ASAP (As Soon As Publishable)

We recently identified N-cadherin as a novel receptor for fibrin and localized complementary binding sites within the fibrin βN-domains and the third and fifth extracellular domains (EC3 and EC5) of N-cadherin. We also hypothesized that the His16 and Arg17 residues of the βN-domains and the (Asp/Glu)-X-(Asp/Glu) motifs present in the EC3 and EC5 domains may play roles in the interaction between fibrin and N-cadherin. The primary objectives of this study were to test these hypotheses and to further clarify the structural basis for this interaction. To test our hypotheses, we first mutated His16 and Arg17 in the recombinant (β15–66)₂ fragment, which mimics the dimeric arrangement of the βN-domains in fibrin, using site-directed mutagenesis. The results revealed that the mutations of both His16 and Arg17 are critical for the interaction. Next, we mutated Asp/Glu residues in the three (Asp/Glu)-X-(Asp/Glu) motifs, M1 (Asp-Phe-Glu), M2 (Glu-Ala-Glu), and M3 (Asp-Tyr-Asp), of the fibrin-binding N-cad(3–5) fragment of N-cadherin. The results showed that Asp292 and Glu294 of M1, and Asp468 and Asp470 of M3, are critical for the interaction. Our molecular modeling of the 3D structure of the EC3-EC4-EC5 domains revealed that these residues are located at the interfaces of EC3-EC4 and EC4-EC5 and that some may also be involved in calcium binding. In conclusion, our study identified amino acid residues in the fibrin βN-domains and the EC3 and EC5 domains of N-cadherin that are critical for the interaction of fibrin with N-cadherin and localized the fibrin-binding residues in the 3D structure of N-cadherin.

There are surprisingly few RNA intramolecular triple helices known in the human transcriptome. The structure has been most well-studied as a stability-element at the 3′ end of lncRNAs such as MALAT1 and NEAT1, but the intrigue remains whether it is indeed as rare as it is understood to be or just waiting for a closer look from a new vantage point. TRIPinRNA, our Python-based in silico platform, allows for a comprehensive sequence-pattern search for potential triplex formation in the human transcriptome─noncoding as well as coding. Using this tool, we report the putative occurrence of homopyrimidine type (canonical) triple helices as well as heteropurine–pyrimidine strand type (noncanonical) triple helices in the human transcriptome and validate the formation of both types of triplexes using biophysical approaches. We find that the occurrence of triplex structures has a strong correlation with local GC content, which might be influencing their formation. By employing a search that encompasses both canonical and noncanonical triplex structures across the human transcriptome, this study enriches the understanding of RNA biology. Lastly, TRIPinRNA can be utilized in finding triplex structures for any organism with an annotated transcriptome.

Dithiolopyrrolone (DTP) natural products are broad-spectrum antimicrobial and anticancer prodrugs. The DTP structure contains a unique bicyclic ene-disulfide that once reduced in the cell, chelates metal ions and disrupts metal homeostasis. In this work we investigate the intracellular activation of the DTPs and their resistance mechanisms in bacteria. We show that the prototypical DTP holomycin is reduced by several bacterial reductases and small-molecule thiols in vitro. To understand how bacteria develop resistance to the DTPs, we generate Staphylococcus aureus mutants that exhibit increased resistance to the hybrid DTP antibiotic thiomarinol. From these mutants we identify loss-of-function mutations in redox genes that are involved in DTP activation. This work advances the understanding of how DTPs are activated and informs development of bioreductive disulfide prodrugs.

Bacteroides are often the most abundant, commensal species in the gut microbiome of industrialized human populations. One of the most commonly detected species is Bacteroides ovatus. It has been linked to benefits like the suppression of intestinal inflammation but is also correlated with some autoimmune disorders, for example irritable bowel disorder (IBD). Bacterial cell surface carbohydrates, like capsular polysaccharides (CPS), may play a role in modulating these varied host interactions. Recent studies have begun to explore the diversity of CPS loci in Bacteroides; however, there is still much unknown. Here, we present structural and functional characterization of a putative polysaccharide deacetylase from Bacteroides ovatus (BoPDA) encoded in a CPS biosynthetic locus. We solved four high resolution crystal structures (1.36–1.56 Å) of the enzyme bound to divalent cations Co²⁺, Ni²⁺, Cu²⁺, or Zn²⁺ and performed carbohydrate binding and deacetylase activity assays. Structural analysis of BoPDA revealed an atypical domain architecture that is unique to this enzyme, with a carbohydrate esterase 4 (CE4) superfamily catalytic domain inserted into a carbohydrate binding module (CBM). Additionally, BoPDA lacks the canonical CE4 His-His-Asp metal binding motif and our structures show it utilizes a noncanonical His-Asp dyad to bind metal ions. BoPDA is the first protein involved in CPS biosynthesis from B. ovatus to be characterized, furthering our understanding of significant biosynthetic processes in this medically relevant gut microbe.

