Allosteric Regulation of Glycogen Phosphorylase by Order/Disorder Transition of the 250′ and 280s Loops

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This article references 52 other publications.

1. 1

   Monod, J.; Changeux, J. P.; Jacob, F. Allosteric proteins and cellular control systems. J. Mol. Biol. 1963, 6, 306– 329,  DOI: 10.1016/s0022-2836(63)80091-1

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   Allosteric proteins and cellular control systems

   Monod, Jacques; Changeux, Jean Pierre; Jacob, Francois

   Journal of Molecular Biology (1963), 6 (), 306-29CODEN: JMOBAK; ISSN:0022-2836.

   A general model schematizing the functional structures of controlling proteins is proposed. These proteins are assumed to possess 2 or more stereospecifically different, nonoverlapping receptor sites. One of these, the active site, binds the substrate and is responsible for the biol. activity of the protein. The other, or allosteric site, is complementary to the structure of another metabolite, the allosteric effector, which it binds specifically and reversibly. The effect of these regulatory metabolites appears to result exclusively from a conformational alteration (allosteric transition) induced in the protein when it binds the agent. It is suggested that this mechanism plays an essential role in the regulation of metabolic activity and also possibly in the specific control of protein synthesis.

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   Monod, J.; Wyman, J.; Changeux, J.-P. On the nature of allosteric transitions: A plausible model. J. Mol. Biol. 1965, 12, 88– 118,  DOI: 10.1016/S0022-2836(65)80285-6

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   On the nature of allosteric transitions: a plausible model

   Monod, Jacques; Wyman, Jeffries; Changeux, Jean Pierre

   Journal of Molecular Biology (1965), 12 (1), 88-118CODEN: JMOBAK; ISSN:0022-2836.

   A math. model is proposed for those regulatory proteins which are oligomeric in nature and the model applied with considerable success to published results on several enzymes or metabolically active proteins.

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   Guo, J.; Zhou, H. X. Protein Allostery and Conformational Dynamics. Chem. Rev. 2016, 116, 6503– 6515,  DOI: 10.1021/acs.chemrev.5b00590

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   Protein Allostery and Conformational Dynamics

   Guo, Jingjing; Zhou, Huan-Xiang

   Chemical Reviews (Washington, DC, United States) (2016), 116 (11), 6503-6515CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)

   The functions of many proteins are regulated through allostery, whereby effector binding at a distal site changes the functional activity (e.g., substrate binding affinity or catalytic efficiency) at the active site. Most allosteric studies have focused on thermodn. properties, in particular, substrate binding affinity. Changes in substrate binding affinity by allosteric effectors have generally been thought to be mediated by conformational transitions of the proteins or, alternatively, by changes in the broadness of the free energy basin of the protein conformational state without shifting the basin min. position. When effector binding changes the free energy landscape of a protein in conformational space, the change affects not only thermodn. properties but also dynamic properties, including the amplitudes of motions on different time scales and rates of conformational transitions. Here we assess the roles of conformational dynamics in allosteric regulation. Two cases are highlighted where NMR spectroscopy and mol. dynamics simulation have been used as complementary approaches to identify residues possibly involved in allosteric communication. Perspectives on contentious issues, for example, the relationship between picosecond-nanosecond local and microsecond-millisecond conformational exchange dynamics, are presented.

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   Wodak, S. J.; Paci, E.; Dokholyan, N. V.; Berezovsky, I. N.; Horovitz, A.; Li, J.; Hilser, V. J.; Bahar, I.; Karanicolas, J.; Stock, G. Allostery in Its Many Disguises: From Theory to Applications. Structure 2019, 27, 566– 578,  DOI: 10.1016/j.str.2019.01.003

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   Allostery in Its Many Disguises: From Theory to Applications

   Wodak, Shoshana J.; Paci, Emanuele; Dokholyan, Nikolay V.; Berezovsky, Igor N.; Horovitz, Amnon; Li, Jing; Hilser, Vincent J.; Bahar, Ivet; Karanicolas, John; Stock, Gerhard; Hamm, Peter; Stote, Roland H.; Eberhardt, Jerome; Chebaro, Yassmine; Dejaegere, Annick; Cecchini, Marco; Changeux, Jean-Pierre; Bolhuis, Peter G.; Vreede, Jocelyne; Faccioli, Pietro; Orioli, Simone; Ravasio, Riccardo; Yan, Le; Brito, Carolina; Wyart, Matthieu; Gkeka, Paraskevi; Rivalta, Ivan; Palermo, Giulia; McCammon, J. Andrew; Panecka-Hofman, Joanna; Wade, Rebecca C.; Di Pizio, Antonella; Niv, Masha Y.; Nussinov, Ruth; Tsai, Chung-Jung; Jang, Hyunbum; Padhorny, Dzmitry; Kozakov, Dima; McLeish, Tom

   Structure (Oxford, United Kingdom) (2019), 27 (4), 566-578CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)

   Allosteric regulation plays an important role in many biol. processes, such as signal transduction, transcriptional regulation, and metab. Allostery is rooted in the fundamental phys. properties of macromol. systems, but its underlying mechanisms are still poorly understood. A collection of contributions to a recent interdisciplinary CECAM (Center Europe´en de Calcul Atomique et Mole´culaire) workshop is used here to provide an overview of the progress and remaining limitations in the understanding of the mechanistic foundations of allostery gained from computational and exptl. analyses of real protein systems and model systems. The main conceptual frameworks instrumental in driving the field are discussed. We illustrate the role of these frameworks in illuminating mol. mechanisms and explaining cellular processes, and describe some of their promising practical applications in engineering mol. sensors and informing drug design efforts.

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   Barford, D.; Johnson, L. N. The allosteric transition of glycogen phosphorylase. Nature 1989, 340, 609– 616,  DOI: 10.1038/340609a0

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   The allosteric transition of glycogen phosphorylase

   Barford, D.; Johnson, L. N.

   Nature (London, United Kingdom) (1989), 340 (6235), 609-16CODEN: NATUAS; ISSN:0028-0836.

   The crystal structure of R-stage glycogen phosphorylase b has been detd. at 2.9 Å resoln. A comparison of T-state and R-state structures of the enzyme explains its cooperative behavior upon ligand binding and the allosteric regulation of its activity. Communication between catalytic sites of the dimer is provided by a change in packing geometry of 2 helixes linking each site with the subunit interface. Activation by AMP or by phosphorylation results in a quaternary conformational change that switches these 2 helixes into the R-state conformation.

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   Barford, D.; Hu, S. H.; Johnson, L. N. Structural mechanism for glycogen phosphorylase control by phosphorylation and AMP. J. Mol. Biol. 1991, 218, 233– 260,  DOI: 10.1016/0022-2836(91)90887-C

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   Structural mechanism for glycogen phosphorylase control by phosphorylation and AMP

   Barford, D.; Hu, S. H.; Johnson, L. N.

   Journal of Molecular Biology (1991), 218 (1), 233-60CODEN: JMOBAK; ISSN:0022-2836.

   The crystal structures of activated R state glycogen phosphorylase a (GPa) and R and T state glycogen phosphorylase b (GPb) complexed with AMP were solved at 2.9, 2.9 and 2.2 Å resoln., resp. The structure of R state GPa was nearly identical to the structure of sulfate-activated R state GPb, except in the region of Ser-4, where there was a covalently attached phosphate group in GPa and a noncovalently attached sulfate group in GPb. The contacts made by the N-terminal tail residues in R state GPa at the subunit interface of the functionally active dimer were similar to those obsd. previously for T state GPa. The quaternary and tertiary structural changes on the T-to-R transition allowed these interactions to be relayed to the catalytic site in R state GPa. The transition from the T state GPb structure to the R state GPa structure resulted in a change in the N-terminal residues from a poorly ordered extended structure that makes intrasubunit contacts to an ordered coiled conformation that makes intersubunit contacts. The distance between Arg-10, the 1st residue to be located from the N-terminus, in R state GPa and T state GPb was 50 Å. One of the important subunit-subunit interactions in the dimer mol. involved contacts between the helix α2 and the cap' (residues 35'-45' that form a loop between the 1st and 2nd α-helixes, α1' and α2', of the other subunit; the prime denotes residues from the other subunit). The interactions made by the N-terminal residues induced structural changes at the cap'/α2 helix interface that led to the creation of a high-affinity AMP site. The tertiary structural changes at the cap (shifted 1.2-2.1 Å for residues 35-45) were partially compensated by the quaternary structural change so that the overall shifts in these residues after the combined tertiary and quaternary changes were at 0.5-1.3 Å. AMP bound to R state GPb with at least 100-fold greater affinity and exhibited 4 addnl. H-bonds, stronger ionic interactions, and more extensive van der Waals' interactions with 116 Å2 greater solvent accessible surface area buried compared with AMP bound to T state GPb. A H-bond obsd. in the R state complex between Asn-4' and N-1 of the adenine moiety of AMP provided a possible explanation for the differences in affinity between AMP and IMP, and the different allosteric properties of the 2 nucleotides. The obsd. correlation between tertiary and quaternary conformational changes form the basis for a structural explanation for allosteric control by phosphorylation and by AMP.

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   Leonidas, D. D.; Zographos, S. E.; Tsitsanou, K. E.; Skamnaki, V. T.; Stravodimos, G.; Kyriakis, E. Glycogen phosphorylase revisited: extending the resolution of the R- and T-state structures of the free enzyme and in complex with allosteric activators. Acta Crystallogr F Struct Biol Commun 2021, 77, 303– 311,  DOI: 10.1107/S2053230X21008542

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   Glycogen phosphorylase revisited: extending the resolution of the R- and T-state structures of the free enzyme and in complex with allosteric activators

   Leonidas, Demetres D.; Zographos, Spyros E.; Tsitsanou, Katerina E.; Skamnaki, Vassiliki T.; Stravodimos, George; Kyriakis, Efthimios

   Acta Crystallographica, Section F: Structural Biology Communications (2021), 77 (9), 303-311CODEN: ACSFEN; ISSN:2053-230X. (International Union of Crystallography)

   The crystal structures of free T-state and R-state glycogen phosphorylase (GP) and of R-state GP in complex with the allosteric activators IMP and AMP are reported at improved resoln. GP is a validated pharmaceutical target for the development of antihyperglycemic agents, and the reported structures may have a significant impact on structure-based drug-design efforts. Comparisons with previously reported structures at lower resoln. reveal the detailed conformation of important structural features in the allosteric transition of GP from the T-state to the R-state. The conformation of the N-terminal segment (residues 7-17), the position of which was not located in previous T-state structures, was revealed to form an α-helix (now termed α0). The conformation of this segment (which contains Ser14, phosphorylation of which leads to the activation of GP) is significantly different between the T-state and the R-state, pointing in opposite directions. In the T-state it is packed between helixes α4 and α16 (residues 104-115 and 497-508, resp.), while in the R-state it is packed against helix α1 (residues 22'-38') and towards the loop connecting helixes α4' and α5' of the neighboring subunit. The allosteric binding site where AMP and IMP bind is formed by the ordering of a loop (residues 313-326) which is disordered in the free structure, and adopts a conformation dictated mainly by the type of nucleotide that binds at this site.

