ABSTRACT
Celiac disease (CD) is a chronic and systemic autoimmune disorder that affects preferentially the small intestine of individuals with a genetic predisposition. CD is promoted by the ingestion of gluten, a storage protein contained in the endosperm of the seeds of wheat, barley, rye, and related cereals. Once in the gastrointestinal (GI) tract, gluten is enzymatically digested with the consequent release of immunomodulatory and cytotoxic peptides, i.e., 33mer and p31-43. In the late 1970s a new group of biologically active peptides, called gluten exorphins (GEs), was discovered and characterized. In particular, these short peptides showed a morphine-like activity and high affinity for the δ-opioid receptor (DOR). The relevance of GEs in the pathogenesis of CD is still unknown. Recently, it has been proposed that GEs could contribute to asymptomatic CD, which is characterized by the absence of symptoms that are typical of this disorder. In the present work, GEs cellular and molecular effects were in vitro investigated in SUP-T1 and Caco-2 cells, also comparing viability effects with human normal primary lymphocytes. As a result, GEs treatments increased tumor cell proliferation by cell cycle and Cyclins activation as well as by induction of mitogenic and pro-survival pathways. Finally, a computational model of GEs interaction with DOR is provided. Altogether, the results might suggest a possible role of GEs in CD pathogenesis and on its associated cancer comorbidities.
Subject(s)
Celiac Disease , Glutens , Humans , Glutens/chemistry , Caco-2 Cells , Peptides/metabolism , Intercellular Signaling Peptides and Proteins , Cell ProliferationABSTRACT
G protein-coupled receptors (GPCRs) play a key role in many cellular signaling mechanisms, and must select among multiple coupling possibilities in a ligand-specific manner in order to carry out a myriad of functions in diverse cellular contexts. Much has been learned about the molecular mechanisms of ligand-GPCR complexes from Molecular Dynamics (MD) simulations. However, to explore ligand-specific differences in the response of a GPCR to diverse ligands, as is required to understand ligand bias and functional selectivity, necessitates creating very large amounts of data from the needed large-scale simulations. This becomes a Big Data problem for the high dimensionality analysis of the accumulated trajectories. Here we describe a new machine learning (ML) approach to the problem that is based on transforming the analysis of GPCR function-related, ligand-specific differences encoded in the MD simulation trajectories into a representation recognizable by state-of-the-art deep learning object recognition technology. We illustrate this method by applying it to recognize the pharmacological classification of ligands bound to the 5-HT2A and D2 subtypes of class-A GPCRs from the serotonin and dopamine families. The ML-based approach is shown to perform the classification task with high accuracy, and we identify the molecular determinants of the classifications in the context of GPCR structure and function. This study builds a framework for the efficient computational analysis of MD Big Data collected for the purpose of understanding ligand-specific GPCR activity.
Subject(s)
Drug Discovery , Ligands , Machine Learning , Quantitative Structure-Activity Relationship , Receptors, G-Protein-Coupled , Algorithms , Binding Sites , Drug Design , Drug Discovery/methods , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Receptor, Serotonin, 5-HT2A/chemistry , Receptor, Serotonin, 5-HT2A/metabolism , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolismABSTRACT
Saccharomyces WEE1 (Swe1), the only "true" tyrosine kinase in budding yeast, is an Hsp90 client protein. Here we show that Swe1(Wee1) phosphorylates a conserved tyrosine residue (Y24 in yeast Hsp90 and Y38 in human Hsp90alpha) in the N domain of Hsp90. Phosphorylation is cell-cycle associated and modulates the ability of Hsp90 to chaperone a selected clientele, including v-Src and several other kinases. Nonphosphorylatable mutants have normal ATPase activity, support yeast viability, and productively chaperone the Hsp90 client glucocorticoid receptor. Deletion of SWE1 in yeast increases Hsp90 binding to its inhibitor geldanamycin, and pharmacologic inhibition/silencing of Wee1 sensitizes cancer cells to Hsp90 inhibitor-induced apoptosis. These findings demonstrate that Hsp90 chaperoning of distinct client proteins is differentially regulated by specific posttranslational modification of a unique subcellular pool of the chaperone, and they provide a strategy to increase the cellular potency of Hsp90 inhibitors.