Sensing of peptidoglycan fragments is essential for inducing downstream signaling in both mammalian and fungal systems. The hexokinases NagK and Hxk1 are crucial enzymes for the phosphorylation of peptidoglycan molecules in order to activate specific cellular responses; however, biochemical characterization of their enzymatic specificity and efficiency has yet to be investigated in depth. Here a mass spectrometry enzymatic screen was implemented to assess substrate specificity, and an ATP coupled assay provided the quantitative kinetic profiles of these two homologous, eukaryotic enzymes. The data show, that while homologous, NagK and Hxk1 have vastly different substrate profiles. NagK accepts a variety of different peptidoglycan-based substrates albeit with reduced efficiency but are still valuable as a tool in large scale chemoenzymatic settings. Conversely, Hxk1 has a smaller substrate scope but can turnover these alternative substrates at similar levels to its natural substrate. These results allow for deeper understanding into the biosynthetic machinery responsible for essential cellular processes including UDP-GlcNAc regulation and immune recognition events in the cell.

During Staphylococcus aureus infections, reactive oxygen species cause DNA damage, including nucleotide base modification. After removal of the defective base, excision repair requires an endonuclease IV (Nfo), which hydrolyzes the phosphodiester bond 5′ to the abasic nucleotide. This class of enzymes, typified by the enzyme from Escherichia coli, contains a catalytic site with three metal ions, previously reported to be all Zn²⁺. The 1.05 Å structure of Nfo from the Gram-positive organism S. aureus (SaNfo) revealed two inner Fe²⁺ ions and one Zn²⁺ as confirmed by dispersive anomalous difference maps. SaNfo has a previously undescribed water molecule liganded to Fe₁ forming an octahedral coordination geometry and hydrogen bonded to Tyr33, an active site residue conserved in many Gram-positive bacteria, but which is Phe in Gram-negative species that coordinate Zn²⁺ at the corresponding site. The 1.9 Å structure of E. coli Nfo (EcNfo), purified without added metals, revealed that metal 2 is Fe²⁺ and not Zn²⁺. Octahedral coordination for the sites occupied by Fe²⁺ suggests a stereoselective mechanism for differentiating between Fe²⁺ and Zn²⁺ in this enzyme class. Kinetics and an inhibitor competition assay of SaNfo reveal product inhibition (or slow product release), especially at low ionic strength, caused in part by a Lys-rich DNA binding loop present in SaNfo and Gram-positive species but not in EcNfo. Biological significance of the slow product release is discussed. Catalytic activity in vitro is optimal at 300 mM NaCl, which is consistent with the halotolerant phenotype of S. aureus.

Phosphatidylinositol phosphates (PIPs) are a family of seven different eukaryotic membrane lipids that have a large role in cell viability, despite their minor concentration in eukaryotic cellular membranes. PIPs tightly regulate cellular processes, such as cellular growth, metabolism, immunity, and development through direct interactions with partner proteins. Understanding the biophysical properties of PIPs in the complex membrane environment is important to understand how PIPs selectively regulate a partner protein. Here, we investigate the structure and dynamics of PIP₃ in lipid bilayers that are simplified models of the natural membrane environment. We probe the effects of the anionic lipid phosphatidylserine (PS) and the divalent cation Ca²⁺ by using full-length lipids in well-formed bilayers. We used solution and solid-state NMR on naturally abundant ¹H, ³¹P, and ¹³C atoms combined with molecular dynamics (MD) simulations to characterize the structure and dynamics of PIPs. ¹H and ³¹P 1D spectra show good resolution at temperatures above the phase transition with isolated peaks in the headgroup, interfacial, and bilayer regions. Site-specific assignment of the chemical shifts of these reporters enables the measurement of the effects of Ca²⁺ and PS at the single atom level. In particular, the resolved ³¹P signals of the PIP₃ headgroup allow for extremely well-localized information about PIP₃ phosphate dynamics, which the MD simulations can further explain. A quantitative assessment of cross-polarization kinetics provides additional dynamics measurements for the PIP₃ headgroups.

Photosystem I (PSI) from Acaryochloris marina utilizes chlorophyll d (Chld) with a formyl group as its primary pigment, which is more red-shifted than chlorophyll a (Chla) in PSI from Thermosynechococcus elongatus. Using the cryo-electron microscopy structure and solving the linear Poisson–Boltzmann equation, here we report the redox potential (E_m) values in A. marina PSI. The E_m(Chld) values at the paired chlorophyll site, [P_AP_B], are nearly identical to the corresponding E_m(Chla) values in T. elongatus PSI, despite Chld having a 200 mV lower reduction power. The accessory chlorophyll site, A_–1, in the B branch exhibits an extensive H-bond network with its ligand water molecule, contributing to E_m(A_–1B) being lower than E_m(A_–1A). The substitution of pheophytin a (Pheoa) with Chla at the electron acceptor site, A₀, decreases E_m(A₀), resulting in an uphill electron transfer from A_–1. The impact of the A_–1 formyl group on E_m(A₀) is offset by the reorientation of the A₀ ester group. It seems likely that Pheoa is necessary for A. marina PSI to maintain the overall electron-transfer cascade characteristic of PSI in its unique light environment.

It is now well-established that microbial genomes carry sparingly expressed biosynthetic gene clusters (BGCs) that need to be induced in order to characterize their products. To do so, we herein subjected two well-known producers, Burkholderia plantarii and Burkholderia gladioli, to high-throughput elicitor screening (HiTES), an emerging approach for accessing the products of these “cryptic” BGCs. Both organisms have previously been examined extensively in liquid cultures. We therefore applied HiTES on agar and found several novel natural products that are only produced in this format and not in liquid cultures. Most notably we found two metabolites, termed burkethyl A and B, that contain an unusual m-ethylbenzoyl group and for which we identified the cognate BGC using bioinformatic and genetic studies. Our results indicate that agar-based HiTES is a promising approach for natural product discovery and are in line with the notion that even “drained” strains remain sources of new metabolites as long as alternative approaches are employed.

