Pushed to extremes: distinct effects of high temperature versus pressure on the structure of STEP.

Protein function hinges on small shifts of three-dimensional structure. Elevating temperature or pressure may provide experimentally accessible insights into such shifts, but the effects of these distinct perturbations on protein structures have not been compared in atomic detail. To quantitatively explore these two axes, we report the first pair of structures at physiological temperature versus. high pressure for the same protein, STEP (PTPN5). We show that these perturbations have distinct and surprising effects on protein volume, patterns of ordered solvent, and local backbone and side-chain conformations. This includes interactions between key catalytic loops only at physiological temperature, and a distinct conformational ensemble for another active-site loop only at high pressure. Strikingly, in torsional space, physiological temperature shifts STEP toward previously reported active-like states, while high pressure shifts it toward a previously uncharted region. Altogether, our work indicates that temperature and pressure are complementary, powerful, fundamental macromolecular perturbations.

Uncovering Protein Ensembles: Automated Multiconformer Model Building for X-ray Crystallography and Cryo-EM.

In their folded state, biomolecules exchange between multiple conformational states that are crucial for their function. Traditional structural biology methods, such as X-ray crystallography and cryogenic electron microscopy (cryo-EM), produce density maps that are ensemble averages, reflecting molecules in various conformations. Yet, most models derived from these maps explicitly represent only a single conformation, overlooking the complexity of biomolecular structures. To accurately reflect the diversity of biomolecular forms, there is a pressing need to shift towards modeling structural ensembles that mirror the experimental data. However, the challenge of distinguishing signal from noise complicates manual efforts to create these models. In response, we introduce the latest enhancements to qFit, an automated computational strategy designed to incorporate protein conformational heterogeneity into models built into density maps. These algorithmic improvements in qFit are substantiated by superior Rfree and geometry metrics across a wide range of proteins. Importantly, unlike more complex multicopy ensemble models, the multiconformer models produced by qFit can be manually modified in most major model building software (e.g. Coot) and fit can be further improved by refinement using standard pipelines (e.g. Phenix, Refmac, Buster). By reducing the barrier of creating multiconformer models, qFit can foster the development of new hypotheses about the relationship between macromolecular conformational dynamics and function.

Pushed to extremes: distinct effects of high temperature vs. pressure on the structure of an atypical phosphatase.

Protein function hinges on small shifts of three-dimensional structure. Elevating temperature or pressure may provide experimentally accessible insights into such shifts, but the effects of these distinct perturbations on protein structures have not been compared in atomic detail. To quantitatively explore these two axes, we report the first pair of structures at physiological temperature vs. high pressure for the same protein, STEP (PTPN5). We show that these perturbations have distinct and surprising effects on protein volume, patterns of ordered solvent, and local backbone and side-chain conformations. This includes novel interactions between key catalytic loops only at physiological temperature, and a distinct conformational ensemble for another active-site loop only at high pressure. Strikingly, in torsional space, physiological temperature shifts STEP toward previously reported active-like states, while high pressure shifts it toward a previously uncharted region. Together, our work argues that temperature and pressure are complementary, powerful, fundamental macromolecular perturbations.

The temperature-dependent conformational ensemble of SARS-CoV-2 main protease (Mpro).

The COVID-19 pandemic, instigated by the SARS-CoV-2 coronavirus, continues to plague the globe. The SARS-CoV-2 main protease, or Mpro, is a promising target for the development of novel antiviral therapeutics. Previous X-ray crystal structures of Mpro were obtained at cryogenic tem-per-ature or room tem-per-ature only. Here we report a series of high-resolution crystal structures of unliganded Mpro across multiple tem-per-atures from cryogenic to physiological, and another at high humidity. We inter-rogate these data sets with parsimonious multiconformer models, multi-copy ensemble models, and isomorphous difference density maps. Our analysis reveals a perturbation-dependent conformational landscape for Mpro, including a mobile zinc ion inter-leaved between the catalytic dyad, mercurial conformational heterogeneity at various sites including a key substrate-binding loop, and a far-reaching intra-molecular network bridging the active site and dimer inter-face. Our results may inspire new strategies for antiviral drug development to aid preparation for future coronavirus pandemics.

A metal ion-dependent conformational switch modulates activity of the Plasmodium M17 aminopeptidase.

