A dynamics based analysis of allosteric modulation in heat shock proteins

The 70 kDa and 90 kDa heat shock proteins (Hsp70 and Hsp90) are molecular chaperones that play central roles in maintaining cellular homeostasis in all organisms of life with the exception of archaea. In addition to their general chaperone function in protein quality control, Hsp70 and Hsp90 cooperate in the regulation and activity of some 200 known natively folded protein clients which include protein kinases, transcription factors and receptors, many of which are implicated as key regulators of essential signal transduction pathways. Both chaperones are considered to be large multi-domain proteins that rely on ATPase activity and co-chaperone interactions to regulate their conformational cycles for peptide binding and release. The unique positioning of Hsp90 at the crossroads of several fundamental cellular pathways coupled with its known association with diverse oncogenic peptide clients has brought the molecular chaperone under increasing interest as a potential anti-cancer target that is crucially implicated with all eight hallmarks of the disease. Current orthosteric drug discovery efforts aimed at the inhibition of the ATPase domain of Hsp90 have been limited due to high levels of associated toxicity. In an effort to circumnavigate this, the combined focus of research efforts is shifting toward alternative approaches such as interference with co-chaperone binding and the allosteric inhibition/activation of the molecular chaperone. The overriding aim of this thesis was to demonstrate how the computational technique of Perturbation response scanning (PRS) coupled with all-atom molecular dynamics simulations (MD) and dynamic residue interaction network (DRN) analysis can be used as a viable strategy to efficiently scan and accurately identify allosteric control element capable of modulating the functional dynamics of a protein. In pursuit of this goal, this thesis also contributes to the current understanding of the nucleotide dependent allosteric mechanisms at play in cellular functionality of both Hsp70 and Hsp90. All-atom MD simulations of E. coli DnaK provided evidence of nucleotide driven modulation of conformational dynamics in both the catalytically active and inactive states. PRS analysis employed on these trajectories demonstrated sensitivity toward bound nucleotide and peptide substrate, and provided evidence of a putative allosterically active intermediate state between the ATPase active and inactive conformational states. Simultaneous binding of ATP and peptide substrate was found to allosterically prime the chaperone for interstate conversion regardless of the transition direction. Detailed analysis of these allosterically primed states revealed select residue sites capable of selecting a coordinate shift towards the opposite conformational state. In an effort to validate these results, the predicted allosteric hot spot sites were cross-validated with known experimental works and found to overlap with functional sites implicated in allosteric signal propagation and ATPase activation in Hsp70. This study presented for the first time, the application of PRS as a suitable diagnostic tool for the elucidation and quantification of the allosteric potential of select residues to effect functionally relevant global conformational rearrangements. The PRS methodology described in this study was packaged within the Python programming environment in the MD-TASK software suite for command-line ease of use and made freely available. Homology modelling techniques were used to address the lack of experimental structural data for the human cytosolic isoform of Hsp90 and for the first time provided accurate full-length structural models of human Hsp90α in fully-closed and partially-open conformations. Long-range all-atom MD simulations of these structures revealed nucleotide driven modulation of conformational dynamics in Hsp90. Subsequent DRN and PRS analysis of these MD trajectories allowed for the quantification and elucidation of nucleotide driven allosteric modulation in the molecular chaperone. A detailed PRS analysis revealed allosteric inter-domain coupling between the extreme terminals of the chaperone in response to external force perturbations at either domain. Furthermore PRS also identified several individual residue sites that are capable of selecting conformational rearrangements towards functionally relevant states which may be considered to be putative allosteric target sites for future drug discovery efforts Molecular docking techniques were employed to investigate the modulation of conformational dynamics of human Hsp90α in response to ligand binding interactions at two identified allosteric sites at the C-terminal. High throughput screening of a small library of natural compounds indigenous to South Africa revealed three hit compounds at these sites: Cephalostatin 17, 20(29)-Lupene-3β isoferulate and 3'-Bromorubrolide F. All-atom MD simulations on these protein-ligand complexes coupled with DRN analysis and several advanced trajectory based analysis techniques provided evidence of selective allosteric modulation of Hsp90α conformational dynamics in response to the identity and location of the bound ligands. Ligands bound at the four-helix bundle presented as putative allosteric inhibitors of Hsp90α, driving conformational dynamics in favour of dimer opening and possibly dimer separation. Meanwhile, ligand interactions at an adjacent sub-pocket located near the interface between the middle and C-terminal domains demonstrated allosteric activation of the chaperone, modulating conformational dynamics in favour of the fully-closed catalytically active conformational state. Taken together, the data presented in this thesis contributes to the understanding of allosteric modulation of conformational dynamics in Hsp70 and Hsp90, and provides a suitable platform for future biochemical and drug discovery studies. Furthermore, the molecular docking and computational identification of allosteric compounds with suitable binding affinity for allosteric sites at the CTD of human Hsp90α provide for the first time “proof-of-principle” for the use of PRS in conjunction with MD simulations and DRN analysis as a suitable method for the rapid identification of allosteric sites in proteins that can be probed by small molecule interaction. The data presented in this section could pave the way for future allosteric drug discovery studies for the treatment of Hsp90 associated pathologies