Prenylation consists of the modification of proteins with either farnesyl diphosphate (FPP) or geranylgeranyl diphosphate (GGPP) at a cysteine near the C-terminus of target proteins to generate thioether-linked lipidated proteins. In recent work, metabolic labeling with alkyne-containing isoprenoid analogues including C15AlkOPP has been used to identify prenylated proteins and track their levels in different diseases. Here, a systematic study of the impact of isoprenoid length on proteins labeled with these probes was performed. Chemical synthesis was used to generate two new analogues, C15hAlkOPP and C20AlkOPP, bringing the total number of compounds to eight used in this study. Enzyme kinetics performed in vitro combined with metabolic labeling in cellulo, resulted in the identification of 8 proteins for C10AlkOPP, 70 proteins for C15AlkOPP, 41 proteins for C15hAlkOPP, and 7 proteins for C20AlkOPP. While C10AlkOPP was the most selective for farnesylated proteins and C20AlkOPP was most selective for geranylgeranylated proteins, the number of proteins identified using those probes was relatively small. In contrast, C15AlkOPP labeled the most proteins including representatives from all classes of prenylated proteins. Functional analysis of these analogues demonstrated that C15AlkOPP was particularly well suited for biological studies since it was efficiently incorporated in cellulo, was able to confer correct plasma membrane localization of H-Ras protein and complement the effects of GGPP depletion in macrophages to yield correct cell polarization and filopodia. Collectively, these results indicate that C15AlkOPP is a biologically functional, universal probe for metabolic labeling experiments that has minimal effects on cellular physiology.

Plants make pyrimidine base substitutions in organellar mRNAs through the action of sequence-specific nuclear-encoded enzymes. Pentatricopeptide repeat (PPR) proteins are essential for ensuring specificity, while the enzymatic DYW domain is often present at the C-terminus of a PPR protein and dependent on the variant possessing C-to-U and/or U-to-C RNA editing activities. Expression of exogenous DYW-KP variant enzymes in bacteria leads to the modification of RNAs suggestive of U-to-C base changes. The modified RNAs could only be purified from the interphase of an acidic guanidinium thiocyanate-phenol-chloroform experiment. It was projected that in bacteria stable RNA-enzyme cross-links form from a lysyl attack. In this study, RNA editing was examined for dual U-to-C/C-to-U editing enzyme KP6 with conserved lysine residues substituted by alanine. A single lysine was found to be essential for U-to-C editing and, based on the crystal structures of DYW domains, would likely be present in the active site. Crystal structures also suggest that the lysine can potentially form an ion pair with the catalytic glutamate critical for C-to-U RNA editing. Mutation of lysine to alanine greatly stimulated the C-to-U RNA editing by KP6. A ∼319 Da adduct observed on DYW-KP proteins could not be detected on the U-to-C-deficient lysine to alanine point mutant enzymes. This work establishes the critical role for a single lysine in the DYW-KP domain specifically for U-to-C editing activity but also highlights a secondary role for the lysine in modulating C-to-U editing through the formation of an inhibitory ion pair with the catalytic glutamate.

Post-transcriptional modifications at the anticodon stem-loop of tRNAs are key to the translation function. Metabolic pathways to these modifications often incorporate complex enzymology. A notable example is the hypermodified nucleoside, queuosine, found at the wobble position of Asn, Asp, His, and Tyr encoding tRNAs. The epoxyqueuosine reductase, QueH, catalyzes the final step in the biosynthetic pathway to queuosine. The metalloenzyme catalyzes a two-electron reduction of epoxyqueuosine to provide the modified tRNA. The structure of QueH from T. maritima has previously been determined and unexpectedly contains two metal binding motifs in the active site. This includes a predicted 4Fe-4S cluster, along with a single-metal binding site coordinated by two cysteines along an aspartate carboxylate. In this report, we describe the structural and biochemical analysis of the QueH metal binding sites along with the chemistry of epoxide deoxygenation. To probe the active-site architecture, enzyme mutants of metal binding residues were structurally and biochemically characterized. In addition, structural and binding experiments were used to probe interactions of QueH with tRNA and the in vivo role of QueH and variants in Q-tRNA synthesis was evaluated. Overall, this work provides insight into the chemical mechanism of the final step of the queuosine biosynthetic pathway.

DNA-encoded libraries hold great potential for discovering small, cyclized peptides with drug potential. Split-intein circular ligation of peptides and proteins (SICLOPPS) is a well-established method for in vivo selection of cyclic peptides targeting specific intracellular components. However, the method has mainly been used in prokaryotic cells. In contrast, selection studies performed directly in eukaryotic cells allow for the identification of cyclic peptides promoting a functional outcome, without the need to define a specific cellular target. Here, we report the construction of a Saccharomyces cerevisiae-specific SICLOPPS library of 80 million members, via careful optimization of several steps to increase the size of the library. Individual library members were shown to be correctly expressed and processed in yeast. High-throughput sequencing was conducted on the randomized primer used for library construction and the pure yeast SICLOPPS library isolated from Escherichia coli. A distinct guanine insertion bias was observed in the peptide-encoding, randomized sequence, which was primarily attributed to the degenerate primer used to introduce the randomized sequence. Moreover, high-throughput sequencing was performed on the library before and after the induction of cyclic peptide expression in yeast. Importantly, expression of the SICLOPPS library in S. cerevisiae caused only a marginal further sequence bias. Our work paves the way for selection studies using a large and diverse library to identify cyclic peptides of therapeutic interest that promote a specific phenotypic outcome in eukaryotic organisms, with yeast representing a beneficial model system due to its high transformation efficiency.