The metal-dependent M17 aminopeptidases are conserved throughout all kingdoms of life. This large enzyme family is characterized by a conserved binuclear metal center and a distinctive homohexameric arrangement. Recently, we showed that hexamer formation in Plasmodium M17 aminopeptidases was controlled by the metal ion environment, although the functional necessity for hexamer formation is still unclear. To further understand the mechanistic role of the hexameric assembly, here we undertook an investigation of the structure and dynamics of the M17 aminopeptidase from Plasmodium falciparum, PfA-M17. We describe a novel structure of PfA-M17, which shows that the active sites of each trimer are linked by a dynamic loop, and loop movement is coupled with a drastic rearrangement of the binuclear metal center and substrate-binding pocket, rendering the protein inactive. Molecular dynamics simulations and biochemical analyses of PfA-M17 variants demonstrated that this rearrangement is inherent to PfA-M17, and that the transition between the active and inactive states is metal dependent and part of a dynamic regulatory mechanism. Key to the mechanism is a remodeling of the binuclear metal center, which occurs in response to a signal from the neighboring active site and serves to moderate the rate of proteolysis under different environmental conditions. In conclusion, this work identifies a precise mechanism by which oligomerization contributes to PfA-M17 function. Furthermore, it describes a novel role for metal cofactors in the regulation of enzymes, with implications for the wide range of metalloenzymes that operate via a two-metal ion catalytic center, including DNA processing enzymes and metalloproteases.

The temperature-dependent conformational ensemble of SARS-CoV-2 main protease (Mpro).

The COVID-19 pandemic, instigated by the SARS-CoV-2 coronavirus, continues to plague the globe. The SARS-CoV-2 main protease, or Mpro, is a promising target for development of novel antiviral therapeutics. Previous X-ray crystal structures of Mpro were obtained at cryogenic temperature or room temperature only. Here we report a series of high-resolution crystal structures of unliganded Mpro across multiple temperatures from cryogenic to physiological, and another at high humidity. We interrogate these datasets with parsimonious multiconformer models, multi-copy ensemble models, and isomorphous difference density maps. Our analysis reveals a temperature-dependent conformational landscape for Mpro, including mobile solvent interleaved between the catalytic dyad, mercurial conformational heterogeneity in a key substrate-binding loop, and a far-reaching intramolecular network bridging the active site and dimer interface. Our results may inspire new strategies for antiviral drug development to counter-punch COVID-19 and combat future coronavirus pandemics.

High avidity drives the interaction between the streptococcal C1 phage endolysin, PlyC, with the cell surface carbohydrates of Group A Streptococcus.

Endolysin enzymes from bacteriophage cause bacterial lysis by degrading the peptidoglycan cell wall. The streptococcal C1 phage endolysin PlyC, is the most potent endolysin described to date and can rapidly lyse group A, C, and E streptococci. PlyC is known to bind the Group A streptococcal cell wall, but the specific molecular target or the binding site within PlyC remain uncharacterized. Here we report for the first time, that the polyrhamnose backbone of the Group A streptococcal cell wall is the binding target of PlyC. We have also characterized the putative rhamnose binding groove of PlyC and found four key residues that were critical to either the folding or the cell wall binding action of PlyC. Based on our results, we suggest that the interaction between PlyC and the cell wall may not be a high-affinity interaction as previously proposed, but rather a high avidity one, allowing for PlyC's remarkable lytic activity. Resistance to our current antibiotics is reaching crisis levels and there is an urgent need to develop the antibacterial agents with new modes of action. A detailed understanding of this potent endolysin may facilitate future developments of PlyC as a tool against the rise of antibiotic resistance.

Mutational and biophysical robustness in a prestabilized monobody.

The fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold, which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyperstable monobody derivative with diagnostic and therapeutic potential. Prestabilization of the scaffold mitigates the stability-function trade-off commonly associated with evolving a protein domain toward biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerization. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the prestabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 °C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a prestabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics and that FN3Con is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics.

qFit 3: Protein and ligand multiconformer modeling for X-ray crystallographic and single-particle cryo-EM density maps.

New X-ray crystallography and cryo-electron microscopy (cryo-EM) approaches yield vast amounts of structural data from dynamic proteins and their complexes. Modeling the full conformational ensemble can provide important biological insights, but identifying and modeling an internally consistent set of alternate conformations remains a formidable challenge. qFit efficiently automates this process by generating a parsimonious multiconformer model. We refactored qFit from a distributed application into software that runs efficiently on a small server, desktop, or laptop. We describe the new qFit 3 software and provide some examples. qFit 3 is open-source under the MIT license, and is available at https://github.com/ExcitedStates/qfit-3.0.

Strategies for Increasing Protein Stability.