Molecular characterization of ebselen binding activity to SARS-CoV-2 main protease

There is an urgent need to repurpose drugs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recent computational-experimental screenings have identified several existing drugs that could serve as effective inhibitors of the virus? main protease, Mpro, which is involved in gene expression and replication. Among these, ebselen (2-phenyl-1,2-benzoselenazol-3-one) appears to be particularly promising. Here, we examine, at a molecular level, the potential of ebselen to decrease Mpro activity. We find that it exhibits a distinct affinity for the catalytic region. Our results reveal a higher-affinity, previously unknown binding site localized between the II and III domains of the protein. A detailed strain analysis indicates that, on such a site, ebselen exerts a pronounced allosteric effect that regulates catalytic site access through surface-loop interactions, thereby inducing a reconfiguration of water hotspots. Together, these findings highlight the promise of ebselen as a repurposed drug against SARS-CoV-2.Fil: Menéndez, Cintia Anabella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Byléhn, Fabian. University of Chicago; Estados UnidosFil: Perez Lemus, Gustavo R.. University of Chicago; Estados UnidosFil: Alvarado, Walter. University of Chicago; Estados UnidosFil: de Pablo, Juan J.. University of Chicago; Estados Unido

Structure Dynamics Guided Enzyme Improvement of ENDO-BETA-1, 4-XYLANASE I

Enzyme structure dynamics has recently been revealed to be essential for structure-function relationship. Among various structure dynamics analysis platforms, hydrogen deuterium exchange mass spectrometry stands as an efficient and high-throughput way to analyze protein dynamics upon ligand binding, protein folding, and enzyme catalysis. HDX-MS can be used to study the regional dynamics of proteins based on the m/z value or percentage of deuterium incorporation for the digested peptides in the HDX experiments. Various software packages have been developed to analyze HDX-MS data. However, for the accurate, enhanced, and explicit statistical analysis of HDX-MS data statistical analysis of software was developed as HDXanalyzer. The capability of HDX-MS analysis for the identification of enzyme structure dynamics was tested by using model catalysis endoxylanase A (XYN I) from Trichoderma longibrachiatum. The HDX data of XYN I revealed a highly dynamic personality of XYN I through the interaction with two substrates. The dynamic data which certainly restricts the targeted regions for the protein engineering efforts provided useful knowledge about the essential structural modifications for the catalysis of XYN I. The obtained knowledge was then employed for the engineering studies in order to improve the certain characteristics of XYN I protein. The high level stabilization of XYN I protein was gathered and the two highly active and moderately thermostable XYN I recombinants were developed based on the HDX-MS data which further confirmed the efficiency of the current strategy for the rational designs of catalytic proteins. A differential dynamics analysis of the two structurally similar catalysts was also performed through HDX-MS. The functionally and sequentially different but structurally highly similar XYN I and endoglucanase (Eg1A) enzymes revealed distinct structure dynamic characteristics. Compared to XYN I, Eg1A from Aspergillus niger indicated quite restricted structural motions. The data clearly postulated that the intrinsic dynamic modifications of during the enzymatic catalysis may not be the only driving force in all cases. In summary, the integration of the structure dynamics knowledge to the current biochemical and biophysical data of catalysts may provide novel insights to further enzyme improvement applications

