structure was maintained in holo-ACBP. The 4 α-helixes were: Ac1 for residues 3-15; Ac2 for residues 20-36; Ac3 for residues 51-62; and Ac4 for residues 65-84. For the 4 α-helixes of the peptide backbone of holo-ACBP, the r.m.s.d. for the C, Cα, and N atoms was 0.42 Å. The binding site for the palmitoyl chain stretched between the N-terminal end of Ac3 where the carboxyl part binds, to the N-terminal of Ac3 where the ω-end of the palmitoyl part binds. The 3'-AMP was bound near residues of each of the 4 helixes in an arrangement where it could form salt bridges and/or H-bonds to either backbone or side-chain atoms of Ala-9, Tyr-28, Lys-32, Lys-54, and Tyr-73. The polar parts of the pantetheine and the pyrophosphate were structured in the bound ligand to form an interface with the solvent. Also, the ligand formed a set of nonpolar intramol. interactions where the adenine, the pantetheine, and the palmitoyl chain were assocd., so that the overall structure of the bound ligand appeared to be organized to protect the lipophilic palmitoyl part from the polar solvent.
- 29Schirmer, T.; Evans, P. R. Structural basis of the allosteric behaviour of phosphofructokinase. Nature 1990, 343, 140– 145, DOI: 10.1038/343140a029https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXpsVarsA%253D%253D&md5=373daba1e0eeedb16a98fd461eb5f694Structural basis of the allosteric behavior of phosphofructokinaseSchirmer, Tilman; Evans, Philip R.Nature (London, United Kingdom) (1990), 343 (6254), 140-5CODEN: NATUAS; ISSN:0028-0836.A review and discussion with 20 refs. A comparison between the crystal structures of low- and high-affinity forms of phosphofructokinase shows a close coupling between the change of quaternary structure and local changes triggered by binding of the allosteric effectors. These concerted changes link all the substrate and effector sites in the tetramer, and explain the change of affinity for the cooperative substrate.
- 30Doyle, D. A.; Lee, A.; Lewis, J.; Kim, E.; Sheng, M.; MacKinnon, R. Crystal structures of a complexed and peptide-free membrane protein-binding domain: molecular basis of peptide recognition by PDZ. Cell 1996, 85, 1067– 1076, DOI: 10.1016/S0092-8674(00)81307-030https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XjvF2iu7s%253D&md5=bcc54fe12efeda2bd672deb5487fb670Crystal structures of a complexed and peptide-free membrane protein-binding domain: molecular basis of peptide recognition by PDZDoyle, Declan A.; Lee, Alice; Lewis, John; Kim, Eunjoon; Sheng, Morgan; MacKinnon, RoderickCell (Cambridge, Massachusetts) (1996), 85 (7), 1067-1076CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Modular PDZ domains, found in many cell junction-assocd. proteins, mediate the clustering of membrane ion channels by binding to their C-terminus. The X-ray crystallog. structures of the third PDZ domain from the synaptic protein PSD-95 in complex with and in the absence of its peptide ligand were detd. at 1.8 Å and 2.3 Å resoln., resp. The structures reveal that a 4-residue C-terminal stretch (X-Thr/Ser-X-Val-COO-) engages the PDZ domain through antiparallel main chain interactions with a β sheet of the domain. Recognition of the terminal carboxylate group of the peptide is conferred by a cradle of main chain amides provided by a Gly-Leu-Gly-Phe loop as well as by an arginine side chain. Specific side chain interactions and a prominent hydrophobic pocket explain the selective recognition of the C-terminal consensus sequence.
- 31Hultqvist, G.; Haq, S. R.; Punekar, A. S.; Chi, C. N.; Engstrom, A.; Bach, A.; Stromgaard, K.; Selmer, M.; Gianni, S.; Jemth, P. Energetic pathway sampling in a protein interaction domain. Structure 2013, 21, 1193– 1202, DOI: 10.1016/j.str.2013.05.01031https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtVehtr3J&md5=57ecfd7d086265edc7b4bd5d77f95a0bEnergetic Pathway Sampling in a Protein Interaction DomainHultqvist, Greta; Haq, S. Raza; Punekar, Avinash S.; Chi, Celestine N.; Engstroem, Aake; Bach, Anders; Stroemgaard, Kristian; Selmer, Maria; Gianni, Stefano; Jemth, PerStructure (Oxford, United Kingdom) (2013), 21 (7), 1193-1202CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)The affinity and specificity of protein-ligand interactions are influenced by energetic crosstalk within the protein domain. However, the mol. details of such intradomain allostery are still unclear. Here, we have exptl. detected and computationally predicted interaction pathways in the postsynaptic d. 95/disks large/zonula occludens 1 (PDZ)-peptide ligand model system using wild-type and circularly permuted PDZ proteins. The circular permutant introduced small perturbations in the tertiary structure and a concomitant rewiring of allosteric pathways, allowing us to describe how subtle changes may reshape energetic signaling. The results were analyzed in the context of other members of the PDZ family, which were found to contain distinct interaction pathways for different peptide ligands. The data reveal a fascinating scenario whereby several energetic pathways are sampled within one single domain and distinct pathways are activated by specific protein ligands.
- 32Petit, C. M.; Zhang, J.; Sapienza, P. J.; Fuentes, E. J.; Lee, A. L. Hidden dynamic allostery in a PDZ domain. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 18249– 18254, DOI: 10.1073/pnas.090449210632https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsVWrsrjO&md5=9ae15474e5320584dbe68dee381b2f5cHidden dynamic allostery in a PDZ domainPetit, Chad M.; Zhang, Jun; Sapienza, Paul J.; Fuentes, Ernesto J.; Lee, Andrew L.Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (43), 18249-18254CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Structure-function relationships in proteins are predicated on the spatial proximity of noncovalently interacting groups of atoms. Thus, structural elements located away from a protein's active site are typically presumed to serve a stabilizing or scaffolding role for the larger structure. Here we report a functional role for a distal structural element in a PDZ domain, even though it is not required to maintain PDZ structure. The third PDZ domain from PSD-95/SAP90 (PDZ3) has an unusual addnl. third alpha helix (α3) that packs in contiguous fashion against the globular domain. Although α3 lies outside the active site and does not make direct contact with C-terminal peptide ligand, removal of α3 reduces ligand affinity by 21-fold. Further investigation revealed that the difference in binding free energies between the full-length and truncated constructs is predominantly entropic in nature and that without α3, picosecond-nanosecond side-chain dynamics are enhanced throughout the domain, as detd. by 2H Me NMR relaxation. Thus, the distal modulation of binding function appears to occur via a delocalized conformational entropy mechanism. Without removal of α3 and characterization of side-chain dynamics, this dynamic allostery would have gone unnoticed. Moreover, what appeared at first to be an artificial modification of PDZ3 has been corroborated by exptl. verified phosphorylation of α3, revealing a tangible biol. mechanism for this novel regulatory scheme. This hidden dynamic allostery raises the possibility of as-yet unidentified or untapped allosteric regulation in this PDZ domain and is a very clear example of function arising from dynamics rather than from structure.