Arginyltransferase 1 (ATE1) catalyzes arginylation, an important posttranslational modification (PTM) in eukaryotes that plays a critical role in cellular homeostasis. The disruption of ATE1 function is implicated in mammalian neurodegenerative disorders and cardiovascular maldevelopment, while posttranslational arginylation has also been linked to the activities of several important human viruses such as SARS-CoV-2 and HIV. Despite the known significance of ATE1 in mammalian cellular function, past biophysical studies of this enzyme have mainly focused on yeast ATE1, leaving the mechanism of arginylation in mammalian cells unclear. In this study, we sought to structurally and biophysically characterize mouse (Mus musculus) ATE1. Using size-exclusion chromatography (SEC), small-angle X-ray scattering (SAXS), and hydrogen–deuterium exchange mass spectrometry (HDX-MS), assisted by AlphaFold modeling, we found that mouse ATE1 is structurally more complex than yeast ATE1. Importantly, our data indicate the existence of an intrinsically disordered region (IDR) in all mouse ATE1 splice variants. However, comparative HDX-MS analyses show that yeast ATE1 does not have such an IDR, consistent with prior X-ray, cryo-EM, and SAXS analyses. Furthermore, bioinformatics approaches reveal that mammalian ATE1 sequences, as well those as in a large majority of other eukaryotes, contain an IDR-like sequence positioned in proximity to the ATE1 GNAT active-site fold. Computational analysis suggests that the IDR facilitates the formation of a complex between ATE1 and tRNA^Arg, adding a new complexity to the ATE1 structure and providing new insights for future studies of ATE1 functions.

Pyridoxal 5′-phosphate (PLP)-dependent enzymes catalyze a diverse array of biochemical transformations, making them invaluable biocatalytic tools for the synthesis of complex bioactive compounds. Here, we report the biochemical characterization of LolC, a PLP-dependent γ-synthase involved in the biosynthesis of loline alkaloids. LolC catalyzes the formation of a Cγ-N bond between O-acetyl--homoserine (OAH) and l-proline, generating a diamino diacid intermediate. Our findings reveal that LolC exhibits strict specificity for proline and its analogues, contrasting with the substrate promiscuity of closely related Cγ-C bond-forming enzyme Fub7. Structural analysis, using an AlphaFold model, identifies key differences in the substrate entrance tunnel of LolC, which is amphiphilic and distinct from the hydrophobic tunnel in Fub7. A mutagenesis study further highlights the functional divergence of a key active site residue between these enzymes. These results provide new insights into the substrate specificity and catalytic function of LolC, offering a valuable comparison to Fub7 and advancing our understanding of PLP-dependent enzymes involved in γ-substitution reactions.

Anaerobilin synthase catalyzes the decyclization of the heme protoporphyrin ring, an O₂-independent reaction that liberates iron and produces the linear tetrapyrrole, anaerobilin. The marine bacterium Aliivibrio fischeri, the enteric pathogen Escherichia coli O157:H7, and the opportunistic oral pathogen Fusobacterium nucleatum encode anaerobilin synthase as part of their heme uptake/utilization operons, designated chu (E. coli O157:H7), hmu (F. nucleatum), and hut (A. fischeri). F. nucleatum and E. coli O157:H7 contain accessory proteins (ChuS, ChuY, and HmuF) encoded in their respective operons that mitigate against the cytotoxicity of labile heme and anaerobilin by functioning in heme trafficking and anaerobilin reduction. However, the hut operon of A. fischeri and other members of the Vibrionaceae family including the enteric pathogen Vibrio cholerae do not contain homologues to these accessory proteins, raising questions as to how members of this family mitigate against anaerobilin and heme toxicity. Herein, we show that HutW (anaerobilin synthase) from A. fischeri produces anaerobilin, but that HutX and HutZ, encoded downstream of HutW, do not catalyze anaerobilin reduction in the presence of excess NAD(P)H, FAD, and FMN. However, we show that HutZ prevents labile heme and anaerobilin cytotoxicity by binding tightly to heme, sequestering it from HutW, and preventing anaerobilin formation. Thus, A. fischeri is seemingly unable to extract iron from heme using the hutWXZ gene products. Our results further suggest that the structurally distinct chu, hmu, and hut operons have functionally converged to protect the cell from anaerobilin accumulation and heme cytotoxicity.

Salivabactin is a newly identified polyketide/nonribosomal peptide (PK/NRP) from a human oral probiotic, possessing a unique chemical structure and outstanding antibiotic activities. Although the biosynthetic gene cluster for salivabactin is known, the enzymatic logic that governs the synthesis of salivabactin has not yet been fully studied. In this work, we dissected the biosynthetic pathway for salivabactin using biochemical analysis. We successfully reconstituted the enzymatic synthesis of salivabactin in vitro, identified the minimal set of enzymes required for its biosynthesis, and revealed an unusual thioesterase domain involved in terminal olefin formation.