Novel Insights into Membrane Transport from Computational Methodologies

Atomic-resolution imaging of the plasma membrane and its constituents has advanced significantly in recent years. However, membrane transport is profoundly reliant on dynamic processes ranging from highly concerted atomic fluctuations to large-scale conformational changes, which cannot be sufficiently described by static structural information. As a consequence, computational methodologies have become a prominent tool to investigate membrane organisation and dynamics. In particular, molecular dynamics simulation has proven to be a pertinent method to investigate how matter is transported through membranes, either directly through the membrane or via integral membrane proteins, in an appropriate level of detail. In this chapter, we will provide a brief overview of molecular dynamics simulations and related methodologies, and use prototypical biological systems to illustrate how these methods have contributed to our understanding of unassisted diffusion through membranes, passive diffusion through ion channels, signalling through receptors and active transport through transporters

Novel mechanisms of eIF2B action and regulation by eIF2alpha phoshorylation

Eukaryotic translation initiation factor 2 (eIF2) is a heterotrimeric G-protein that plays a critical role in protein synthesis regulation. eIF2-GTP binds Met-tRNAi to form the eIF2-GTP:Met-tRNAi ternary complex (TC), that is recruited to the 40S ribosomal subunit. Following GTP hydrolysis, eIF2-GDP is recycled back to TC by its guanine nucleotide exchange factor (GEF), eIF2B. Phosphorylation of the eIF2α subunit in response to various cellular stresses converts eIF2 into a competitive inhibitor of eIF2B, triggering the integrated stress response. Dysregulation of eIF2B activity is associated with a number of pathologies, including neurodegenerative diseases, metabolic disorders, and cancer. However, despite decades of research, the underlying molecular mechanisms remain unknown. This is due in large part to the absence of a structural understanding of the eIF2B assembly and of the eIF2B:eIF2 interaction. Common methods, such as yeast genetics, have been unable to unambiguously determine these mechanisms. Meanwhile, expanded interest in the integrated stress response has uncovered a diverse array of pathologies for which therapeutic modulation of the eIF2B:eIF2 interaction may ameliorate or overcome disease states. In this dissertation, a combination of structural and biochemical techniques is employed to elucidate the mechanisms of eIF2B action and its regulation by eIF2α phosphorylation. The aim is to provide a direct, unambiguous, structural understanding of eIF2B assembly and of eIF2B’s interactions with phosphorylated and unphosphorylated eIF2α. The work described here was among the first to challenge the widely held notion of a pentameric eIF2B assembly, as eventually confirmed by the recent publication of eIF2B’s crystal structure. The work further aims to overturn another long-standing assumption regarding the nature of inhibition of eIF2B activity: that competitive inhibition is mediated by a “direct effect” of the negatively charged phosphate group on the eIF2α:eIF2B interaction. Instead, we present evidence for an “indirect effect,” whereby phosphorylation disrupts a novel intramolecular interface within eIF2α, exposing an eIF2α surface that binds eIF2B and is responsible for inhibition of eIF2B. In the end, we combine a structural model of the eIF2B:eIF2 complex with our novel mechanism of inhibition, placing them within the larger thermodynamic context of eIF2-GDP recycling by eIF2B.2017-09-08T00:00:00

All-scale structural analysis of biomolecules through dynamical graph partitioning

From femtosecond bond vibrations to millisecond domain motions, the dynamics of biomolecules spans a wide range of time and length scales. This hierarchy of overlapping scales links the molecular and biophysical details to key aspects of their functionality. However, the span of scales combined with their intricate coupling rapidly drives atomic simulation methods to their limits, thereby often resulting in the need for coarse-graining techniques which cannot take full account of the biochemical details. To overcome this tradeoff, a graph-theoretical framework inspired by multiscale community detection methods and stochastic processes is here introduced for the analysis of protein and DNA structures. Using biophysical force fields, we propose a general mapping of the 3D atomic coordinates onto an energy-weighted network that includes the physico-chemical details of interatomic bonds and interactions.Making use of a dynamics-based approach for community detection on networks, optimal partitionings of the structure are identified which are biochemically relevant over different scales. The structural organisation of the biomolecule is shown to be recovered bottom-up over the entire range of chemical, biochemical and biologically meaningful scales, directly from the atomic information of the structure, and without any reparameterisation. This methodology is applied and discussed in five proteins and an ensemble of DNA quadruplexes. In each case, multiple conformations associated with different states of the biomolecule or stages of the underlying catalytic reaction are analysed. Experimental observations are shown to be correctly captured, including the functional domains, regions of the protein with coherent dynamics such as rigid clusters, and the spontaneous closure of some enzymes in the absence of substrate. A computational mutational analysis tool is also derived which identifies both known and new residues with a significant impact on ligand binding. In large multimeric structures, the methodology highlights patterns of long range communication taking place between subunits. In the highly dynamic and polymorphic DNA quadruplexes, key structural features for their physical stability and signatures of their unfolding pathway are identified in the static structure.Open Acces