Subject(s)
Cell Cycle Proteins/physiology , HSP90 Heat-Shock Proteins/metabolism , Protein-Tyrosine Kinases/physiology , Saccharomyces cerevisiae Proteins/physiology , Tyrosine/metabolism , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Line, Tumor , Dimerization , HSP90 Heat-Shock Proteins/physiology , Humans , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Nuclear Proteins/physiology , Phosphorylation , Proteasome Endopeptidase Complex/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , RNA Interference , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae Proteins/genetics , UbiquitinationABSTRACT
Although intensively studied, the high-resolution crystal structure of the peptide DFNKF, the core-segment of human calcitonin, has never been described. Here we report how the use of iodination as a strategy to promote crystallisation and facilitate phase determination, allowed us to solve, for the first time, the single-crystal X-ray structure of a DFNKF derivative. Computational studies suggest that both the iodinated and the wild-type peptides populate very similar conformations. Furthermore, the conformer found in the solid-state structure is one of the most populated in solution, making the crystal structure a reliable model for the peptide in solution. The crystal structure of DFNKF(I) confirms the overall features of the amyloid cross-ß spine and highlights how aromatic-aromatic interactions are important structural factors in the self-assembly of this peptide. A detailed analysis of such interactions is reported.
Subject(s)
Calcitonin/chemistry , Phenylalanine/chemistry , Amino Acid Sequence , Calcitonin/metabolism , Crystallography, X-Ray , Humans , Molecular Dynamics Simulation , Protein Structure, SecondaryABSTRACT
Herein we investigate the potential of novel methods of molecular dynamics analysis to provide information on the key factors that underlie the preferential localization and the effects of mutations modulating protein activities. Epidermal growth factor receptor (EGFR) kinases are selected as a test case. The combined analysis of protein energetics and internal dynamics indicates a clear polarization in the native protein, whereby a highly stable and ordered scaffold in one domain, namely the C-lobe, is combined to a flexible and loosely stabilized domain, the N-lobe. The subdivision in two portions with different properties directs the presence of point mutations mainly to the N-lobe. This allows modulating protein flexibility so that the protein can more efficiently sample the conformations necessary for substrate recognition, while leaving the stability of the protein unperturbed. In this context, comparative simulations of EGFR in the wild type sequence and in the presence of the activating oncogenic mutation G719S reveal flexibility changes in several key regions, involving in particular the part of the kinase devoted to the regulation of substrate recognition (regulatory core) and an increase in the number of stabilizing interactions in the N-lobe for the activated mutant. Our approaches represent a promising and simple strategy toward rationalizing the effects of mutations in modulating enzymatic activities.
Subject(s)
ErbB Receptors/chemistry , ErbB Receptors/metabolism , Molecular Dynamics Simulation , Mutation , Allosteric Regulation , Enzyme Activation , Enzyme Stability , ErbB Receptors/genetics , Protein Structure, Tertiary , ThermodynamicsABSTRACT
The recent advances in the in meso crystallization technique for the structural characterization of G-protein coupled receptor (GPCR) proteins have established the usefulness of the lipidic-cubic phases (LCPs) in the field of crystallography of membrane proteins. It is surprising that despite the success of the approach, the molecular mechanisms of the in meso method are still not well understood. Therefore, the approach must rely on extensive screening for a suitable protein construct, for host and additive lipids, and for the appropriate precipitants and temperature. To shed light on the in meso crystallization mechanisms, we used extensive coarse-grained molecular dynamics simulations to study, in molecular detail, LCPs under different conditions (compositions and temperatures relevant to crystallogenesis) and their interactions with different types of GPCR constructs. The results presented show how the modulation of the lattice constant of the LCP (triggered by the addition of precipitant during the in meso assay), or of the host lipid type, can destabilize monomeric proteins in the bilayer of the LCP and thus drive their aggregation into the stacked lamellae, where the residual hydrophobic mismatch between the protein and the membrane can drive the formation of lateral contacts leading to nucleation and crystal growth. Moreover, we demonstrate how particular protein designs (such as transmembrane proteins engineered to contain large polar regions) can promote protein stacking interactions in the third, out-of-plane, dimension. The insights provided by the new aspects of the specific molecular mechanisms responsible for protein-protein interactions inside the cubic phase presented here should be helpful in guiding the rational design of future in meso trials with successful outcomes.