Deoxyribonucleic acid (DNA) nanomachines have been widely exploited in enzyme activity regulation, protein crystallization, protein assembly, and control of the protein–protein interaction (PPI). Yet, the fundamental biophysical framework of DNA nanomachines in the case of regulating protein–protein interactions remains elusive. Here, we established a DNA nanospring-mCherry model with mCherry homodimers of different K_d. Using size exclusion chromatography and fluorescence polarization, we profiled the DNA nanospring-mediated manipulation of PPI as an entropy-reducing process. The energy transfer efficiency was a function of the length of the complementary sequence and the geometry of the DNA nanospring construction. With basic force analysis and physical chemistry calculation, we proposed a unified model of the correlation between the dissociation constant, local concentration, construction of DNA nanospring, and kinetics of protein dimerization. Overall, we demonstrated that the DNA nanospring-mCherry conjugate was a simple and practical model to analyze DNA-controlled protein–protein interaction.

Radical S-adenosyl-l-methionine (SAM) enzymes catalyze radical mediated chemical transformations notable for their diversity. The radical mediated reactions that take place in their catalytic cycles can be characterized with respect to one or more phases of initiation, propagation, and termination. Mechanistic models abound regarding these three phases of catalysis being regularly informed and updated by new discoveries that offer insights into their detailed workings. However, questions continue to be raised that touch on fundamental aspects of their mechanistic enzymology. Radical SAM enzymes are consequently far from fully understood, and this Perspective aims to outline some of the current models of radical SAM chemistry with an emphasis on lines of investigation that remain to be explored.

Organophosphate flame retardants have been widely used in plastic products since the early 2000s. Unfortunately, these compounds leach out of the plastics over time and are carcinogenic, developmental toxins, and endocrine disruptors. Due to the high usage levels and stable nature of the compounds, widespread contamination of the environment has now been observed. Despite their recent introduction into the environment, bacteria from the Sphingomonadaceae family have evolved a three-step hydrolytic pathway to utilize these compounds. The second step in this pathway in Sphingobium sp. TCM1 is catalyzed by Sb-PDE, which is a member of the polymerase and histidinol phosphatase (PHP) family of phosphatases. This enzyme is only the second case of a PHP-family enzyme capable of hydrolyzing phosphodiesters. Bioinformatics analysis has now been used to identify a second PHP diesterase from Novosphingobium sp. EMRT-2 (No-PDE). Kinetic characterization of Sb-PDE and No-PDE with authentic organophosphate flame-retardant diesters demonstrates that these enzymes are true diesterases with more than 1000-fold selectivity for the diesterase activity seen in some cases. Synthesis of a wide array of authentic flame-retardant diesters has allowed the substrate specificity of these enzymes to be determined, and mutagenic analysis of the active site residues has identified key residues that give rise to the high levels of diesterase activity. Despite high sequence identity, No-PDE is found to have a broader substrate specificity against flame-retardant derived diesters, and k_cat/K_m values greater than 10⁴ M^–1 s^–1 are seen with the best substrates.

Dihydroorotate dehydrogenases (DHODs) catalyze the transfer of electrons between dihydroorotate and specific oxidant substrates. Class 1B DHODs (DHODBs) use NAD⁺ as the oxidant substrate and have a heterodimeric structure that incorporates two active sites, each with a flavin cofactor. One Fe₂S₂ center lies roughly equidistant between the flavin isoalloxazine rings. This arrangement allows for simultaneous association of reductant and oxidant substrates. Here we describe a series of experiments designed to reveal sequences and contingencies in DHODB chemistry. From these data it was concluded that the resting state of the enzyme is FAD•Fe₂S₂•FMN. Reduction by either NADH or DHO results in two electrons residing on the FMN cofactor that has a 47 mV higher reduction potential than the FAD. The FAD•Fe₂S₂•FMNH₂ state accumulates with a bisemiquinone state that is an equilibrium accumulation formed from a partial transfer of one electron to the FAD. Pyrimidine reduction is reliant on the availability of the Cys135 proton, as the C135S variant slows orotate reduction by ∼40-fold. The rate of pyrimidine reduction is modulated by occupancy of the FAD site; NADH•FAD•Fe₂S₂•FMNH₂•orotate complex can reduce the pyrimidine at 16 s^–1, while NAD⁺•FAD•Fe₂S₂•FMNH₂•orotate complex reduces the pyrimidine at 5.4 s^–1 and the FAD•Fe₂S₂•FMNH₂•orotate complex at 0.6 s^–1. This set of effector states account for the apparent discrepancy in the slowest rate observed in transient state single turnover reactions with limiting NADH and the limiting rate observed in steady state.

Targeting G-quadruplexes, which have distinctive structures, to regulate biological reactions in cells has attracted interest due to the many disease-related genes that possess G-quadruplex-forming sequences. To achieve regulation of gene expression using G-quadruplexes, their folding kinetics and time scales should be well understood. However, the G-quadruplex folding kinetics is highly dependent on its nucleotide sequence as well as its surrounding environment, and thus a general folding mechanism is difficult to propose. Moreover, the effects of G-quadruplex folding kinetics on biological functions such as transcription inhibition are not represented yet. Here, we investigated the folding kinetics and mechanism of G-quadruplexes by focusing on the loop region. Kinetic analyses showed that the hairpin structure in the second loop region significantly accelerated G4 folding, suggesting that it served as a nucleation site for the subsequent folding process. The hairpin in the second loop adopted an intermediate state, an antiparallel G4 structure, in the folding process. Moreover, T7 polymerase assay demonstrated that faster G4 folding resulted in more efficient transcription inhibition. These findings demonstrate the importance of hairpin in the G4 folding kinetics and mechanism and a new strategy for developing G4-targeting small molecules.