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   Fletterick, R. J.; Sprang, S. R. Glycogen phosphorylase structures and function. Acc. Chem. Res. 1982, 15, 361– 369,  DOI: 10.1021/ar00083a004

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   Glycogen phosphorylase structures and function

   Fletterick, Robert J.; Sprang, Stephen R.

   Accounts of Chemical Research (1982), 15 (11), 361-9CODEN: ACHRE4; ISSN:0001-4842.

   A review and discussion with 56 refs.

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   Buchbinder, J. L.; Fletterick, R. J. Role of the active site gate of glycogen phosphorylase in allosteric inhibition and substrate binding. J. Biol. Chem. 1996, 271, 22305– 22309,  DOI: 10.1074/jbc.271.37.22305

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   Role of the active site gate of glycogen phosphorylase in allosteric inhibition and substrate binding

   Buchbinder, Jenny L.; Fletterick, Robert J.

   Journal of Biological Chemistry (1996), 271 (37), 22305-22309CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)

   The functional role in allosteric regulation of a flexible loop (residues 280-288) located near the active site of muscle glycogen phosphorylase was investigated. Mutations were made in residues 283-285 based on crystallog. studies that indicate that the loop functions as a gate controlling access of substrates to the active site and that these specific residues play distinct roles in mimicking the substrate and binding inhibitors when the enzyme is in an inactive conformation. Substitution of Ala or Asn for Asp-283, the putative substrate mimic, results in a 15-fold decrease in Vmax, a 10-fold decrease in the S0.5 for glucose 1-phosphate, a 10-fold increase in the Kα for AMP, and a 10-20-fold increase in the Ki for glucose. Substitution of Ala for Asn-284, which normally forms a hydrogen bond with the inhibitor glucose, reduces Vmax 10-fold, elevates the Ki for glucose 10-fold, decreases AMP cooperativity, but has little effect on the affinity of AMP or the cooperativity and binding of glucose 1-phosphate. Substitution of Leu for Phe-285, which forms arom. stacking interactions with purine inhibitors, reduces Vmax 2-fold, decreases the affinity for caffeine at least 10-fold, raises the Kα for AMP 3-fold, and decreases AMP cooperativity but has little effect on glucose 1-phosphate binding or cooperativity. The results of the mutagenesis studies show the importance of the 280's loop for inhibitor binding and modulation of substrate affinity and suggest a role for the loop in allosteric activation. The propagation of allosteric effects across the domain interface may depend upon specific contacts between residues of the 280's loop and the C-terminal domain.

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10. 10

    Mueller, M.; Nidetzky, B. Orthophosphate binding at the dimer interface of Corynebacterium callunae starch phosphorylase: mutational analysis of its role for activity and stability of the enzyme. BMC Biochemistry 2010, 11, 8,  DOI: 10.1186/1471-2091-11-8

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    Orthophosphate binding at the dimer interface of Corynebacterium callunae starch phosphorylase: mutational analysis of its role for activity and stability of the enzyme

    Mueller Mario; Nidetzky Bernd

    BMC biochemistry (2010), 11 (), 8 ISSN:.

    BACKGROUND: Orthophosphate recognition at allosteric binding sites is a key feature for the regulation of enzyme activity in mammalian glycogen phosphorylases. Protein residues co-ordinating orthophosphate in three binding sites distributed across the dimer interface of a non-regulated bacterial starch phosphorylase (from Corynebacterium callunae) were individually replaced by Ala to interrogate their unknown function for activity and stability of this enzyme. RESULTS: While the mutations affected neither content of pyridoxal 5'-phosphate cofactor nor specific activity in phosphorylase preparations as isolated, they disrupted (Thr28-->Ala, Arg141-->Ala) or decreased (Lys31-->Ala, Ser174-->Ala) the unusually strong protective effect of orthophosphate (10 or 100 mM) against inactivation at 45 degrees C and subunit dissociation enforced by imidazole, as compared to wild-type enzyme. Loss of stability in the mutated phosphorylases appeared to be largely due to weakened affinity for orthophosphate binding. Binding of sulphate mimicking the crystallographically observed "non-covalent phosphorylation" of the phosphorylase at the dimer interface did not have an allosteric effect on the enzyme activity. CONCLUSIONS: The phosphate sites at the subunit-subunit interface of C. callunae starch phosphorylase appear to be cooperatively functional in conferring extra kinetic stability to the native dimer structure of the active enzyme. The molecular strategy exploited for quaternary structure stabilization is to our knowledge novel among dimeric proteins. It can be distinguished clearly from the co-solute effect of orthophosphate on protein thermostability resulting from (relatively weak) interactions of the ligand with protein surface residues.

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11. 11

    Buller, A. R.; van Roye, P.; Cahn, J. K. B.; Scheele, R. A.; Herger, M.; Arnold, F. H. Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble. J. Am. Chem. Soc. 2018, 140, 7256– 7266,  DOI: 10.1021/jacs.8b03490

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    Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble

    Buller, Andrew R.; van Roye, Paul; Cahn, Jackson K. B.; Scheele, Remkes A.; Herger, Michael; Arnold, Frances H.

    Journal of the American Chemical Society (2018), 140 (23), 7256-7266CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)

    Allosteric enzymes contain a wealth of catalytic diversity that remains distinctly underutilized for biocatalysis. Tryptophan synthase is a model allosteric system and a valuable enzyme for the synthesis of non-canonical amino acids (ncAA). Previously, we evolved the β-subunit from Pyrococcus furiosus, PfTrpB, for ncAA synthase activity in the absence of its native partner protein PfTrpA. However, the precise mechanism by which mutation activated TrpB to afford a stand-alone catalyst remained enigmatic. Here, we show that directed evolution caused a gradual change in the rate-limiting step of the catalytic cycle. Concomitantly, the steady-state distribution of intermediates shifts to favor covalently bound Trp adducts, which is assocd. with increased thermodn. stability of these species. The biochem. properties of these evolved, stand-alone TrpBs converge on those induced in the native system by allosteric activation. High resoln. crystal structures of the wild-type enzyme, an intermediate in the lineage, and the final variant, encompassing five distinct chem. states, show that activating mutations have only minor structural effects on their immediate environment. Instead, mutation stabilizes the large-scale motion of a sub-domain to favor an otherwise transiently populated closed conformational state. This increase in stability enabled the first structural description of Trp covalently bound in a catalytically active TrpB, confirming key features of catalysis. These data combine to show that sophisticated models of allostery are not a prerequisite to recapitulating its complex effects via directed evolution, opening the way to engineering stand-alone versions of diverse allosteric enzymes.

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12. 12

    Wellens, A.; Lahmann, M.; Touaibia, M.; Vaucher, J.; Oscarson, S.; Roy, R.; Remaut, H.; Bouckaert, J. The Tyrosine Gate as a Potential Entropic Lever in the Receptor-Binding Site of the Bacterial Adhesin FimH. Biochemistry 2012, 51, 4790– 4799,  DOI: 10.1021/bi300251r

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    The Tyrosine Gate as a Potential Entropic Lever in the Receptor-Binding Site of the Bacterial Adhesin FimH

    Wellens, Adinda; Lahmann, Martina; Touaibia, Mohamed; Vaucher, Jonathan; Oscarson, Stefan; Roy, Rene; Remaut, Han; Bouckaert, Julie

    Biochemistry (2012), 51 (24), 4790-4799CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)

    Uropathogenic Escherichia coli (UPEC) are the major causative agents of urinary tract infections. During infection, UPEC adhere to mannosylated glycoreceptors on the urothelium via the FimH adhesin located at the tip of type 1 pili. Synthetic FimH antiadhesives such as alkyl and Ph α-d-mannopyranosides are thus ideal candidates for the chem. interception of this crucial step in pathogenesis. The crystal structures of the FimH lectin domain in its ligand-free form and in complexes with eight medium- and high-affinity mannopyranoside inhibitors are presented. The thermodn. profiles of the FimH-inhibitor interactions indicate that the binding of FimH to α-D-mannopyranose is enthalpy-driven and has a neg. entropic change. Addn. of a hydrophobic aglycon influences the binding enthalpy and can induce a favorable entropic change. The alleviation of the entropic cost is at least in part explained by increased dynamics in the tyrosine gate (Tyr48 and Tyr137) of the FimH receptor-binding site upon binding of the ligand. Ligands with a Ph group directly linked to the anomeric oxygen of α-D-mannose introduce the largest dynamics into the Tyr48 side chain, because conjugation with the anomeric oxygen of α-d-mannose forces the arom. aglycon into a conformation that comes into close contact (≈2.65 Å) with Tyr48. A propargyl group in this position predetermines the orientation of the aglycon and significantly decreases affinity. FimH has the highest affinity for α-D-mannopyranosides substituted with hydrophobic aglycons that are compatible in shape and electrostatic properties to the tyrosine gate, such as heptyl α-D-mannose.

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13. 13

    Fan, Y.; Cross, P. J.; Jameson, G. B.; Parker, E. J. Exploring modular allostery via interchangeable regulatory domains. Proc. Natl. Acad. Sci. U. S. A. 2018, 115, 3006– 3011,  DOI: 10.1073/pnas.1717621115

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    Exploring modular allostery via interchangeable regulatory domains

    Fan, Yifei; Cross, Penelope J.; Jameson, Geoffrey B.; Parker, Emily J.

    Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (12), 3006-3011CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

    Most proteins comprise two or more domains from a limited suite of protein families. These domains are often rearranged in various combinations through gene fusion events to evolve new protein functions, including the acquisition of protein allostery through the incorporation of regulatory domains. The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) is the first enzyme of arom. amino acid biosynthesis and displays a diverse range of allosteric mechanisms. DAH7PSs adopt a common architecture with a shared (β/α)8 catalytic domain which can be attached to an ACT-like or a chorismate mutase regulatory domain that operates via distinct mechanisms. These resp. domains confer allosteric regulation by controlling DAH7PS function in response to ligand Tyr or prephenate. Starting with contemporary DAH7PS proteins, two protein chimeras were created, with interchanged regulatory domains. Both engineered proteins were catalytically active and delivered new functional allostery with switched ligand specificity and allosteric mechanisms delivered by their nonhomologous regulatory domains. This interchangeability of protein domains represents an efficient method not only to engineer allostery in multidomain proteins but to create a new bifunctional enzyme.