Subject(s)
Crystallization/methods , Lipids/chemistry , Membrane Proteins/chemistry , Molecular Dynamics Simulation , X-Ray DiffractionABSTRACT
Hsp70 belongs to a family of molecular chaperones ubiquitous through organisms that assist client protein folding and prevent aggregation. It works through a tightly ATP-regulated allosteric cycle mechanism, which organizes its two NBD and SBD into alternate open and closed arrangements that facilitate loading and unloading of client proteins. The two cytosolic human isoforms Hsc70 and HspA1 are relevant targets for neurodegenerative diseases and cancer. Illuminating the molecular details of Hsp70 functional dynamics is essential to rationalize differences among the well-characterized bacterial homologue DnaK and the less explored human forms and develop subtype- or species-selective allosteric drugs. We present here a molecular dynamics-based analysis of the conformational dynamics of HspA1. By using an "allosterically impaired" mutant for comparison, we can reconstruct the impact of the ADP-ATP swap on interdomain contacts and dynamic coordination in full-length HspA1, supporting previous predictions that were, however, limited to the NBD. We model the initial onset of the conformational cycle by proposing a sequence of structural steps, which reveal the role of a specific human sequence insertion at the linker, and a modulation of the angle formed by the two NBD lobes during the progression of docking. Our findings pinpoint functionally relevant conformations and set the basis for a selective structure-based drug discovery approach targeting allosteric sites in human Hsp70.
Subject(s)
Adenosine Diphosphate , Adenosine Triphosphate , HSP70 Heat-Shock Proteins , Molecular Dynamics Simulation , Mutation , Humans , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/genetics , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/chemistry , Adenosine Diphosphate/metabolism , Adenosine Diphosphate/chemistry , Protein ConformationABSTRACT
Understanding how local protein modifications, such as binding small-molecule ligands, can trigger and regulate large-scale motions of large protein domains is a major open issue in molecular biology. We address various aspects of this problem by analyzing and comparing atomistic simulations of Hsp90 family representatives for which crystal structures of the full length protein are available: mammalian Grp94, yeast Hsp90 and E.coli HtpG. These chaperones are studied in complex with the natural ligands ATP, ADP and in the Apo state. Common key aspects of their functional dynamics are elucidated with a novel multi-scale comparison of their internal dynamics. Starting from the atomic resolution investigation of internal fluctuations and geometric strain patterns, a novel analysis of domain dynamics is developed. The results reveal that the ligand-dependent structural modulations mostly consist of relative rigid-like movements of a limited number of quasi-rigid domains, shared by the three proteins. Two common primary hinges for such movements are identified. The first hinge, whose functional role has been demonstrated by several experimental approaches, is located at the boundary between the N-terminal and Middle-domains. The second hinge is located at the end of a three-helix bundle in the Middle-domain and unfolds/unpacks going from the ATP- to the ADP-state. This latter site could represent a promising novel druggable allosteric site common to all chaperones.