The stability of wild-type proteins is often a hurdle to their practical use in research, industry, and medicine. The route to engineering stability of a protein of interest lies largely with the available data. Where high-resolution structural data is available, rational design, based on fundamental principles of protein chemistry, can improve protein stability. Recent advances in computational biology and the use of nonnatural amino acids have also provided novel rational methods for improving protein stability. Likewise, the explosion of sequence and structural data available in public databases, in combination with improvements in freely available computational tools, has produced accessible phylogenetic approaches. Trawling modern sequence databases can identify the thermostable homologs of a target protein, and evolutionary data can be quickly generated using available phylogenetic tools. Grafting features from those thermostable homologs or ancestors provides stability improvement through a semi-rational approach. Further, molecular techniques such as directed evolution have shown great promise in delivering designer proteins. These strategies are well documented and newly accessible to the molecular biologist, allowing for rapid enhancements of protein stability.

Crystal structure of the inhibitor-free form of the serine protease kallikrein-4.

Kallikrein 4 (KLK4) is a serine protease that is predominantly expressed in the prostate and is overexpressed in prostate cancer. As such, it has gained attention as an attractive target for prostate cancer therapeutics. Currently, only liganded structures of KLK4 exist in the Protein Data Bank. Until now, inferences about the subtle structural changes in KLK4 upon ligand binding have been made by comparison to other liganded forms, rather than to an apo form. In this study, an inhibitor-free form of KLK4 was crystallized. The crystals obtained belonged to space group P1, contained four molecules in the asymmetric unit and diffracted to 1.64 Šresolution. Interestingly, a nonstandard rotamer of the specificity-determining residue Asp189 was observed in all chains. This model will provide a useful unliganded structure for the future structure-guided design of KLK4 inhibitors.

The Role of Conformational Dynamics in Abacavir-Induced Hypersensitivity Syndrome.

Abacavir is an antiretroviral drug used to reduce human immunodeficiency virus (HIV) replication and decrease the risk of developing acquired immune deficiency syndrome (AIDS). However, its therapeutic value is diminished by the fact that it is associated with drug hypersensitivity reactions in up to 8% of treated patients. This hypersensitivity is strongly associated with patients carrying human leukocyte antigen (HLA)-B*57:01, but not patients carrying closely related alleles. Abacavir's specificity to HLA-B*57:01 is attributed to its binding site within the peptide-binding cleft and subsequent influence of the repertoire of peptides that can bind HLA-B*57:01. To further our understanding of abacavir-induced hypersensitivity we used molecular dynamics (MD) to analyze the dynamics of three different peptides bound to HLA-B*57:01 in the presence and absence of abacavir or abacavir analogues. We found that abacavir and associated peptides bind to HLA-B*57:01 in a highly diverse range of conformations that are not apparent from static crystallographic snapshots, but observed no difference in either the conformations, nor degree of flexibility when compared to abacavir-unbound systems. Our results support hypersensitivity models in which abacavir-binding alters the conformational ensemble of neopeptides, so as to favour exposed peptide surfaces that are no longer recognized as self by circulating CD8+ T cells, and are conducive to TCR binding. Our findings highlight the need to also consider the role of dynamics in understanding drug-induced hypersensitivities at the molecular and mechanistic level. This additional insight can help inform the chemical modification of abacavir to prevent hypersensitivity reactions in HLA-B*57:01+ HIV patients whilst retaining potent antiretroviral activity.

Wavelength-Dependent Fluorescent Immunosensors via Incorporation of Polarity Indicators near the Binding Interface of Antibody Fragments.

Herein, we describe a fluorescent immunosensor designed by incorporating an unnatural amino acid fluorophore into the binding site of an EGFR-specific antibody fragment, resulting in quantifiable EGFR-dependent changes in peak fluorescence emission wavelength. To date, immunosensor design strategies have relied on binding-induced changes in fluorescence intensity that are prone to excitation source fluctuations and sample-dependent noise. In this study, we used a rational design approach to incorporate a polarity indicator (Anap) into specific positions of an anti-EGFR single chain antibody to generate an emission wavelength-dependent immunosensor. We found that when incorporated within the topological neighborhood of the antigen binding interface, the Anap emission wavelength is blue-shifted by EGFR-binding in a titratable manner, up to 20 nm, with nanomolar detection limits. This approach could be applicable to other antibody/antigen combinations for integration into a wide range of assay platforms (including homogeneous, solid-phase assay, or microfluidic assays) for one-step protein quantification.

KLK4 Inhibition by Cyclic and Acyclic Peptides: Structural and Dynamical Insights into Standard-Mechanism Protease Inhibitors.