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14. 14

    Hoover, D. J.; Lefkowitz-Snow, S.; Burgess-Henry, J. L.; Martin, W. H.; Armento, S. J.; Stock, I. A.; McPherson, R. K.; Genereux, P. E.; Gibbs, E. M.; Treadway, J. L. Indole-2-carboxamide Inhibitors of Human Liver Glycogen Phosphorylase. J. Med. Chem. 1998, 41, 2934– 2938,  DOI: 10.1021/jm980264k

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    Indole-2-carboxamide inhibitors of human liver glycogen phosphorylase

    Hoover, Dennis J.; Lefkowitz-Snow, Sheri; Burgess-Henry, Jana L.; Martin, William H.; Armento, Sandra J.; Stock, Ingrid A.; McPherson, R. Kirk; Genereux, Paul E.; Gibbs, E. Michael; Treadway, Judith L.

    Journal of Medicinal Chemistry (1998), 41 (16), 2934-2938CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)

    Indole-2-carboxamide derivs. (I; X = Cl, F, Br, H, OMe; R = Ph, cyclohexyl, H, F; Y = CONMe2, CONHMe, CO2Me, CO2H, CH2OH, CONH2, etc.) were prepd. I are potent inhibitors of human liver glycogen phosphorylase which are active in cells, and produce hypoglycemic activity on oral administration in a rodent model of type 2 diabetes. I [CP-320626; X = Cl, R = F, Y = CO(1-piperidin-4-ol)] produced oral activity at 10 mg/kg.

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15. 15

    Oikonomakos, N. G. Glycogen phosphorylase as a molecular target for type 2 diabetes therapy. Curr. Protein Pept. Sci. 2002, 3, 561– 586,  DOI: 10.2174/1389203023380422

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    Glycogen phosphorylase as a molecular target for type 2 diabetes therapy

    Oikonomakos, Nikos G.

    Current Protein and Peptide Science (2002), 3 (6), 561-586CODEN: CPPSCM; ISSN:1389-2037. (Bentham Science Publishers Ltd.)

    A review. The regulation of the hepatic glucose output through glycogenolysis is an important target for type 2 diabetes therapy. Glycogenolysis is catalyzed in liver, muscle and brain by tissue specific isoforms of glycogen phosphorylase (GP). Because of its central role in glycogen metab., GP has been exploited as a model for structure-assisted design of potent inhibitors, which may be relevant to the control of blood glucose concns. in type 2 diabetes. Several regulatory binding sites have been identified in GP, such as the catalytic, the allosteric, and the inhibitor binding sites. Protein crystallog. has contributed significant structural information on the specificity and interactions that distinguish the binding sites, and also revealed a new unexpected binding site (new allosteric site). In this review, the kinetic, crystallog. binding, and physiol. studies of a no. of compds., inhibitors of GP, are described, and the essential inhibitory and binding properties of specific compds. are analyzed in an effort to provide rationalizations for the affinities of these compds. and to exploit the mol. interactions that might give rise to a better inhibitor. These studies have given new insights into fundamental structural aspects of the enzyme enhancing our understanding of how the enzyme recognizes and specifically binds ligands, that could be of potential therapeutic value in the treatment of type 2 diabetes.

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16. 16

    Zois, C. E.; Harris, A. L. Glycogen metabolism has a key role in the cancer microenvironment and provides new targets for cancer therapy. J Mol Med (Berl) 2016, 94, 137– 154,  DOI: 10.1007/s00109-015-1377-9

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    16

    Glycogen metabolism has a key role in the cancer microenvironment and provides new targets for cancer therapy

    Zois Christos E; Harris Adrian L

    Journal of molecular medicine (Berlin, Germany) (2016), 94 (2), 137-54 ISSN:.

    Metabolic reprogramming is a hallmark of cancer cells and contributes to their adaption within the tumour microenvironment and resistance to anticancer therapies. Recently, glycogen metabolism has become a recognised feature of cancer cells since it is upregulated in many tumour types, suggesting that it is an important aspect of cancer cell pathophysiology. Here, we provide an overview of glycogen metabolism and its regulation, with a focus on its role in metabolic reprogramming of cancer cells under stress conditions such as hypoxia, glucose deprivation and anticancer treatment. The various methods to detect glycogen in tumours in vivo as well as pharmacological modulators of glycogen metabolism are also reviewed. Finally, we discuss the therapeutic value of targeting glycogen metabolism as a strategy for combinational approaches in cancer treatment.

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17. 17

    Brown, A. M.; Ransom, B. R. Astrocyte glycogen and brain energy metabolism. Glia 2007, 55, 1263– 1271,  DOI: 10.1002/glia.20557

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    17

    Astrocyte glycogen and brain energy metabolism

    Brown Angus M; Ransom Bruce R; Brown Angus M

    Glia (2007), 55 (12), 1263-1271 ISSN:0894-1491.

    The brain contains glycogen but at low concentration compared with liver and muscle. In the adult brain, glycogen is found predominantly in astrocytes. Astrocyte glycogen content is modulated by a number of factors including some neurotransmitters and ambient glucose concentration. Compelling evidence indicates that astrocyte glycogen breaks down during hypoglycemia to lactate that is transferred to adjacent neurons or axons where it is used aerobically as fuel. In the case of CNS white matter, this source of energy can extend axon function for 20 min or longer. Likewise, during periods of intense neural activity when energy demand exceeds glucose supply, astrocyte glycogen is degraded to lactate, a portion of which is transferred to axons for fuel. Astrocyte glycogen, therefore, offers some protection against hypoglycemic neural injury and ensures that neurons and axons can maintain their function during very intense periods of activation. These emerging principles about the roles of astrocyte glycogen contradict the long held belief that this metabolic pool has little or no functional significance.

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18. 18

    Oikonomakos, N. G.; Skamnaki, V. T.; Tsitsanou, K. E.; G Gavalas, N. G.; Johnson, L. N. A new allosteric site in glycogen phosphorylase b as a target for drug interactions. Structure 2000, 8, 575– 584,  DOI: 10.1016/s0969-2126(00)00144-1

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    18

    A new allosteric site in glycogen phosphorylase b as a target for drug interactions

    Oikonomakos, Nikos G.; Skamnaki, Vicky T.; Tsitsanou, Katerina E.; Gavalas, Nikos G.; Johnson, Louise N.

    Structure (London) (2000), 8 (6), 575-584CODEN: STRUE6; ISSN:0969-2126. (Elsevier Science Ltd.)

    Background: In muscle and liver, glycogen concns. are regulated by the coordinated activities of glycogen phosphorylase (GP) and glycogen synthase. GP exists in two forms: the dephosphorylated low-activity form GPb and the phosphorylated high-activity form GPa. In both forms, allosteric effectors can promote equil. between a less active T state and a more active R state. GP is a possible target for drugs that aim to prevent unwanted glycogen breakdown and to stimulate glycogen synthesis in non-insulin-dependent diabetes. As a result of a data bank search, 5-chloro-1H-indole-2-carboxylic acid (1-(4-fluorobenzyl)-2-(4-hydroxypiperidin-1-yl)-2-oxoethyl)amide, CP320626, was identified as a potent inhibitor of human liver GP. Structural studies have been carried out in order to establish the mechanism of this unusual inhibitor. Results: The structure of the cocrystd. GPb-CP320626 complex has been detd. to 2.3 Å resoln. CP320626 binds at a site located at the subunit interface in the region of the central cavity of the dimeric structure. The site has not previously been obsd. to bind ligands and is some 15 Å from the AMP allosteric site and 33 Å from the catalytic site. The contacts between GPb and CP320626 comprise six hydrogen bonds and extensive van der Waals interactions that create a tight binding site in the T-state conformation of GPb. In the R-state conformation of GPa these interactions are significantly diminished. Conclusions: CP320626 inhibits GPb by binding at a new allosteric site. Although over 30 Å from the catalytic site, the inhibitor exerts its effects by stabilizing the T state at the expense of the R state and thereby shifting the allosteric equil. between the two states. The new allosteric binding site offers a further recognition site in the search for improved GP inhibitors.

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19. 19

    Barford, D.; Hu, S. H.; Johnson, L. N. Structural mechanism for glycogen phosphorylase control by phosphorylation and AMP. J. Mol. Biol. 1991, 218, 233– 260,  DOI: 10.1016/0022-2836(91)90887-c

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    19

    Structural mechanism for glycogen phosphorylase control by phosphorylation and AMP

    Barford, D.; Hu, S. H.; Johnson, L. N.

    Journal of Molecular Biology (1991), 218 (1), 233-60CODEN: JMOBAK; ISSN:0022-2836.

    The crystal structures of activated R state glycogen phosphorylase a (GPa) and R and T state glycogen phosphorylase b (GPb) complexed with AMP were solved at 2.9, 2.9 and 2.2 Å resoln., resp. The structure of R state GPa was nearly identical to the structure of sulfate-activated R state GPb, except in the region of Ser-4, where there was a covalently attached phosphate group in GPa and a noncovalently attached sulfate group in GPb. The contacts made by the N-terminal tail residues in R state GPa at the subunit interface of the functionally active dimer were similar to those obsd. previously for T state GPa. The quaternary and tertiary structural changes on the T-to-R transition allowed these interactions to be relayed to the catalytic site in R state GPa. The transition from the T state GPb structure to the R state GPa structure resulted in a change in the N-terminal residues from a poorly ordered extended structure that makes intrasubunit contacts to an ordered coiled conformation that makes intersubunit contacts. The distance between Arg-10, the 1st residue to be located from the N-terminus, in R state GPa and T state GPb was 50 Å. One of the important subunit-subunit interactions in the dimer mol. involved contacts between the helix α2 and the cap' (residues 35'-45' that form a loop between the 1st and 2nd α-helixes, α1' and α2', of the other subunit; the prime denotes residues from the other subunit). The interactions made by the N-terminal residues induced structural changes at the cap'/α2 helix interface that led to the creation of a high-affinity AMP site. The tertiary structural changes at the cap (shifted 1.2-2.1 Å for residues 35-45) were partially compensated by the quaternary structural change so that the overall shifts in these residues after the combined tertiary and quaternary changes were at 0.5-1.3 Å. AMP bound to R state GPb with at least 100-fold greater affinity and exhibited 4 addnl. H-bonds, stronger ionic interactions, and more extensive van der Waals' interactions with 116 Å2 greater solvent accessible surface area buried compared with AMP bound to T state GPb. A H-bond obsd. in the R state complex between Asn-4' and N-1 of the adenine moiety of AMP provided a possible explanation for the differences in affinity between AMP and IMP, and the different allosteric properties of the 2 nucleotides. The obsd. correlation between tertiary and quaternary conformational changes form the basis for a structural explanation for allosteric control by phosphorylation and by AMP.