Subject(s)
HSP90 Heat-Shock Proteins/chemistry , HSP90 Heat-Shock Proteins/ultrastructure , Models, Chemical , Models, Molecular , Computer Simulation , Motion , Protein ConformationABSTRACT
Investigating ligand-regulated allosteric coupling between protein domains is fundamental to understand cell-life regulation. The Hsp70 family of chaperones represents an example of proteins in which ATP binding and hydrolysis at the Nucleotide Binding Domain (NBD) modulate substrate recognition at the Substrate Binding Domain (SBD). Herein, a comparative analysis of an allosteric (Hsp70-DnaK) and a non-allosteric structural homolog (Hsp110-Sse1) of the Hsp70 family is carried out through molecular dynamics simulations, starting from different conformations and ligand-states. Analysis of ligand-dependent modulation of internal fluctuations and local deformation patterns highlights the structural and dynamical changes occurring at residue level upon ATP-ADP exchange, which are connected to the conformational transition between closed and open structures. By identifying the dynamically responsive protein regions and specific cross-domain hydrogen-bonding patterns that differentiate Hsp70 from Hsp110 as a function of the nucleotide, we propose a molecular mechanism for the allosteric signal propagation of the ATP-encoded conformational signal.
Subject(s)
HSP70 Heat-Shock Proteins/metabolism , Molecular Dynamics Simulation , Allosteric Regulation , Amino Acid Sequence , Binding Sites , HSP70 Heat-Shock Proteins/chemistry , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence Homology, Amino AcidABSTRACT
Isoxazoline-carbocyclic monophosphate nucleotides were designed and synthesized through the chemistry of nitrosocarbonyl intermediates and stable anthracenenitrile oxide. Docking and molecular dynamics studies were first conducted for determining the best candidate for polymerase SARS-CoV-2 inhibition. The setup phosphorylation protocol afforded the nucleotides available for the biological tests. Preliminary inhibition and cytotoxicity assays were then performed, and the results showed a moderate activity of the nucleotides accompanied by cytotoxicity.
ABSTRACT
Class A (rhodopsin-like) G protein-coupled receptors (GPCRs) are constitutive phospholipid scramblases as evinced after their reconstitution into liposomes. Yet phospholipid scrambling is not detectable in the resting plasma membrane of mammalian cells that is replete with GPCRs. We considered whether cholesterol, a prominent component of the plasma membrane, limits the ability of GPCRs to scramble lipids. Our previous Markov State Model (MSM) analysis of molecular dynamics simulations of membrane-embedded opsin indicated that phospholipid headgroups traverse a dynamically revealed hydrophilic groove between transmembrane helices (TM) 6 and 7 while their tails remain in the bilayer. Here, we present comparative MSM analyses of 150-µs simulations of opsin in cholesterol-free and cholesterol-rich membranes. Our analyses reveal that cholesterol inhibits phospholipid scrambling by occupying the TM6/7 interface and stabilizing the closed groove conformation while itself undergoing flip-flop. This mechanism may explain the inability of GPCRs to scramble lipids at the plasma membrane.
Subject(s)
Phospholipid Transfer Proteins , Receptors, G-Protein-Coupled , Animals , Biological Transport , Cholesterol , Lipid Bilayers , Mammals/metabolism , Opsins/metabolism , Phospholipid Transfer Proteins/genetics , Phospholipid Transfer Proteins/metabolism , Phospholipids/metabolism , Receptors, G-Protein-Coupled/metabolismABSTRACT
Molecular switching and ligand-based modulation of the 90-kDa heat-shock protein (Hsp90) chaperone activity may ultimately facilitate conformational coupling to the ATPase cycle along with activation and recruitment of the broad range of client proteins. We present an atomic resolution analysis of the Hsp90 N-terminal domain (NTD) binding energy landscape by simulating protein dynamics with a range of binding partners. We show that the activity of the molecular chaperone may be linked to (i) local folding-unfolding transitions and conformational switching of the "active site lid" upon binding and (ii) differences in the underlying protein dynamics as a function of the binding partner. This study suggests that structural plasticity of the Hsp90 NTD can be exploited by the molecular chaperone machinery to modulate enhanced structural rigidity during ATP binding and increased protein flexibility as a consequence of the inhibitor binding. The present study agrees with the experimental structural data and provides a plausible molecular model for understanding mechanisms of modulation of molecular chaperone activities by binding partners.