G protein-coupled receptors (GPCRs) play a pivotal role in regulating numerous physiological processes through their interactions with two key effectors: G proteins and β-arrestins (βarrs). This makes them crucial targets for therapeutic drug development. Interestingly, the evolving concept of biased signaling where ligands selectively activate either the G proteins or the βarrs has not only refined our understanding of segregation of physiological responses downstream of GPCRs but has also revolutionized drug discovery, offering the potential for treatments with enhanced efficacy and minimal side effects. This Review explores the mechanisms behind biased agonism, exploring it through various lenses, including ligand, receptor, cellular systems, location, and tissue-specific biases. It also offers structural insights into both orthosteric and allosteric ligand-binding pockets, structural rearrangements associated with the loops, and how ligand-engineering can contribute to biased signaling. Moreover, we also discuss the unique conformational signature in an intrinsically biased GPCR, which currently remains relatively less explored and adds a new dimension in biased signaling. Lastly, we address the translational challenges and practical considerations in characterizing bias, emphasizing its therapeutic potential and the latest advancements in drug development. By designing ligands that target specific signaling pathways, biased signaling presents a transformative approach to creating safer and more effective therapies. This Review focuses on our current understanding of GPCR-biased signaling, discussing potential mechanisms that lead to bias, the effect of bias on GPCR structures at a molecular level, recent advancements, and its profound potential to drive innovation in drug discovery.

Human iodotyrosine deiodinase (hIYD) catalyzes the reductive deiodination of iodotyrosine using a flavin mononucleotide cofactor to maintain the iodine concentration in the body. Mutations in the hIYD gene are linked to human hypothyroidism, emphasizing its role in thyroid function regulation. The present work employs microsecond-scale molecular dynamics simulations and quantum chemical calculations to elucidate the conformational dynamics and reactivity in the active site at various stages of hIYD enzymatic cycle. The flavin is found to employ a unique butterfly motion of its isoalloxazine ring accompanied by a novel active-and-resting state of its ribose 2′-OH group at different stages of the enzymatic cycle. The flavin dynamics are found to control substrate binding affinity, the active site lid closure, and NADPH recognition. The predicted hIYD model shows enhanced stabilization of NADPH due to additional interactions with the N-terminal and intermediate domains. The enzyme uses a group of basic residues (R100, R101, R104, K182, and R279) to stabilize flavin in different stages of catalysis, suggesting potential mutations to control enzyme activity. The reactivity descriptors and stereoelectronic analysis predict the N5 nitrogen of flavin as a proton source during the reductive deiodination, while the anisotropic charge distribution on the halogen atom has negligible structural and electronic effects. The present findings provide key insights into the molecular basis of hIYD activity and lay the groundwork for future research aimed at therapeutic interventions and industrial applications.

Engrailed homeodomain (EngHD), a highly charged transcription factor regulating over 200 genes, is a fast-folding protein. Recent studies have shown that the abundant charged residues in EngHD not only facilitate protein–DNA interactions but also influence the conformational disorder of its native structure. However, the mechanisms by which electrostatic interactions modulate the folding of EngHD remain unclear. Here, we employ a coarse-grained structure-based model that incorporates the salt-dependent Debye–Hückel model to investigate the (un)folding behavior of EngHD under various salt concentrations. Our findings demonstrate that increasing salt concentrations enhance both folding stability and cooperativity, while the folding barrier height remains relatively constant due to the distinct electrostatic effects on individual residues. By modulating the energetic balance between local and nonlocal interactions, we shift the folding of EngHD from a downhill process to a two-state process. Notably, we observe a nonmonotonic relationship between the strength of local interactions and residue-level coupling degree during (un)folding, likely attributed to the repulsive electrostatic interactions present in the native structure of EngHD. Additionally, we identify a critical turning point in the dependence of folding cooperativity on salt concentration, classified by the energetic balance of local and nonlocal interactions. Our results provide valuable insights into how electrostatic interactions influence the folding of EngHD, contributing to the theoretical framework for engineering highly charged proteins.

Understanding the factors that contribute to specificity of protein–protein interactions allows for design of orthosteric small molecules. Within this environment, a small molecule requires both structural and electrostatic complementarity. While the structural contribution to protein–drug–protein specificity is well characterized, electrostatic contributions require more study. To this end, we used a series of protein complexes involving Arf1 bound to guanine nucleotide exchange factors (GEFs) that are sensitive or resistant to the small molecule brefeldin A (BFA). By comparing BFA-sensitive Arf1-Gea1p and Arf1-ARNO with different combinations of four BFA sensitizing ARNO mutations (ARNOwt, ARNO1M, ARNO3M, and ARNO4M), we describe how electrostatic environments at each interface guide BFA binding specificity. We labeled Arf1 with cyanocysteine at several interfacial sites and measured by nitrile adsorption frequencies to map changes in electric field at each interface using the linear Stark equation. Temperature dependence of nitrile vibrational spectra was used to investigate differences in hydrogen bonding environments. These comparisons showed that interfacial electric field at the surface of Arf1 varied substantially depending on the GEF. The greatest differences were seen between Arf1-ARNOwt and Arf1-ARNO4M, suggesting a greater change in electric field is required for BFA binding to Arf1-ARNO. Additionally, rigidity of the interface of the Arf1-ARNO complex correlated strongly with BFA sensitivity, indicating that flexible interfaces are sensitive to disruption upon orthosteric small molecule binding. These findings demonstrate a qualitatively consistent electrostatic environment for Arf1 binding and more subtle differences preventing BFA specificity. We discuss how these results will guide improved design of other small molecules that can target protein–protein interfaces.