Sunflower trypsin inhibitor (SFTI-1) is a 14 amino acid serine protease inhibitor. The dual antiparallel ß-sheet arrangement of SFTI-1 is stabilized by an N-terminal-C-terminal backbone cyclization and a further disulfide bridge to form a final bicyclic structure. This constrained structure is further rigidified by an extensive network of internal hydrogen bonds. Thus, the structure of SFTI-1 in solution resembles the protease-bound structure, reducing the entropic penalty upon protease binding. When cleaved at the scissile bond, it is thought that the rigidifying features of SFTI-1 maintain its structure, allowing the scissile bond to be reformed. The lack of structural plasticity for SFTI-1 is proposed to favor initial protease binding and continued occupancy in the protease active site, resulting in an equilibrium between the cleaved and uncleaved inhibitor in the presence of a protease. We have determined, at 1.15 Å resolution, the X-ray crystal structures of complexes between human kallikrein-related peptidase 4 (KLK4) and SFTI-FCQR(Asn14) and between KLK4 and an acyclic form of the same inhibitor, SFTI-FCQR(Asn14)[1,14], with the latter displaying a cleaved scissile bond. Structural analysis and MD simulations together reveal the roles of the altered contact sequence, intramolecular hydrogen bonding network, and backbone cyclization in altering the state of SFTI's scissile bond ligation at the protease active site. Taken together, the data presented reveal insights into the role of dynamics in the standard-mechanism inhibition and suggest that modifications on the non-contact strand may be a useful, underexplored approach for generating further potent or selective SFTI-based inhibitors against members of the serine protease family.

Reactive centre loop dynamics and serpin specificity.

Serine proteinase inhibitors (serpins), typically fold to a metastable native state and undergo a major conformational change in order to inhibit target proteases. However, conformational lability of the native serpin fold renders them susceptible to misfolding and aggregation, and underlies misfolding diseases such as α1-antitrypsin deficiency. Serpin specificity towards its protease target is dictated by its flexible and solvent exposed reactive centre loop (RCL), which forms the initial interaction with the target protease during inhibition. Previous studies have attempted to alter the specificity by mutating the RCL to that of a target serpin, but the rules governing specificity are not understood well enough yet to enable specificity to be engineered at will. In this paper, we use conserpin, a synthetic, thermostable serpin, as a model protein with which to investigate the determinants of serpin specificity by engineering its RCL. Replacing the RCL sequence with that from α1-antitrypsin fails to restore specificity against trypsin or human neutrophil elastase. Structural determination of the RCL-engineered conserpin and molecular dynamics simulations indicate that, although the RCL sequence may partially dictate specificity, local electrostatics and RCL dynamics may dictate the rate of insertion during protease inhibition, and thus whether it behaves as an inhibitor or a substrate. Engineering serpin specificity is therefore substantially more complex than solely manipulating the RCL sequence, and will require a more thorough understanding of how conformational dynamics achieves the delicate balance between stability, folding and function required by the exquisite serpin mechanism of action.

Potent, multi-target serine protease inhibition achieved by a simplified ß-sheet motif.

Engagement of an extended ß-sheet is a common substrate/inhibitor interaction at the active site of serine proteases and is an important feature of Laskowski mechanism inhibitors that present a substrate-like loop to a target protease. This loop is cleaved but subsequently relegated forming a stable inhibitor/protease complex. Laskowski inhibitors are ubiquitous in nature and are used extensively in serine protease inhibitor design. However, most studies concentrate on introducing new sidechain interactions rather than the direct contributions of the substrate-like ß-sheet to enzyme inhibition. Here we report the crystal structure of an simplified ß-sheet inhibitory motif within the Sunflower Trypsin Inhibitor (SFTI) in complex with trypsin. We show that the intramolecular hydrogen bond network of this SFTI variant (SFTI-TCTR) engages the inhibitor sidechains that would normally interact with a target protease, giving mainchain interactions a more prominent role in complex formation. Despite having reduced sidechain interactions, this SFTI variant is remarkably potent and inhibits a diverse range of serine proteases. Crystal structural analysis and molecular modelling of SFTI-TCTR complexes again indicates an interface dominated by ß-sheet interactions, highlighting the importance of this motif and the adaptability of SFTI as a scaffold for inhibitor design.

Mapping the Pathway and Dynamics of Bestatin Inhibition of the Plasmodium falciparum M1 Aminopeptidase PfA-M1.