Subject(s)
HSP90 Heat-Shock Proteins/chemistry , HSP90 Heat-Shock Proteins/metabolism , Computer Simulation , Humans , Ligands , Models, Molecular , Pliability , Protein Structure, Secondary , Protein Structure, Tertiary , Structure-Activity Relationship , ThermodynamicsABSTRACT
An increasing number of functional studies of proteins have shown that sequence and structural similarities alone may not be sufficient for reliable prediction of their interaction properties. This is particularly true for proteins recognizing specific antibodies, where the prediction of antibody-binding sites, called epitopes, has proven challenging. The antibody-binding properties of an antigen depend on its structure and related dynamics. Aiming to predict the antibody-binding regions of a protein, we investigate a new approach based on the integrated analysis of the dynamical and energetic properties of antigens, to identify nonoptimized, low-intensity energetic interaction networks in the protein structure isolated in solution. The method is based on the idea that recognition sites may correspond to localized regions with low-intensity energetic couplings with the rest of the protein, which allows them to undergo conformational changes, to be recognized by a binding partner, and to tolerate mutations with minimal energetic expense. Upon analyzing the results on isolated proteins and benchmarking against antibody complexes, it is found that the method successfully identifies binding sites located on the protein surface that are accessible to putative binding partners. The combination of dynamics and energetics can thus discriminate between epitopes and other substructures based only on physical properties. We discuss implications for vaccine design.
Subject(s)
Epitope Mapping/methods , Proteins/chemistry , Proteins/immunology , Antibodies/immunology , Binding Sites , Chemical Phenomena , Databases, Protein , Epitopes/chemistry , Epitopes/immunology , Humans , Molecular Dynamics Simulation , Protein Conformation , Protein Stability , Proteins/isolation & purification , Reproducibility of Results , Structure-Activity Relationship , ThermodynamicsABSTRACT
Human immunodeficiency virus type 1 protease (HIV-1 PR) is an essential enzyme in the HIV-1 life cycle. As such, this protein represents a major drug target in AIDS therapy, but emerging resistance to antiretroviral inhibitor cocktails, caused by high viral mutation rates, represents a significant challenge in AIDS treatment. Many mutations are not located within the active site or binding pocket, nor they do significantly modify the three-dimensional structural organization of the enzyme; hence, the mechanism(s) by which they alter inhibitor affinity for the protease remains uncertain. In this article, we present an all-atom computational analysis of the dynamic residue-residue coordination between the active site residues and the rest of the protein and of the energetic properties of different HIV-1 PR complexes. We analyze both the wild-type form and mutated forms that induce drug resistance. In particular, the results show differences between the wild type and the mutants in their mechanism of dynamic coordination, in the signal propagation between the active site residues and the rest of the protein, and in the energy networks responsible for the stabilization of the bound inhibitor conformation. Finally, we propose a dynamic and energetic explanation for HIV-1 protease drug resistance, and, through this model, we identify a possible new site that could be helpful in the design of a new family of HIV-1 PR allosteric inhibitors.