RNA Structural Dynamics As Captured by Molecular Simulations: A Comprehensive Overview

With both catalytic and genetic functions, ribonucleic acid (RNA) is perhaps the most pluripotent chemical species in molecular biology, and its functions are intimately linked to its structure and dynamics. Computer simulations, and in particular atomistic molecular dynamics (MD), allow structural dynamics of biomolecular systems to be investigated with unprecedented temporal and spatial resolution. We here provide a comprehensive overview of the fast-developing field of MD simulations of RNA molecules. We begin with an in-depth, evaluatory coverage of the most fundamental methodological challenges that set the basis for the future development of the field, in particular, the current developments and inherent physical limitations of the atomistic force fields and the recent advances in a broad spectrum of enhanced sampling methods. We also survey the closely related field of coarse-grained modeling of RNA systems. After dealing with the methodological aspects, we provide an exhaustive overview of the available RNA simulation literature, ranging from studies of the smallest RNA oligonucleotides to investigations of the entire ribosome. Our review encompasses tetranucleotides, tetraloops, a number of small RNA motifs, A-helix RNA, kissing-loop complexes, the TAR RNA element, the decoding center and other important regions of the ribosome, as well as assorted others systems. Extended sections are devoted to RNA-ion interactions, ribozymes, riboswitches, and protein/RNA complexes. Our overview is written for as broad of an audience as possible, aiming to provide a much-needed interdisciplinary bridge between computation and experiment, together with a perspective on the future of the field

Computational Approaches: Drug Discovery and Design in Medicinal Chemistry and Bioinformatics

This book is a collection of original research articles in the field of computer-aided drug design. It reports the use of current and validated computational approaches applied to drug discovery as well as the development of new computational tools to identify new and more potent drugs

Investigation of physiological and pathological vascular functions using engineered systems

The vasculature is a highly complex, hierarchical system that performs a variety of functions in both physiological and pathological contexts. To maintain tissue homeostasis for example, the endothelium which lines all vascular structures generates a semi-permeable barrier that controls the exchange of fluids, ions, and solutes between the blood and tissue. During phases of tissue growth and wound repair, the vasculature undergoes angiogenesis, the development of new blood vessels, to provide adequate oxygen and nutrients to the new and healing tissues. In pathological situations such as cancer, blood vessels have been demonstrated to support tumor growth and provide access to the circulatory system for metastatic progression. This dissertation focuses on elucidating new mechanisms that are involved in regulating these three dynamic functions of the vasculature. In Chapter 2, we discuss preliminary work connecting the Notch signaling pathway with the ability for endothelial cells to mechanically couple to their substrate, a property that is known to regulate endothelial barrier function. Using traditional methods in two-dimensional traction force microscopy, we observed reductions in traction stresses generated by endothelial monolayers treated with a Notch inhibitor. This was accompanied by a decrease in cell- matrix tethering through focal adhesions. In Chapter 3, we utilized an engineered model of angiogenesis to probe the role of endothelial cell contractility in the formation of new vascular sprouts. Through these studies, we established an essential role of non-muscle myosin II in maintaining multicellularity during sprout morphogenesis. And in Chapter 4, we described the adaptation of a cranial window model for studying melanoma brain metastases and demonstrated the utility of this system to monitor dynamic interactions between cancer cells and the brain vasculature. Together, the work in this dissertation provides new insights into and techniques for probing outstanding questions regarding various key functions of the vasculature.2021-02-28T00:00:00