The study focuses on the interaction between canonical uracil and its rare tautomers with Mg²⁺ and MgCl₂ in the microcosmic water environment and aims to elucidate how ions interact with nucleobase and the cation–anion correlation effect involved using density functional theory calculations. The structures of the Ura–Mg²⁺(H₂O)_0–6 and Ura–MgCl₂(H₂O)_0–6 clusters are characterized and show that the water molecules preferentially interact with Mg²⁺/MgCl₂, and Mg²⁺ adopts a hexacoordination pattern in both Ura–Mg²⁺(H₂O)_0–6 and Ura–MgCl₂(H₂O)_0–6 clusters. When uracil interacts with Mg²⁺ in (H₂O)_0–6 environments, it tends to favor the formation of keto–enol structures. However, in the presence of Cl^– cooperating with Mg²⁺, the Ura–MgCl₂(H₂O)_0–6 complexes prefer to form diketo structures. The proton transfer mechanism shows that the initial solvation can promote the change from the keto–enol structure to the diketo structure, which is strengthened by the analysis of the Ura–Mg²⁺(H₂O)₆ and Ura–MgCl₂(H₂O)₆ structures in the aqueous phase using the PCM model. Additionally, reduced density gradient, atom in molecules, and energy decomposition analysis combined with charge transfer analysis were carried out to obtain the variation law of the interactions between Mg²⁺ and Ura with the water number increasing, thereby revealing the interaction mechanism of uracil with magnesium ion and the effect of Cl^– on the interaction between Mg²⁺ and uracil.

Proteases are a privileged class of enzymes due to their catalysis of an irreversible post translational modification, namely cleavage of substrate proteins. Protease activity is essential for human pathways including inflammation, blood clotting, and apoptosis. Proteases are also essential for the propagation of many viruses due to their role in cleavage of the viral polyprotein. For these reasons, proteases are an attractive and highly exploited class of drug targets. To fully harness the power of proteases as drug targets, it is essential that their presence and function are detectable throughout the course of the protease lifetime, from inactive zymogen to the fully cleaved (mature) protease. A number of methods for detection of proteases have been developed, however, many rely on catalytic activity, so are not useful throughout the proteolytic life cycle. Here, we build on our observation that the MH1 family of benzofuran-aminothiazolopyridine inhibitors of Zika virus protease (ZVP) undergo a unique FRET interaction with tryptophan residues in the protease. The full FRET signal is only observed in higher potency binding interactions. Moreover, this approach can distinguish two inactive variants of ZVP based on their folded or unfolded state. These studies also probe the physicochemical basis of the FRET signal. Exploiting these types of FRET interactions may offer an orthogonal approach for detection of this protease, which takes advantage of the relationship between the novel ligand and the core of the protein and is therefore useful throughout the protease maturation cycle. Depending on chemical properties, this approach may be applicable in other proteases and other protein classes.

Nicotinamide-containing cofactors play an essential role in many enzymes that catalyze two-electron redox reactions. However, it is difficult to engineer nicotinamide binding sites into proteins due to the extended nature of the cofactor–protein interface and the precise orientation of the nicotinamide moiety required for efficient electron transfer to or from the substrate. To address these challenges, we genetically encoded a noncanonical amino acid (ncAA) bearing a nicotinamide side chain in bacteria. This redox-active amino acid, termed Nic1, exhibits similar electrochemical properties to the natural cofactor nicotinamide adenine dinucleotide (NAD⁺). Nic1 can be reversibly reduced and oxidized using chemical reagents both free in solution and when incorporated into a model protein. This genetically encodable cofactor can be introduced into proteins in a site-specific fashion and may serve as a tool to study electron-transfer mechanisms in enzymes and to engineer redox-active proteins.

The RhiE and RhiF proteins work together as RhiEF and function as a thiamine pyrophosphate (TPP)-dependent phosphonopyruvate decarboxylase to produce phosphonoacetaldehyde in the rhizocticin biosynthesis pathway. In this study, we determined the crystal structure of the RhiEF complexed with TPP and Mg²⁺. RhiEF forms a dimer of heterodimers, and the cofactor TPP is bound at the heterotetrameric subunit interface. Structural analysis of RhiEF revealed that the RhiE and RhiF moieties correspond to the pyrimidine-binding (PYR) and pyrophosphate-binding (PP) domains commonly found in TPP-dependent enzymes, respectively, as predicted by amino acid sequence alignment analysis. In contrast to other TPP-dependent enzymes with known structures, RhiEF has no domains other than the PYR and PP domains. Furthermore, structure-based evolutionary and sequence-based phylogenetic analyses have suggested that heteromultimeric enzymes such as RhiEF are ancestral types. These results indicate that RhiEF is one of the smallest and most ancient TPP-dependent decarboxylases. Based on the structural comparisons of RhiEF with other TPP-dependent decarboxylases, we identified the amino acid residues responsible for the catalytic mechanism of TPP-dependent decarboxylation in RhiEF.