Subject(s)
HIV Protease Inhibitors/chemistry , HIV Protease/chemistry , Computational Biology , Drug Resistance, Viral , HIV Infections/drug therapy , HIV Protease/metabolism , HumansABSTRACT
BACKGROUND: Protein-DNA recognition underlies fundamental biological processes ranging from transcription to replication and modification. Herein, we present a computational study of the sequence modulation of internal dynamic properties and of intraprotein networks of aminoacid interactions that determine the stability and specificity of protein-DNA complexes. RESULTS: To this aim, we apply novel theoretical approaches to analyze the dynamics and energetics of biological systems starting from MD trajectories. As model system, we chose different sequences of Zinc Fingers (ZF) of the Zif268 family bound with different sequences of DNA. The complexes differ for their experimental stability properties, but share the same overall 3 D structure and do not undergo structural modifications during the simulations. The results of our analysis suggest that the energy landscape for DNA binding may be populated by dynamically different states, even in the absence of major conformational changes. Energetic couplings between residues change in response to protein and/or DNA sequence variations thus modulating the selectivity of recognition and the relative importance of different regions for binding. CONCLUSIONS: The results show differences in the organization of the intra-protein energy-networks responsible for the stabilization of the protein conformations recognizing and binding DNA. These, in turn, are reflected into different modulation of the ZF's internal dynamics. The results also show a correlation between energetic and dynamic properties of the different proteins and their specificity/selectivity for DNA sequences. Finally, a dynamic and energetic model for the recognition of DNA by Zinc Fingers is proposed.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/chemistry , DNA/metabolism , Early Growth Response Protein 1/chemistry , Early Growth Response Protein 1/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , Crystallography, X-Ray , DNA-Binding Proteins/chemistry , Models, Molecular , Molecular Dynamics Simulation , Nucleic Acid Conformation , Protein Binding , Protein Conformation , Substrate Specificity , Zinc FingersABSTRACT
Hsp90 is a molecular chaperone essential for protein folding and activation in normal homeostasis and stress response. ATP binding and hydrolysis facilitate Hsp90 conformational changes required for client activation. Hsp90 plays an important role in disease states, particularly in cancer, where chaperoning of the mutated and overexpressed oncoproteins is important for function. Recent studies have illuminated mechanisms related to the chaperone function. However, an atomic resolution view of Hsp90 conformational dynamics, determined by the presence of different binding partners, is critical to define communication pathways between remote residues in different domains intimately affecting the chaperone cycle. Here, we present a computational analysis of signal propagation and long-range communication pathways in Hsp90. We carried out molecular dynamics simulations of the full-length Hsp90 dimer, combined with essential dynamics, correlation analysis, and a signal propagation model. All-atom MD simulations with timescales of 70 ns have been performed for complexes with the natural substrates ATP and ADP and for the unliganded dimer. We elucidate the mechanisms of signal propagation and determine "hot spots" involved in interdomain communication pathways from the nucleotide-binding site to the C-terminal domain interface. A comprehensive computational analysis of the Hsp90 communication pathways and dynamics at atomic resolution has revealed the role of the nucleotide in effecting conformational changes, elucidating the mechanisms of signal propagation. Functionally important residues and secondary structure elements emerge as effective mediators of communication between the nucleotide-binding site and the C-terminal interface. Furthermore, we show that specific interdomain signal propagation pathways may be activated as a function of the ligand. Our results support a "conformational selection model" of the Hsp90 mechanism, whereby the protein may exist in a dynamic equilibrium between different conformational states available on the energy landscape and binding of a specific partner can bias the equilibrium toward functionally relevant complexes.
Subject(s)
HSP90 Heat-Shock Proteins/chemistry , HSP90 Heat-Shock Proteins/ultrastructure , Models, Chemical , Models, Molecular , Binding Sites , Computer Simulation , Dimerization , Ligands , Molecular Chaperones/chemistry , Protein Binding , Protein ConformationABSTRACT
A fundamental requirement to predict the native conformation, address questions of sequence design and optimization, and gain insights into the folding mechanisms of proteins lies in the definition of an unbiased reaction coordinate that reports on the folding state without the need to compare it to reference values, which might be unavailable for new (designed) sequences. Here, we introduce such a reaction coordinate, which does not depend on previous structural knowledge of the native state but relies solely on the energy partition within the protein: the spectral gap of the pair nonbonded energy matrix (ENergy Gap, ENG). This quantity can be simply calculated along unbiased MD trajectories. We show that upon folding the gap increases significantly, while its fluctuations are reduced to a minimum. This is consistently observed for a diverse set of systems and trajectories. Our approach allows one to promptly identify residues that belong to the folding core as well as residues involved in non-native contacts that need to be disrupted to guide polypeptides to the folded state. The energy gap and fluctuations criteria are then used to develop an automatic detection system which allows us to extract and analyze folding transitions from a generic MD trajectory. We speculate that our method can be used to detect conformational ensembles in dynamic and intrinsically disordered proteins, revealing potential preorganization for binding.