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20. 20

    Martin, J.; Veluraja, K.; Ross, K.; Johnson, L.; Fleet, G.; Ramsden, N.; Bruce, I.; Orchard, M.; Oikonomakos, N. Glucose analog inhibitors of glycogen phosphorylase: The design of potential drugs for diabetes. Biochemistry 1991, 30, 10101– 10116,  DOI: 10.1021/bi00106a006

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    20

    Glucose analog inhibitors of glycogen phosphorylase: the design of potential drugs for diabetes

    Martin, J. L.; Veluraja, K.; Ross, K.; Johnson, L. N.; Fleet, G. W. J.; Ramsden, N. G.; Bruce, I.; Orchard, M. G.; Oikonomakos, N. G.; et al.

    Biochemistry (1991), 30 (42), 10101-16CODEN: BICHAW; ISSN:0006-2960.

    The T-state crystal structure of the glucose-phosphorylase b complex has been used as a model for the design of glucose analog inhibitors that may be effective in the regulation of blood glucose levels. Modeling studies indicated room for addnl. atoms attached at the C1-β position of glucose and some scope for addnl. atoms at the C1-α position. Kinetic parameters were detd. for α-D-glucose: Ki = 1.7 mM, Hill coeff. n = 1.5, and α (synergism with caffeine) = 0.2. For β-D-glucose, Ki = 7.4 mM, n = 1.5, and α = 0.4. More than 20 glucose analogs have been synthesized and tested in kinetic expts. Most were less effective inhibitors than glucose itself and the best inhibitor was α-hydroxymethyl-1-deoxy-D-glucose (Ki = 1.5 mM, n = 1.3, α = 0.4). The binding of 14 glucose analogs to glycogen phosphorylase b in the crystal has been studied at 2.4-Å resoln. and the structures have been refined to crystallog. R values of less than 0.20. The kinetic and crystallog. studies have been combined to provide rationalizations for the apparent affinities of glucose and the analogs. The results show the discrimination against β-D-glucose in favor of α-D-glucose is achieved by an addnl. hydrogen bond made in the α-glucose complex through water to a protein group and an unfavorable environment for a polar group in the β pocket. The compd. α-hydroxymethyl-1-deoxy-D-glucose has an affinity similar to that of glucose and makes a direct hydrogen bond to a protein group. Comparison of analogs with substituent atoms that have flexible geometry (e.g., 1-hydroxyethyl β-D-glucoside) with those whose substituent atoms are more rigid (e.g., β-azidomethyl-1-deoxyglucose or β-cyanomethyl-1-deoxyglucose) indicates that although all three compds. make similar polar interactions with the enzyme, those with more rigid substituent groups are better inhibitors. In another example, α-azidomethyl-1-deoxyglucose was a poor inhibitor. In the crystal structure the compd. made several favorable interactions with the enzyme but bound in an unfavorable conformation, thus providing an explanation for its poor inhibition. Attempts to utilize a contact to a buried aspartate group were partially successful for a no. of compds. (β-aminoethyl, β-mesylate, and β-azidomethyl analogs). The β pocket was shown to bind gentiobiose (6-O-β-D-glucopyranosyl-D-glucose), indicating scope for binding of larger side groups for future studies.

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21. 21

    Huang, J.; Chu, X.; Luo, Y.; Wang, Y.; Zhang, Y.; Zhang, Y.; Li, H. Insights into Phosphorylation-Induced Protein Allostery and Conformational Dynamics of Glycogen Phosphorylase via Integrative Structural Mass Spectrometry and In Silico Modeling. ACS Chem. Biol. 2022, 17, 1951– 1962,  DOI: 10.1021/acschembio.2c00393

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    21

    Insights into Phosphorylation-Induced Protein Allostery and Conformational Dynamics of Glycogen Phosphorylase via Integrative Structural Mass Spectrometry and In Silico Modeling

    Huang, Jing; Chu, Xiakun; Luo, Yuxiang; Wang, Yong; Zhang, Ying; Zhang, Yu; Li, Huilin

    ACS Chemical Biology (2022), 17 (7), 1951-1962CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)

    Allosteric regulation plays a fundamental role in innumerable biol. processes. Understanding its dynamic mechanism and impact at the mol. level is of great importance in disease diagnosis and drug discovery. Glycogen phosphorylase (GP) is a phosphoprotein responding to allosteric regulation and has significant biol. importance to glycogen metab. Although the at. structures of GP were previously solved, the conformational dynamics of GP related to allostery regulation remain largely elusive due to its macromol. size (~ 196 kDa). Here, the authors integrated native top-down mass spectrometry (nTD-MS), hydrogen-deuterium exchange MS (HDX-MS), protection factor (PF) anal., mol. dynamics (MD) simulations, and allostery signaling anal. to examine the structural basis and dynamics for the allosteric regulation of GP by phosphorylation. nTD-MS reveals differences in structural stability as well as oligomeric state between the unphosphorylated (GPb) and phosphorylated (GPa) forms. HDX-MS, PF anal., and MD simulations further pinpoint the structural differences between GPb and GPa involving the binding interfaces (the N-terminal and tower-tower helixes), catalytic site, and PLP-binding region. More importantly, it also allowed the authors to complete the missing link of the long-range communication process from the N-terminal tail to the catalytic site caused by phosphorylation. This integrative MS and in silico-based platform is highly complementary to biophys. approaches and yields valuable insights into protein structures and dynamic regulation.

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22. 22

    Yip, K. M.; Fischer, N.; Paknia, E.; Chari, A.; Stark, H. Atomic-resolution protein structure determination by cryo-EM. Nature 2020, 587, 157– 161,  DOI: 10.1038/s41586-020-2833-4

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    22

    Atomic-resolution protein structure determination by cryo-EM

    Yip, Ka Man; Fischer, Niels; Paknia, Elham; Chari, Ashwin; Stark, Holger

    Nature (London, United Kingdom) (2020), 587 (7832), 157-161CODEN: NATUAS; ISSN:0028-0836. (Nature Research)

    Single-particle electron cryo-microscopy (cryo-EM) is a powerful method for solving the three-dimensional structures of biol. macromols. The technol. development of transmission electron microscopes, detectors and automated procedures in combination with user-friendly image processing software and ever-increasing computational power have made cryo-EM a successful and expanding technol. over the past decade1. At resolns. better than 4 Å, at. model building starts to become possible, but the direct visualization of true at. positions in protein structure detn. requires much higher (better than 1.5 Å) resoln., which so far has not been attained by cryo-EM. The direct visualization of atom positions is essential for understanding the mechanisms of protein-catalyzed chem. reactions, and for studying how drugs bind to and interfere with the function of proteins2. Here we report a 1.25 Å-resoln. structure of apoferritin obtained by cryo-EM with a newly developed electron microscope that provides, to our knowledge, unprecedented structural detail. Our apoferritin structure has almost twice the 3D information content of the current world record reconstruction (at 1.54 Å resoln.3). We can visualize individual atoms in a protein, see d. for hydrogen atoms and image single-atom chem. modifications. Beyond the nominal improvement in resoln., we also achieve a substantial improvement in the quality of the cryo-EM d. map, which is highly relevant for using cryo-EM in structure-based drug design.

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23. 23

    Papageorgiou, A. C.; Poudel, N.; Mattsson, J. Protein Structure Analysis and Validation with X-Ray Crystallography. Methods Mol Biol 2021, 2178, 377– 404,  DOI: 10.1007/978-1-0716-0775-6_25

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    23

    Protein structure analysis and validation with X-ray crystallography

    Papageorgiou, Anastassios C.; Poudel, Nirmal; Mattsson, Jesse

    Methods in Molecular Biology (New York, NY, United States) (2021), 2178 (Protein Downstream Processing), 377-404CODEN: MMBIED; ISSN:1940-6029. (Springer)

    X-ray crystallog. is the main technique for the detn. of protein structures. About 85% of all protein structures known to date have been elucidated using X-ray crystallog. Knowledge of the three-dimensional structure of proteins can be used in various applications in biotechnol., biomedicine, drug design, and basic research and as a validation tool for protein modifications and ligand binding. Moreover, the requirement for pure, homogeneous, and stable protein solns. in crystns. makes X-ray crystallog. beneficial in other fields of protein research as well. Here, we describe the technique of X-ray protein crystallog. and the steps involved for a successful three-dimensional crystal structure detn.

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24. 24

    Dau, H.; Haumann, M. Time-resolved X-ray spectroscopy leads to an extension of the classical S-state cycle model of photosynthetic oxygen evolution. Photosynth. Res. 2007, 92, 327– 343,  DOI: 10.1007/s11120-007-9141-9

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    24

    Time-resolved X-ray spectroscopy leads to an extension of the classical S-state cycle model of photosynthetic oxygen evolution

    Dau, Holger; Haumann, Michael

    Photosynthesis Research (2007), 92 (3), 327-343CODEN: PHRSDI; ISSN:0166-8595. (Springer)

    In oxygenic photosynthesis, a complete water oxidn. cycle requires absorption of four photons by the chlorophylls of photosystem II (PSII). The photons can be provided successively by applying short flashes of light. Already in 1970, Kok and coworkers [Photochem Photobiol 11:457-475, 1970] developed a basic model to explain the flash-no. dependence of O2 formation. The third flash applied to dark-adapted PSII induces the S3 → S4 → S0 transition, which is coupled to dioxygen formation at a protein-bound Mn4Ca complex. The sequence of events leading to dioxygen formation and the role of Kok's enigmatic S4-state are only incompletely understood. The authors have shown by time-resolved X-ray spectroscopy that in the S3 S0 transition an intermediate is formed, prior to the onset of O-O bond formation. The exptl. results of the time-resolved X-ray expts. are discussed. The identity of the reaction intermediate is considered and the question is addressed how the novel intermediate is related to the S4-state proposed in 1970 by Bessel Kok. This lead the authors to an extension of the classical S-state cycle towards a basic model which describes sequence and interplay of electron and proton abstraction events at the donor side of PSII.

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25. 25

    Escobedo-Hinojosa, W.; Wissner, J. L.; Hauer, B. A real-time (31)P-NMR-based approach for the assessment of glycerol kinase catalyzed monophosphorylations. MethodsX 2021, 8, 101285,  DOI: 10.1016/j.mex.2021.101285

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    25

    A real-time 31P-NMR-based approach for the assessment of glycerol kinase catalyzed monophosphorylations

    Escobedo-Hinojosa, Wendy; Wissner, Julian L.; Hauer, Bernhard

    MethodsX (2021), 8 (), 101285CODEN: METHC8; ISSN:2215-0161. (Elsevier B.V.)