The M1 metallo-aminopeptidase from Plasmodium falciparum, PfA-M1, is an attractive drug target for the design of new antimalarials. Bestatin, a broad-spectrum metalloprotease inhibitor, is a moderate inhibitor of PfA-M1, and has been used to provide structure-activity relationships to inform drug design. The crystal structure of PfA-M1 with bestatin bound within its active site has been determined; however, dynamics of the inhibitor and the association or dissociation pathway have yet to be characterized. Here we present an all-atom molecular dynamics study where we have generated a hidden Markov state model from 2.3 µs of molecular dynamics simulation. Our hidden Markov state model identifies five macrostates that clearly show the events involved in bestatin dissociation from the PfA-M1 active site. The results show for the first time that bestatin can escape the substrate specificity pockets of the enzyme, primarily due to weak interactions within the pockets. Our approach identifies relevant conformational sampling of the inhibitor inside the enzyme and the protein dynamics that could be exploited to produce potent and selective inhibitors that can differentiate between similar members of the M1 aminopeptidase superfamily.

Previously Hidden Dynamics at the TCR-Peptide-MHC Interface Revealed.

A structural characterization of the interaction between αß TCRs and cognate peptide-MHC (pMHC) is central to understanding adaptive T cell-mediated immunity. X-ray crystallography, although the source of much structural data, traditionally provides only a static snapshot of the protein. Given the emerging evidence for the important role of conformational dynamics in protein function, we interrogated 309 crystallographic structures of pMHC complexes using ensemble refinement, a technique that can extract dynamic information from the x-ray data. Focusing on a subset of human pMHC class I systems, we found that in many cases, ensemble methods were able to uncover previously hidden evidence of significant conformational plasticity, thereby revealing additional information that can build upon and significantly enhance functional interpretations that are based on a single static structure. Notable examples include the interpretation of differences in the disease association of HLA subtypes, the relationship between peptide prominence and TCR recognition, the role of conformational flexibility in vaccine design, and the discrimination between induced fit and conformational selection models of TCR binding. We show that the currently widespread practice of analyzing pMHC interactions via the study of a single crystallographic structure does not make use of pertinent and easily accessible information from x-ray data concerning alternative protein conformations. This new analysis therefore not only highlights the capacity for ensemble methods to significantly enrich the interpretation of decades of structural data but also provides previously missing information concerning the dynamics of existing characterized TCR-pMHC interactions.

Generation of AMBER force field parameters for zinc centres of M1 and M17 family aminopeptidases.

The M1 and M17 aminopeptidases are metallo-exopeptidases that rely on the presence of divalent cations, usually zinc, in their active site for proteolytic activity. They are from separate protease superfamilies, however, members often have overlapping substrate specificity. Inhibitors of one or both enzymes can be used to modulate hypertension, reduce proliferation of certain types of cancers and control malaria parasites. Current inhibitors act to chelate the zinc ions in the active site, locking the enzymes in an inactive transition state. We were interested in using a computational approach to understand the structure and dynamics of the M1 and M17 aminopeptidases, however, the presence of the essential metal ions in the proteases presents a challenge to classical molecular dynamics (MD) simulation. The zinc amber force field does not contain applicable descriptions of the zinc coordination environment present in either of these two protease families. To provide tools for the study of these two enzymes, we have used the metal centre parameter builder to generate new hybrid bonded/nonbonded force field (FF) parameters to correctly describe the active site architecture for each enzyme. The new parameters were evaluated by fitting the normal mode frequencies of molecular mechanics to the quantum mechanics frequencies and validated by performing short MD simulations. The new FF parameters now enable more accurate and reliable MD simulations for any member of the M1 or M17 aminopeptidase superfamilies.

Direct and indirect mechanisms of KLK4 inhibition revealed by structure and dynamics.

The kallikrein-related peptidase (KLK) family of proteases is involved in many aspects of human health and disease. One member of this family, KLK4, has been implicated in cancer development and metastasis. Understanding mechanisms of inactivation are critical to developing selective KLK4 inhibitors. We have determined the X-ray crystal structures of KLK4 in complex with both sunflower trypsin inhibitor-1 (SFTI-1) and a rationally designed SFTI-1 derivative to atomic (~1 Å) resolution, as well as with bound nickel. These structures offer a structural rationalization for the potency and selectivity of these inhibitors, and together with MD simulation and computational analysis, reveal a dynamic pathway between the metal binding exosite and the active site, providing key details of a previously proposed allosteric mode of inhibition. Collectively, this work provides insight into both direct and indirect mechanisms of inhibition for KLK4 that have broad implications for the enzymology of the serine protease superfamily, and may potentially be exploited for the design of therapeutic inhibitors.