An attractive strategy for combating antibacterial resistance involves the development of new antibiotics whose mechanisms differ from those of existing ones in the clinic. Elfamycin antibiotics, whose prototypes include kirromycin and aurodox, are illustrative examples based on their ability to target EF-Tu, an essential component for protein translation in bacteria. Our efforts to revisit this antibiotic class were enabled by two developments. First, we produced L-681,217, an understudied member of this polyketide family harboring a terminal carboxylic acid in place of a hydroxypyridone ring, and synthesized a biotinylated derivative with comparable activity to the natural product. Second, we established a sensitive cell-free protein synthesis (CFPS) assay in which superfolder green fluorescent protein (sfGFP) production was inhibited by L-681,217. Biotinyl-L-681,217 was used to drain the CFPS system of endogenous EF-Tu, allowing replenishment with orthologs to interrogate pathogen selectivity and propensity toward resistance. Comparative in vitro analysis of kirromycin and L-681,217 showed that, while both antibiotics are equipotent in CFPS assays, they interact distinctly with purified EF-Tu, a feature that presumably correlates with prior observations that kirromycin enhances GTP hydrolysis by EF-Tu whereas L-681,217 does not. Analysis of L-681,217 and kirromycin accumulation in selected mutant E. coli strains also revealed that antibiotic import and efflux contributed to resistance. The promise of L-681,217 as a medicinal lead was underscored by the observation that, unlike aurodox, this polyketide does not inhibit adenylosuccinate synthase.

Nucleic acid mimics (NAMs) have demonstrated high potential as antibacterial drugs. However, very few studies have assessed their possible diffusion across the bacterial envelope. In this work, we studied NAMs’ diffusion in lipid bilayer systems that mimic the bacterial outer membrane using molecular dynamics (MD) simulations. Additionally, we examined the interactions of a NAM sequence with lipid membranes and ascertained the partition constants (K_p) through MD and spectroscopic investigations. The NAM sequences were composed of locked nucleic acid (LNA) and 2′-O-methyl (2′-OMe) residues, whereas the membrane models were composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) phospholipids. The parametrization protocol followed was validated against literature data and demonstrated the reliability of our approach for simulating NAM sequences. Investigation into the interaction of the sequences with zwitterionic and anionic membranes revealed a preference for hydrogen bond formation with the anionic model over the zwitterionic one. Additionally, potential of mean force (PMF) calculations unveiled a lower free energy barrier for translocation across the zwitterionic bilayer model. Contrarily, the partition constants derived suggested a slightly higher partitioning within the anionic membrane, emphasizing a nuanced interplay of factors. Finally, spectroscopic partition measurements with liposomes presented challenges in quantifying the partition of NAMs due to minimal signal variations. However, a tendency for quenching in anionic vesicles suggested a potential, albeit small, partitioning effect that warrants further investigation. In summary, our study revealed that NAMs will not, in principle, be able to cross an intact bacterial outer membrane by passive diffusion.

The main protease (M^pro) of SARS-CoV-2 is essential for viral replication and is, therefore, an important drug target. Here, we investigate two flexible loops in M^pro that play a role in catalysis. Using all-atom molecular dynamics simulations, we analyze the structural ensemble of M^pro in an apo state and substrate-bound state. We find that the flexible loops can adopt open, intermediate (partly open), and closed conformations in solution, which differs from the partially closed state observed in crystal structures of M^pro. When the loops are in closed or intermediate states, the catalytic residues are more likely to be in close proximity, which is crucial for catalysis. Additionally, we find that substrate binding to one protomer of the homodimer increases the frequency of intermediate states in the bound protomer while also affecting the structural propensity of the apo protomer’s flexible loops. Using dynamic network analysis, we identify multiple allosteric pathways connecting the two active sites of the homodimer. Common to these pathways is an allosteric hotspot involving the N-terminus, a critical region that comprises part of the binding pocket. Taken together, the results of our simulation study provide detailed insight into the relationships between the flexible loops and substrate binding in a prime drug target for COVID-19.

The crowded milieu of a biological cell significantly impacts protein aggregation and interactions. Understanding the effects of macromolecular crowding on the aggregation and fibrillation of amyloidogenic proteins is crucial for the treatment of many amyloid-related disorders. Most in vitro studies of protein amyloid formation and its inhibition by small molecules are conducted in dilute buffers, which do not mimic the complexity of the cellular environment. In this study, we used PEGs to simulate macromolecular crowding and examined the inhibitory effects of flavones DHF, baicalein, and luteolin on human lysozyme (HuL) aggregation at pH 2. Naturally occurring flavones have been effective inhibitors of amyloid formation in some proteins. Our findings indicate that while flavones inhibit HuL aggregation and fibrillation in dilute buffer solutions, complete inhibition is observed with a combination of flavones and PEGs, as shown by ThT fluorescence, light scattering, TEM, and AFM studies. The species formed in the presence of PEG 8000 and flavones were less hydrophobic, less toxic, and α-helix-rich compared to control samples, which were hydrophobic and β-sheet-rich, as demonstrated by ANS hydrophobicity, MTT assay, and CD spectroscopy. Fluorescence titration studies of flavones with HuL showed a significant increase in binding constant values under crowding conditions. These findings highlight the importance of macromolecular crowding in modulating protein aggregation and amyloid inhibition. Further studies using disease-causing mutants of HuL and other amyloidogenic proteins are needed to explore the role of macromolecular crowding in small-molecule-mediated modulation and inhibition of protein aggregation and amyloid formation.