    Phosphorous-NMR is scarcely employed to evaluate enzyme kinetics of kinase driven monophosphorylations, despite of being a powerful and reliable tool to undoubtedly detect the actual phosphoryl transfer to the targeted substrate. Another advantage is that an external supplementation source of the NMR active isotope is not required, since 31P is highly abundant in nature. Glycerol kinase (GlpK) from E. coli is an exemplary ATP-dependent kinase/phosphotransferase model to illustrate the value and usefulness of a 31P-NMR-based approach to assess the enzymically driven monophosphorylation of glycerol. Moreover, the described approach offers an alternative to the indirect coupled glycerol kinase enzyme assays. Herein, we provided a real time 31P-NMR-based method customized for the direct assessment of the glycerol kinase enzyme activity. Real-time detection for phosphoryl group dynamics in the GlpK driven reactionDirect assessment of product formation (glycerol-monophosphate)Parallel detn. of cosubstrate (ATP) consumption and coproduct (ADP) generationMethod validation was performed via31P-NMR for each phosphorylated mol. involved in the reaction in order to assist in the mol. assignments.

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26. 26

    Somssich, M.; Ma, Q.; Weidtkamp-Peters, S.; Stahl, Y.; Felekyan, S.; Bleckmann, A.; Seidel, C. A.; Simon, R. Real-time dynamics of peptide ligand-dependent receptor complex formation in planta. Sci Signal 2015, 8, ra76,  DOI: 10.1126/scisignal.aab0598

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    There is no corresponding record for this reference.
27. 27

    Zinck, N.; Stark, A.-K.; Wilson, D. J.; Sharon, M. An Improved Rapid Mixing Device for Time-Resolved Electrospray Mass Spectrometry Measurements. ChemistryOpen 2014, 3, 109– 114,  DOI: 10.1002/open.201402002

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    27

    An Improved Rapid Mixing Device for Time-Resolved Electrospray Mass Spectrometry Measurements

    Zinck, Nicholas; Stark, Ann-Kathrin; Wilson, Derek J.; Sharon, Michal

    ChemistryOpen (2014), 3 (3), 109-114CODEN: CHOPCK; ISSN:2191-1363. (Wiley-VCH Verlag GmbH & Co. KGaA)

    Time series data can provide valuable insight into the complexity of biol. reactions. Such information can be obtained by mass-spectrometry-based approaches that measure pre-steady-state kinetics. These methods are based on a mixing device that rapidly mixes the reactants prior to the online mass measurement of the transient intermediate steps. Here, we describe an improved continuous-flow mixing app. for real-time electrospray mass spectrometry measurements. Our setup was designed to minimize metal-soln. interfaces and provide a sheath flow of nitrogen gas for generating stable and continuous spray that consequently enhances the signal-to-noise ratio. Moreover, the device was planned to enable easy mounting onto a mass spectrometer replacing the com. electrospray ionization source. We demonstrate the performance of our app. by monitoring the unfolding reaction of cytochrome C, yielding improved signal-to-noise ratio and reduced exptl. repeat errors.

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28. 28

    Keppel, T. R.; Howard, B. A.; Weis, D. D. Mapping Unstructured Regions and Synergistic Folding in Intrinsically Disordered Proteins with Amide H/D Exchange Mass Spectrometry. Biochemistry 2011, 50, 8722– 8732,  DOI: 10.1021/bi200875p

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    28

    Mapping Unstructured Regions and Synergistic Folding in Intrinsically Disordered Proteins with Amide H/D Exchange Mass Spectrometry

    Keppel, Theodore R.; Howard, Brent A.; Weis, David D.

    Biochemistry (2011), 50 (40), 8722-8732CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)

    Mapping the structured and disordered regions and identifying disorder-to-order transitions are essential to understanding intrinsically disordered proteins (IDPs). One technique that can provide such information is H/D exchange coupled with mass spectrometry (H/D-MS). To explore the feasibility of H/D-MS for mapping disordered and ordered regions in IDPs, the authors undertook a systematic evaluation of an unstructured protein, a molten globular protein, and the well-folded complex of the two proteins. Most segments of the unstructured protein, ACTR (activator of thyroid and retinoid receptors, NCOA3_HUMAN, residues 1018-1088), exchange at rates consistent with its assignment as an unstructured protein, but there is slight protection in regions that become helical in the ACTR-CBP complex. The molten globular protein, CBP (the nuclear coactivator binding domain of the CREB binding protein, CBP_MOUSE, residues 2059-2117), is moderately protected from exchange, and the protection is nearly uniform across the length of the protein. The uniformity arises because of rapid interconversion between an ensemble of folded conformers and an ensemble of unstructured conformers. Rapid interconversion causes the H/D exchange kinetics to be dominated by exchange by mols. in unstructured conformations. For the folded ACTR-CBP complex, the exchange data provide a qual. accurate description of the complex. The authors' results provide a useful framework to use in the interpretation of H/D-MS data of intrinsically disordered proteins.

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29. 29

    Beveridge, R.; Calabrese, A. N. Structural Proteomics Methods to Interrogate the Conformations and Dynamics of Intrinsically Disordered Proteins. Front Chem 2021, 9, 603639,  DOI: 10.3389/fchem.2021.603639

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    29

    Structural proteomics methods to interrogate the conformations and dynamics of intrinsically disordered proteins

    Beveridge, Rebecca; Calabrese, Antonio n.

    Frontiers in Chemistry (Lausanne, Switzerland) (2021), 9 (), 603639CODEN: FCLSAA; ISSN:2296-2646. (Frontiers Media S.A.)

    A review. Intrinsically disordered proteins (IDPs) and regions of intrinsic disorder (IDRs) are abundant in proteomes and are essential for many biol. processes. Thus, they are often implicated in disease mechanisms, including neurodegeneration and cancer. The flexible nature of IDPs and IDRs provides many advantages, including (but not limited to) overcoming steric restrictions in binding, facilitating posttranslational modifications, and achieving high binding specificity with low affinity. IDPs adopt a heterogeneous structural ensemble, in contrast to typical folded proteins, making it challenging to interrogate their structure using conventional tools. Structural mass spectrometry (MS) methods are playing an increasingly important role in characterizing the structure and function of IDPs and IDRs, enabled by advances in the design of instrumentation and the development of new workflows, including in native MS, ion mobility MS, top-down MS, hydrogen-deuterium exchange MS, crosslinking MS, and covalent labeling. Here, we describe the advantages of these methods that make them ideal to study IDPs and highlight recent applications where these tools have underpinned new insights into IDP structure and function that would be difficult to elucidate using other methods.

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30. 30

    Seetaloo, N.; Zacharopoulou, M.; Stephens, A. D.; Kaminski Schierle, G. S.; Phillips, J. J. Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry Approach to Correlate Local Structure and Aggregation in alpha-Synuclein. Anal. Chem. 2022, 94, 3183,  DOI: 10.1021/acs.analchem.2c03183

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31. 31

    Kish, M.; Smith, V.; Lethbridge, N.; Cole, L.; Bond, N. J.; Phillips, J. J. Online Fully Automated System for Hydrogen/Deuterium-Exchange Mass Spectrometry with Millisecond Time Resolution. Anal. Chem. 2023,  DOI: 10.1021/acs.analchem.2c05310

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    There is no corresponding record for this reference.
32. 32

    Al-Naqshabandi, M. A.; Weis, D. D. Quantifying Protection in Disordered Proteins Using Millisecond Hydrogen Exchange-Mass Spectrometry and Peptic Reference Peptides. Biochemistry 2017, 56, 4064– 4072,  DOI: 10.1021/acs.biochem.6b01312

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    31

    Quantifying Protection in Disordered Proteins Using Millisecond Hydrogen Exchange-Mass Spectrometry and Peptic Reference Peptides

    Al-Naqshabandi, Mohammed A.; Weis, David D.

    Biochemistry (2017), 56 (31), 4064-4072CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)

    The extent and location of transient structure in intrinsically disordered proteins (IDPs) provide valuable insights into their conformational ensembles and can lead to a better understanding of coupled binding and folding. Millisecond amide hydrogen exchange (HX) can provide such information, but it is difficult to quantify the degree of transient structuring. One reason is that transiently disordered proteins undergo HX at rates only slightly slower than the rate of amide HX by an unstructured random coil, the chem. HX rate. In this work, we evaluate several different methods to obtain an accurate model for the chem. HX rate suitable for millisecond hydrogen exchange-mass spectrometry (HX-MS) anal. of disordered proteins: (1) calcns. using the method of Englander [Bai, et al., Proteins 1993, 17, 75-86], (2) measurement of HX in the presence of 6 M urea or 3 M guanidinium chloride, and (3) measurement of HX by peptide fragments derived directly from the proteins of interest. First, using unstructured model peptides and disordered domains of the activator for thyroid and retinoid receptors (ACTR) and the CREB binding protein (CBP) as the model IDPs, we show that the Englander method has slight inaccuracies that lead to under-estn. of the chem. exchange rate. Second, HX-MS measurements of model peptides show that HX rates are changed dramatically by high concns. of denaturant. Third, we find that measurements of HX by ref. peptides from the proteins of interest provides the most accurate approach for quantifying the extent of transient structure in disordered proteins by millisecond HX-MS.

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33. 33

    Kan, Z. Y.; Walters, B. T.; Mayne, L.; Englander, S. W. Protein hydrogen exchange at residue resolution by proteolytic fragmentation mass spectrometry analysis. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 16438– 16443,  DOI: 10.1073/pnas.1315532110

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    32

    Protein hydrogen exchange at residue resolution by proteolytic fragmentation mass spectrometry analysis

    Kan, Zhong-Yuan; Walters, Benjamin T.; Mayne, Leland; Englander, S. Walter

    Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (41), 16438-16443CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

    Hydrogen exchange technol. provides a uniquely powerful instrument for measuring protein structural and biophys. properties, quant. and in a nonperturbing way, and detg. how these properties are implemented to produce protein function. A developing hydrogen exchange-mass spectrometry method (HX MS) is able to analyze large biol. important protein systems while requiring only minuscule amts. of exptl. material. The major remaining deficiency of the HX MS method is the inability to deconvolve HX results to individual amino acid residue resoln. To pursue this goal we used an iterative optimization program (HDsite) that integrates recent progress in multiple peptide acquisition together with previously unexamd. isotopic envelope-shape information and a site-resolved back-exchange correction. To test this approach, residue-resolved HX rates computed from HX MS data were compared with extensive HX NMR measurements, and analogous comparisons were made in simulation trials. These tests found excellent agreement and revealed the important computational determinants.

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34. 34

    Chetty, P. S.; Nguyen, D.; Nickel, M.; Lund-Katz, S.; Mayne, L.; Englander, S. W.; Phillips, M. C. Comparison of apoA-I helical structure and stability in discoidal and spherical HDL particles by HX and mass spectrometry. J. Lipid Res. 2013, 54, 1589– 1597,  DOI: 10.1194/jlr.M034785

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    Comparison of apoA-I helical structure and stability in discoidal and spherical HDL particles by HX and mass spectrometry

    Chetty, Palaniappan Sevugan; Nguyen, David; Nickel, Margaret; Lund-Katz, Sissel; Mayne, Leland; Englander, S. Walter; Phillips, Michael C.