Subject(s)
Molecular Dynamics Simulation , Proteins/chemistry , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/metabolism , Protein Folding , Proteins/metabolism , ThermodynamicsABSTRACT
SARS-CoV-2 is a health threat with dire socioeconomical consequences. As the crucial mediator of infection, the viral glycosylated spike protein (S) has attracted the most attention and is at the center of efforts to develop therapeutics and diagnostics. Herein, we use an original decomposition approach to identify energetically uncoupled substructures as antibody binding sites on the fully glycosylated S. Crucially, all that is required are unbiased MD simulations; no prior knowledge of binding properties or ad hoc parameter combinations is needed. Our results are validated by experimentally confirmed structures of S in complex with anti- or nanobodies. We identify poorly coupled subdomains that are poised to host (several) epitopes and potentially involved in large functional conformational transitions. Moreover, we detect two distinct behaviors for glycans: those with stronger energetic coupling are structurally relevant and protect underlying peptidic epitopes, and those with weaker coupling could themselves be prone to antibody recognition.
Subject(s)
Epitopes/chemistry , Molecular Dynamics Simulation , Spike Glycoprotein, Coronavirus/chemistry , Algorithms , Betacoronavirus/chemistry , Binding Sites, Antibody , Glycosylation , Humans , Models, Molecular , Molecular Conformation , Peptides/chemistry , Polysaccharides/chemistry , SARS-CoV-2ABSTRACT
Understanding the selectivity of a small molecule for its target(s) in cells is an important goal in chemical biology and drug discovery. One powerful way to address this question is with dominant negative (DN) mutants, in which an active site residue in the putative target is mutated. While powerful, this approach is less straightforward for allosteric sites. Here, we introduce tryptophan scanning mutagenesis as an expansion of this idea. As a test case, we focused on the challenging drug target, heat shock cognate protein 70 (Hsc70), and its allosteric inhibitor JG-98. Structure-based modelling predicted that mutating Y149W in human Hsc70 or Y145W in the bacterial ortholog DnaK would place an indole side chain into the allosteric pocket normally occupied by the compound. Indeed, we found that the tryptophan mutants acted as if they were engaged with JG-98. We then used DnaK Y145W to suggest that this protein may be an anti-bacterial target. Indeed, we found that DnaK inhibitors have minimum inhibitory concentration (MIC) values <0.125 µg mL-1 against several pathogens, including multidrug-resistant Staphylococcus aureus (MRSA) strains. We propose that tryptophan scanning mutagenesis may provide a distinct way to address the important problem of target engagement.
ABSTRACT
Molecular chaperones HSP90 and HSP70 are essential regulators of the folding and activation of a disparate ensemble of client proteins. They function through ATP hydrolysis and the assembly of multiprotein complexes with cochaperones and clients. While their therapeutic relevance is recognized, important details underlying the links between ATP-dependent conformational dynamics and clients/cochaperones recruitment remain elusive. Allosteric modulators represent fundamental tools to obtain molecular insights into functional regulation. By selective perturbation of different aspects of HSP90/HSP70 activities, allosteric drugs can tune rather than completely inhibit signaling cascades, providing information on the relationships between structure-dynamics and function. Herein, we review advances in the design of HSP90 and HSP70 allosteric modulators. We consider inhibitors and activators in different biochemical and disease models. We discuss these compounds as probes to decipher the complexity of the chaperone machinery and that at the same time represent starting leads for the development of drugs against cancer and neurodegeneration.