    Journal of Lipid Research (2013), 54 (6), 1589-1597CODEN: JLPRAW; ISSN:0022-2275. (American Society for Biochemistry and Molecular Biology, Inc.)

    Elucidation of apoA-I secondary structure in spherical plasma HDL particles is essential for understanding HDL structure and function at the mol. level. To provide this information, we have applied hydrogen exchange (HX) and mass spectrometry methods to compare apoA-I secondary structure in discoidal (two apoA-I mols./particle) and spherical (five apoA-I mols./particle) HDL particles. The HX kinetics indicate that the locations of helical segments within the apoA-I mols. are the same in both discoidal and spherical HDL particles (approx. 10 nm hydrodynamic diam.). Helix stabilities in both types of particles are 3-5 kcal/mol, consistent with the apoA-I mols. being in a highly dynamic state with helical segments unfolding and refolding in seconds. For the spherical HDL, apoA-I fragments corresponding to residues 115-158 exhibit bimodal HX kinetics consistent with this segment adopting an inter-converting (on the timescale of tens of minutes) helix-loop configuration. The segment adopting this configuration in the 10 nm disk is shorter because the surface area available to each apoA-I mol. is apparently larger. Loop formation in the central region of the apoA-I mol. contributes to the ability of the protein to adapt to changes in available space on the HDL particle surface. Overall, apoA-I secondary structure is largely unaffected by a change in HDL particle shape from disk to sphere.

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35. 35

    Bai, Y.; Milne, J. S.; Mayne, L.; Englander, S. W. Primary structure effects on peptide group hydrogen exchange. Proteins 1993, 17, 75– 86,  DOI: 10.1002/prot.340170110

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    34

    Primary structure effects on peptide group hydrogen exchange

    Bai, Yawen; Milne, John S.; Mayne, Leland; Englander, S. Walter

    Proteins: Structure, Function, and Genetics (1993), 17 (1), 75-86CODEN: PSFGEY; ISSN:0887-3585.

    The rate of exchange of peptide group NH hydrogens with the hydrogens of aq. solvent is sensitive to neighboring side chains. To evaluate the effects of protein side chains, all 20 naturally occurring amino acids were studied using dipeptide models. Both inductive and steric blocking effects are apparent. The additivity of nearest-neighbor blocking and inductive effects was tested in oligo and polypeptides and, surprisingly, confirmed. Ref. rates for alanine-contg. peptides were detd. and effects of temp. considered. These results provide the information necessary to evaluate measured protein NH to ND exchange rates by comparing them with rates to be expected for the same amino acid sequence is unstructured oligo- and polypeptides. The application of this approach to protein studies is discussed.

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36. 36

    Bai, Y.; Milne, J. S.; Mayne, L.; Englander, S. W. Protein stability parameters measured by hydrogen exchange. Proteins 1994, 20, 4– 14,  DOI: 10.1002/prot.340200103

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    35

    Protein stability parameters measured by hydrogen exchange

    Bai, Yawen; Milne, John S.; Mayne, Leland; Englander, S. Walter

    Proteins: Structure, Function, and Genetics (1994), 20 (1), 4-14CODEN: PSFGEY; ISSN:0887-3585.

    The hydrogen exchange (HX) rates of the slowest peptide group NH hydrogens in oxidized cytochrome c (equine) are controlled by the transient global unfolding equil. These rates can be measured by one-dimensional NMR and used to det. the thermodn. parameters of global unfolding at mild soln. conditions well below the melting transition. The free energy for global unfolding measured by hydrogen exchange can differ from values found by std. denaturation methods, most notably due to the slow cis-trans isomerization of the prolyl peptide bond. This difference can be quant. calcd. from basic principles. Even with these corrections, HX expts. at low denaturant concn. measure a free energy of protein stability that rises above the usual linear extrapolation from denaturation data, as predicted by the denaturant binding model of Tanford.

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37. 37

    Chetty, P. S.; Mayne, L.; Lund-Katz, S.; Stranz, D.; Englander, S. W.; Phillips, M. C. Helical structure and stability in human apolipoprotein A-I by hydrogen exchange and mass spectrometry. Proc Natl Acad Sci U S A 2009, 106, 19005– 19010,  DOI: 10.1073/pnas.0909708106

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    36

    Helical structure and stability in human apolipoprotein A-I by hydrogen exchange and mass spectrometry

    Chetty, Palaniappan Sevugan; Mayne, Leland; Lund-Katz, Sissel; Stranz, David; Englander, S. Walter; Phillips, Michael C.

    Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (45), 19005-19010, S19005/1-S19005/11CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

    Apolipoprotein A-I (apoA-I) stabilizes anti-atherogenic high d. lipoprotein particles (HDL) in the circulation and governs their biogenesis, metab., and functional interactions. To decipher these important structure-function relationships, it will be necessary to understand the structure, stability, and plasticity of the apoA-I mol. Biophys. studies show that lipid-free apoA-I contains a large amt. of α-helical structure but the location of this structure and its properties are not established. We used hydrogen-deuterium exchange coupled with a fragmentation-sepn. method and mass spectrometric anal. to study human lipid-free apoA-I in its physiol. pertinent monomeric form. The acquisition of ≈ 100 overlapping peptide fragments that redundantly cover the 243-residue apoA-I polypeptide made it possible to define the positions and stabilities of helical segments and to draw inferences about their interactions and dynamic properties. Residues 7-44, 54-65, 70-78, 81-115, and 147-178 form α-helixes, accounting for a helical content of 48 ± 3%, in agreement with CD measurements (49%). At 3 to 5 kcal/mol in free energy of stabilization, the helixes are far more stable than could be achieved in isolation, indicating mutually stabilizing helix bundle interactions. However the helical structure is dynamic, unfolding and refolding in seconds, allowing facile apoA-I reorganization during HDL particle formation and remodeling.

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38. 38

    Hageman, T. S.; Weis, D. D. Reliable Identification of Significant Differences in Differential Hydrogen Exchange-Mass Spectrometry Measurements Using a Hybrid Significance Testing Approach. Anal. Chem. 2019, 91, 8008– 8016,  DOI: 10.1021/acs.analchem.9b01325

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    37

    Reliable Identification of Significant Differences in Differential Hydrogen Exchange-Mass Spectrometry Measurements Using a Hybrid Significance Testing Approach

    Hageman, Tyler S.; Weis, David D.

    Analytical Chemistry (Washington, DC, United States) (2019), 91 (13), 8008-8016CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)

    Differential hydrogen exchange-mass spectrometry (HX-MS) measurements are valuable for identification of differences in the higher order structures of proteins. Typically, the data sets are large with many differential HX values corresponding to many peptides monitored at several labeling times. To eliminate subjectivity and reliably identify significant differences in HX-MS measurements, a statistical anal. approach is needed. In this work, the authors performed null HX-MS measurements (i.e., no meaningful differences) on maltose binding protein and infliximab, a monoclonal antibody, to evaluate the reliability of different statistical anal. approaches. Null measurements are useful for directly evaluating the risk (i.e., falsely classifying a difference as significant) and power (i.e., failing to classify a true difference as significant) assocd. with different statistical anal. approaches. With null measurements, the authors identified weaknesses in the approaches commonly used. Individual tests of significance were prone to false positives due to the problem of multiple comparisons. Incorporation of Bonferroni correction led to unacceptably large limits of detection, severely decreasing the power. Anal. methods using a globally estd. significance limit also led to an over-estn. of the limit of detection, leading to a loss of power. Here, the authors demonstrate a hybrid statistical anal., based on volcano plots, that combines individual significance testing with an estd. global significance limit, simultaneously decreased the risk of false positives and retained superior power. Furthermore, the authors highlight the utility of null HX-MS measurements to explicitly evaluate the criteria used to classify a difference in HX as significant.

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39. 39

    Livanova, N. B.; Chebotareva, N. A.; Eronina, T. B.; Kurganov, B. I. Pyridoxal 5’-phosphate as a catalytic and conformational cofactor of muscle glycogen phosphorylase B. Biochemistry (Mosc) 2002, 67, 1089– 1098,  DOI: 10.1023/a:1020978825802

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    38

    Pyridoxal 5'-phosphate as a catalytic and conformational cofactor of muscle glycogen phosphorylase b

    Livanova, N. B.; Chebotareva, N. A.; Eronina, T. B.; Kurganov, B. I.

    Biochemistry (Moscow, Russian Federation)(Translation of Biokhimiya (Moscow, Russian Federation)) (2002), 67 (10), 1089-1098CODEN: BIORAK; ISSN:0006-2979. (MAIK Nauka/Interperiodica Publishing)

    A review, which summarizes data on the structure of muscle glycogen phosphorylase b (I) and the role of the cofactor, pyridoxal 5'-phosphate (PLP), in catalysis and stabilizing the native conformation of the enzyme. Specific attention is paid to the stabilizing role of PLP upon denaturation of I. The stability of holo-I, apo-I, and I reduced by NaBH4 has been compared.

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40. 40

    Barford, D.; Johnson, L. N. The molecular mechanism for the tetrameric association of glycogen phosphorylase promoted by protein phosphorylation. Protein science : a publication of the Protein Society 1992, 1, 472– 493,  DOI: 10.1002/pro.5560010403

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    The molecular mechanism for the tetrameric association of glycogen phosphorylase promoted by protein phosphorylation

    Barford D; Johnson L N

    Protein science : a publication of the Protein Society (1992), 1 (4), 472-93 ISSN:0961-8368.

    The allosteric transition of glycogen phosphorylase promoted by protein phosphorylation is accompanied by the association of a pair of functional dimers to form a tetramer. The conformational changes within the dimer that lead to the creation of a protein recognition surface have been analyzed from a comparison of the crystal structures of T-state dimeric phosphorylase b and R-state tetrameric phosphorylase a. Regions of the structure that participate in the tetramer interface are situated within structural subdomains. These include the glycogen storage subdomain, the C-terminal subdomain and the tower helix. The subdomains undergo concerted conformational transitions on conversion from the T to the R state (overall r.m.s. shifts between 1 and 1.7 A) and, together with the quaternary conformational change within the functional dimer, create the tetramer interface. The glycogen storage subdomain and the C-terminal subdomain are distinct from those regions that contribute to the dimer interface, but shifts in the subdomains are correlated with the allosteric transitions that are mediated by the dimer interface. The structural properties of the tetramer interface are atypical of an oligomeric protein interface and are more similar to protein recognition surfaces observed in protease inhibitors and antibody-protein antigen complexes. There is a preponderance of polar and charged residues at the tetramer interface and a high number of H-bonds per surface area (one H-bond per 130 A2). In addition, the surface area made inaccessible at the interface is relatively small (1,142 A2 per subunit on dimer to tetramer association compared with 2,217 A2 per subunit on monomer-to-dimer association).

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    Johnson, L. N. Glycogen phosphorylase: control by phosphorylation and allosteric effectors. Faseb j 1992, 6, 2274– 2282,  DOI: 10.1096/fasebj.6.6.1544539

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    Glycogen phosphorylase: control by phosphorylation and allosteric effectors

    Johnson, L. N.

    FASEB Journal (1992), 6 (6), 2274-82CODEN: FAJOEC; ISSN:0892-6638.

    A review with 58 refs. Structural studies of muscle glycogen phosphorylase during the last two decades have provided a detailed mechanism for the mol. basis of the control by phosphorylation and by allosteric effectors and the catalytic mechanism. Control by phosphorylation is effected by a disorder to order transition of the NH2-terminal residues that promotes localized changes in the structure of the protein at the region of subunit-subunit contacts and larger changes in the quaternary structure. The covalently attached phosphate group acts like an allosteric effector but the full manifestation of the response is also dependent on the NH2-terminal tail residues. The noncovalently bound allosteric effectors produce similar shifts in the structural states although these are bound at sites that are remote from the serine-phosphate site. The communication from these sites to the catalytic sites is through long-range interactions that result in activation of the enzyme through opening access to the buried catalytic site and through creation of the substrate phosphate recognition site by an interchange of an acidic group with a basic group. Recent advances in expression systems have opened the way to a study of properties both for the muscle and other isoenzymes and other species that should illuminate the different regulatory roles of the enzyme in different tissues and organisms. The allosteric mechanism of activation of phosphorylase by phosphorylation may be relevant to other enzymes although it is now known that other mechanisms such as electrostatic steric blocking mechanisms also exist.

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42. 42

    Lorek, A.; Wilson, K. S.; Sansom, M. S. P.; Stuart, D. I.; Stura, E. A.; Jenkins, J. A.; Zanotti, G.; Hajdu, J.; Johnson, L. N. Allosteric interactions of glycogen phosphorylase b. A crystallographic study of glucose 6-phosphate and inorganic phosphate binding to di-imidate-cross-linked phosphorylase b. Biochem. J. 1984, 218, 45– 60,  DOI: 10.1042/bj2180045

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    41

    Allosteric interactions of glycogen phosphorylase b. A crystallographic study of glucose 6-phosphate and inorganic phosphate binding to diimidate-crosslinked phosphorylase b

    Lorek, Ann; Wilson, Keith S.; Sansom, Mark S. P.; Stuart, David I.; Stura, Enrico A.; Jenkins, John A.; Zanotti, Guiseppe; Hajdu, Janos; Johnson, Louise N.

    Biochemical Journal (1984), 218 (1), 45-60CODEN: BIJOAK; ISSN:0264-6021.

    The binding to glycogen phosphorylase b (I) of glucose 6-phosphate and inorg. phosphate (resp. allosteric inhibitor and substrate/activator of the enzyme) were studied in the crystal at 0.3 nm (3 Å) resoln. Glucose 6-phosphate binds in the α-configuration at a site that is close to the AMP-allosteric-effector site at the subunit-subunit interface and promotes several conformational changes. The phosphate-binding site of the enzyme for glucose 6-phosphate involves contacts to 2 cationic residues, arginine-309 and lysine-247. This site is also occupied in the inorg.-phosphate-binding studies and is therefore identified as a high-affinity phosphate-binding site. It is distinct from the weaker phosphate-binding site of the enzyme for AMP, which is 0.27 nm (2.7 Å) away. The glucose moiety of glucose 6-phosphate and the adenosine moiety of AMP do not overlap. The results provide a structural explanation for the kinetic observations that glucose 6-phosphate inhibition of AMP activation of I is partially competitive and highly cooperative. Apparently, the transmission of allosteric conformational changes involves an increase in affinity at phosphate-binding sites and relative movements of α-helixes. In order to study glucose 6-phosphate and phosphate binding, it was necessary to crosslink the crystals. The use of di-Me malondiimidate as a new crosslinking reagent in protein crystallog. is discussed.

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43. 43

    Zographos, S. E.; Oikonomakos, N. G.; Tsitsanou, K. E.; Leonidas, D. D.; Chrysina, E. D.; Skamnaki, V. T.; Bischoff, H.; Goldmann, S.; Watson, K. A.; Johnson, L. N. The structure of glycogen phosphorylase b with an alkyldihydropyridine-dicarboxylic acid compound, a novel and potent inhibitor. Structure 1997, 5, 1413– 1425,  DOI: 10.1016/S0969-2126(97)00292-X

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    The structure of glycogen phosphorylase b with an alkyl-dihydropyridine-dicarboxylic acid compound, a novel and potent inhibitor

    Zographos, Spyros E.; Oikonomakos, Nikos G.; Tsitsanou, Katerina E.; Leonidas, Demetrios D.; Chrysina, Evangelia D.; Skamnaki, Vicky T.; Bischoff, Hilmar; Goldmann, Siegfried; Watson, Kimberly A.; Johnson, Louise N.

    Structure (London) (1997), 5 (11), 1413-1425CODEN: STRUE6; ISSN:0969-2126. (Current Biology Ltd.)

    In muscle and liver, glycogen concns. are regulated by the reciprocal activities of glycogen phosphorylase (I) and glycogen synthase. Bay W 1807 [(-)-(S)-3-isopropyl-4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methylpyridine-3,5,6-tricarboxylate] (II), an alkyldihydropyridinedicarboxylic acid, was found to be a potent inhibitor of I, and as such has potential to contribute to the regulation of glycogen metab. in the non-insulin-dependent type II diabetes diseased state. II had no structural similarity to natural regulators of I. Here, the authors carried out structural studies in order to elucidate the mechanism of inhibition. Kinetic studies with rabbit muscle I-b showed that II had a Ki of 1.6 nM and was a competitive inhibitor with respect to AMP. The structure of the cocrystd. I-b·II complex was detd. at 100K to 2.3 Å resoln. and refined to an R factor of 0.198 (Rfree = 0.287). II bound at the I-b allosteric effector site, the site which binds AMP, glucose 6-phosphate, and a no. of other phosphorylated ligands, and induced conformational changes that were characteristic of those obsd. with the naturally occurring allosteric inhibitor, glucose 6-phosphate. The dihydropyridine-5,6-dicarboxylate groups mimicked the phosphate group of ligands that bind to the allosteric site and contact 3 Arg residues. The high affinity of II for I-b appears to arise from the numerous nonpolar interactions made between the ligand and the protein. Its potency as an inhibitor resulted from the induced conformational changes that locked I-b in a conformation known as the T' state. Allosteric enzymes, such as I, offer a new strategy for structure-based drug design in which the allosteric site can be exploited. The results reported here may have important implications in the design of new therapeutic compds.

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44. 44

    Oikonomakos, N. G.; Schnier, J. B.; Zographos, S. E.; Skamnaki, V. T.; Tsitsanou, K. E.; Johnson, L. N. Flavopiridol inhibits glycogen phosphorylase by binding at the inhibitor site. J. Biol. Chem. 2000, 275, 34566– 34573,  DOI: 10.1074/jbc.m004485200

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    43

    Flavopiridol inhibits glycogen phosphorylase by binding at the inhibitor site

    Oikonomakos, Nikos G.; Schnier, Joachim B.; Zographos, Spyros E.; Skamnaki, Vicky T.; Tsitsanou, Katerina E.; Johnson, Louise N.

    Journal of Biological Chemistry (2000), 275 (44), 34566-34573CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)

    Flavopiridol (L86-8275) ((-)-cis-5,7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl)-piperidinyl]-4H-benzopyran-4-one), a potential antitumor drug, currently in phase II trials, has been shown to be an inhibitor of muscle glycogen phosphorylase (GP) and to cause glycogen accumulation in A549 non-small cell lung carcinoma cells (Kaiser, A., Nishi, K., Gorin, F.A., Walsh, D.A., Bradbury, E.M., and Schnier, J.B., unpublished data). Kinetic expts. reported here show that flavopiridol inhibits GPb with an IC50 = 15.5 μM. The inhibition is synergistic with glucose resulting in a redn. of IC50 for flavopiridol to 2.3 μM and mimics the inhibition of caffeine. In order to elucidate the structural basis of inhibition, we detd. the structures of GPb complexed with flavopiridol, GPb complexed with caffeine, and GPa complexed with both glucose and flavopiridol at 1.76-, 2.30-, and 2.23-Å resoln., and refined to crystallog. R values of 0.216 (Rfree = 0.247), 0.189 (Rfree = 0.219), and 0.195 (Rfree = 0.252), resp. The structures provide a rational for flavopiridol potency and synergism with glucose inhibitory action. Flavopiridol binds at the allosteric inhibitor site, situated at the entrance to the catalytic site, the site where caffeine binds. Flavopiridol intercalates between the two arom. rings of Phe285 and Tyr613. Both flavopiridol and glucose promote the less active T-state through localization of the closed position of the 280s loop which blocks access to the catalytic site, thereby explaining their synergistic inhibition. The mode of interactions of flavopiridol with GP is different from that of des-chloro-flavopiridol with CDK2, illustrating how different functional parts of the inhibitor can be used to provide specific and potent binding to two different enzymes.

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45. 45

    Oikonomakos, N. G.; Zographos, S. E.; Skamnaki, V. T.; Archontis, G. The 1.76 Å resolution crystal structure of glycogen phosphorylase B complexed with glucose, and CP320626, a potential antidiabetic drug. Bioorg. Med. Chem. 2002, 10, 1313– 1319,  DOI: 10.1016/s0968-0896(01)00394-7

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    44

    The 1.76 Å resolution crystal structure of glycogen phosphorylase b complexed with glucose, and CP 320626, a potential antidiabetic drug

    Oikonomakos, Nikos G.; Zographos, Spyros E.; Skamnaki, Vicky T.; Archontis, Georgios

    Bioorganic & Medicinal Chemistry (2002), 10 (5), 1313-1319CODEN: BMECEP; ISSN:0968-0896. (Elsevier Science Ltd.)

    CP 320626, a potential antidiabetic drug, inhibits glycogen phosphorylase (I) in synergism with glucose. To elucidate the structural basis of synergistic inhibition, the authors detd. the crystal structure of muscle I complexed with both glucose and CP 320626 at 1.76 Å resoln., and refined it to a crystallog. R value of 0.211 (Rfree = 0.235). CP 320626 was found to bind at a novel allosteric site, which was ∼33 Å from the catalytic site, where glucose binds. The high-resoln. structure allowed unambiguous definition of the conformation of the 1-acetyl-4-hydroxy-piperidine ring supported by theor. energy calcns. Both CP 320626 and glucose promoted the less active T-state, thereby explaining their synergistic inhibition. Structural comparison of the I·glucose·CP 320626 complex with liver glycogen phosphorylase a (II) complexed with a related compd. (CP 403700) showed that the ligand binding site was conserved in II.

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    Sprang, S. R.; Acharya, K. R.; Goldsmith, E. J.; Stuart, D. I.; Varvill, K.; Fletterick, R. J.; Madsen, N. B.; Johnson, L. N. Structural changes in glycogen phosphorylase induced by phosphorylation. Nature 1988, 336, 215– 221,  DOI: 10.1038/336215a0

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    45

    Structural changes in glycogen phosphorylase induced by phosphorylation

    Sprang, S. R.; Acharya, K. R.; Goldsmith, E. J.; Stuart, D. I.; Varvill, K.; Fletterick, R. J.; Madsen, N. B.; Johnson, L. N.

    Nature (London, United Kingdom) (1988), 336 (6196), 215-21CODEN: NATUAS; ISSN:0028-0836.

    A comparison of the refined crystal structures of dimeric glycogen phosphorylase b and a reveals structural changes that represent the 1st step in the activation of the enzyme. On phosphorylation of serine-14, the N-terminus of each subunit assumes an ordered helical conformation and binds to the surface of the dimer. The consequent structural changes at the N- and C-terminal regions lead to strengthened interactions between subunits and alter the binding sites for allosteric effectors and substrates.

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47. 47

    Sprang, S. R.; Goldsmith, E. J.; Fletterick, R. J.; Withers, S. G.; Madsen, N. B. Catalytic site of glycogen phosphorylase: structure of the T state and specificity for .alpha.-D-glucose. Biochemistry 1982, 21, 5364– 5371,  DOI: 10.1021/bi00264a038

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    46

    Catalytic site of glycogen phosphorylase: structure of the T state and specificity for α-D-glucose

    Sprang, Stephen R.; Goldsmith, Elizabeth J.; Fletterick, Robert J.; Withers, Stephen G.; Madsen, Neil B.

    Biochemistry (1982), 21 (21), 5364-71CODEN: BICHAW; ISSN:0006-2960.

    α-D-Glucose (I) inhibits glycogen phosphorylase a (II) by binding at the catalytic site of the inactive conformer (T state) at the same position as does the substrate, α-D-glucose 1-phosphate (III), to the active (R state) enzyme. Crystallog. anal. of the I-II complex and anal. of inhibition by a variety of I analogs were used to study the nature and specificity of the recognition of the glucosyl group by the T-state enzyme. The catalytic site at which I is bound is located at the confluence of the N- and C-terminal domains. Each is an α/β structure consisting of a β-sheet core surrounded by a double tier of α-helixes. The active-site residues are located on flexible loops of polypeptide chain emanating from the domain boundaries. I participates in ≥5 well-defined H-bonds with these residues and presents a complementary mol. surface to the active site at the H-bonded positions of the ligand. Inhibition and model-building studies show that changes in chirality or substitution at any of the I hydroxyl groups can abolish or drastically reduce the binding affinity of the ligand. Absence or low activity in I analogs can be rationalized as a redn. in H-bonding capacity and(or) the induction of steric conflicts with the enzyme. Although there are substantial differences between T- and R-state II with respect to active-site conformation, both conformers exhibit specific binding of the glucosyl moiety of I on the one hand (T) and III or half-chair glycosyl analogs (which mimic the proposed carbonium ion intermediates or transition state) on the other (R). A structural interpretation of these observations is presented. By means of inhibition studies with several III analogs and also by inspection of the crystal structure, it is demonstrated that the substrate-binding site, in the R-state enzyme, comprises adjacent phosphate and glycosyl subsites. Analogs of the substrate which differ substantially in their carbohydrate moiety demonstrate competitive inhibition by occupation of the phosphate subsite alone.

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48. 48

    Dombrádi, V. Structural aspects of the catalytic and regulatory function of glycogen phosphorylase. Int. J. Biochem. 1981, 13, 125– 139,  DOI: 10.1016/0020-711X(81)90147-6

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    Structural aspects of the catalytic and regulatory function of glycogen phosphorylase

    Dombradi, Viktor

    International Journal of Biochemistry (1981), 13 (2), 125-39CODEN: IJBOBV; ISSN:0020-711X.

    A review with 183 refs.

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    Johnson, L. N. Glycogen phosphorylase: A multifaceted enzyme. Carlsberg Res. Commun. 1989, 54, 203,  DOI: 10.1007/BF02910457

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    Glycogen phosphorylase: a multifaceted enzyme

    Johnson, Louise N.

    Carlsberg Research Communications (1989), 54 (6), 203-29CODEN: CRCODS; ISSN:0105-1938.

    A review with 78 refs., on x-ray crystallog. studies of the title enzyme as related to catalytic and allosteric mechanisms, and to oligosaccharide recognition.

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50. 50

    Rath, V. L.; Ammirati, M.; LeMotte, P. K.; Fennell, K. F.; Mansour, M. N.; Danley, D. E.; Hynes, T. R.; Schulte, G. K.; Wasilko, D. J.; Pandit, J. Activation of human liver glycogen phosphorylase by alteration of the secondary structure and packing of the catalytic core. Molecular cell 2000, 6, 139– 148,  DOI: 10.1016/s1097-2765(05)00006-7

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    Activation of human liver glycogen phosphorylase by alteration of the secondary structure and packing of the catalytic core

    Rath, Virginia L.; Ammirati, Mark; LeMotte, Peter K.; Fennell, Kimberly F.; Mansour, Mahmoud N.; Danley, Dennis E.; Hynes, Thomas R.; Schulte, Gayle K.; Wasilko, David J.; Pandit, Jayvardhan

    Molecular Cell (2000), 6 (1), 139-148CODEN: MOCEFL; ISSN:1097-2765. (Cell Press)

    Glycogen phosphorylases catalyze the breakdown of glycogen to glucose-1-phosphate, which enters glycolysis to fulfill the energetic requirements of the organism. Maintaining control of blood glucose levels is crit. in minimizing the debilitating effects of diabetes, making liver glycogen phosphorylase a potential therapeutic target. To support inhibitor design, we detd. the crystal structures of the active and inactive forms of human liver glycogen phosphorylase a. During activation, forty residues of the catalytic site undergo order/disorder transitions, changes in secondary structure, or packing to reorganize the catalytic site for substrate binding and catalysis. Knowing the inactive and active conformations of the liver enzyme and how each differs from its counterpart in muscle phosphorylase provides the basis for designing inhibitors that bind preferentially to the inactive conformation of the liver isoenzyme.

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51. 51

    Oikonomakos, N. G.; Acharya, K. R.; Melpidou, A. E.; Stuart, D. I.; Johnson, L. N. The binding of β-glycerophosphate to glycogen phosphorylase b in the crystal. Arch. Biochem. Biophys. 1989, 270, 62– 68,  DOI: 10.1016/0003-9861(89)90007-6

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    The binding of β-glycerophosphate to glycogen phosphorylase b in the crystal

    Oikonomakos, N. G.; Acharya, K. R.; Melpidou, A. E.; Stuart, D. I.; Johnson, L. N.

    Archives of Biochemistry and Biophysics (1989), 270 (1), 62-8CODEN: ABBIA4; ISSN:0003-9861.

    The binding of β-glycerophosphate (glycerol-2-P) to glycogen phosphorylase b in the crystal has been studied by x-ray diffraction at 3 Å resoln. Glycerol-2-P binds to the allosteric effector site in a position close to that of AMP, glucose-6-phosphate, UDP-glucose (Glc), and phosphate. In this position, glycerol-2-P is stabilized through interactions of its phosphate moiety with the guanidinium groups of arginine (Arg) 309 and Arg 310 which undergo conformational changes, and the hydroxyl group of tyrosine 75, while the same residues and solvent are involved in van der Waals interactions with the remaining part of the mol. Kinetic expts. indicate that glycerol-2-P partially competes with both the activator (AMP) and the inhibitor (glucose-6-phosphate) of phosphorylase b. A comparison of the positions of glycerol-2-P, AMP, glucose 6-phosphate, UDP-Glc, and phosphate at the allosteric site is presented.

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52. 52

    Oikonomakos, n. g.; Acharya, k. R.; Stuart, d. i.; Melpidou, a. e.; McLAUGHLIN, p. j.; Johnson, l. n. Uridine(5’)diphospho(1)-alpha-d-glucose. A binding study to glycogen phosphorylase b in the crystal. Eur. J. Biochem. 1988, 173, 569– 578,  DOI: 10.1111/j.1432-1033.1988.tb14037.x

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    Uridine(5')diphospho(1)-α-D-glucose. A binding study to glycogen phosphorylase b in the crystal

    Oikonomakos, Nikos G.; Acharya, K. Ravindra; Stuart, David I.; Melpidou, Angeliki E.; McLaughlin, Paul J.; Johnson, Louise N.

    European Journal of Biochemistry (1988), 173 (3), 569-78CODEN: EJBCAI; ISSN:0014-2956.

    UDP-glucose is an R-state inhibitor of glycogen phosphorylase b, competitive with the substrate, glucose 1-phosphate and noncompetitive with the allosteric activator, AMP. The diffusion of 100 mM UDP-glucose into crystals of phosphorylase b resulted in a difference Fourier synthesis at 0.3-nm resoln. that showed 2 peaks: (a) binding at the allosteric site and (b) binding at the catalytic site. At the allosteric site, the whole of the UDP-glucose mol. could be located. It was in a well-defined folded conformation with its uracil portion in a similar position to that obsd. for the adenine of AMP. The uracil and the glucose moieties stacked against the arom. side-chains of tyrosine (Tyr)-75 and phenylalanine-196, resp. The phosphates of the pyrophosphate component interacted with arginine (Arg)-242, Arg-309, and Arg-310. At the catalytic site, the glucose 1-phosphate component of UDP-glucose was firmly bound in a position similar to that obsd. for glucose 1-phosphate. The pyrophosphate was also well located with the glucose phosphate interacting with the main-chain NH groups at the start of the glycine-loop α helix and the uridine phosphate interacting through a water mol. with the 5'-phosphate of the cofactor, pyridoxal phosphate, and with the side-chains of residues Tyr-573, lysine-574, and probably Arg-569. However the position of the uridine could not be located although anal. by TLC showed that no degrdn. had taken place. The binding of UDP-glucose to the catalytic site promoted extensive conformational changes. Loop 279-288, which linked the catalytic site to the nucleoside inhibitor site, was displaced and became mobile. Concomitant movements of histidine-571, Arg-569, and loop 378-383, together with the major loop displacement, resulted in an open channel to the catalytic site. Comparison with other structural results showed that these changes form an essential feature of the T-to-R transition. They allow formation of the phosphate recognition site at the catalytic site and destroy the nucleoside inhibitor site. Kinetic expts. demonstrated that UDP-glucose activates the enzyme in the presence of high concns. of the weak activator, IMP, because of its ability to decrease the affinity of IMP for the inhibitor site.

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