Role of Hsp70 ATPase Domain Intrinsic Dynamics and Sequence Evolution in Enabling its Functional Interactions with NEFs

Catalysis of ADP-ATP exchange by nucleotide exchange factors (NEFs) is central to the activity of Hsp70 molecular chaperones. Yet, the mechanism of interaction of this family of chaperones with NEFs is not well understood in the context of the sequence evolution and structural dynamics of Hsp70 ATPase domains. We studied the interactions of Hsp70 ATPase domains with four different NEFs on the basis of the evolutionary trace and co-evolution of the ATPase domain sequence, combined with elastic network modeling of the collective dynamics of the complexes. Our study reveals a subtle balance between the intrinsic (to the ATPase domain) and specific (to interactions with NEFs) mechanisms shared by the four complexes. Two classes of key residues are distinguished in the Hsp70 ATPase domain: (i) highly conserved residues, involved in nucleotide binding, which mediate, via a global hinge-bending, the ATPase domain opening irrespective of NEF binding, and (ii) not-conserved but co-evolved and highly mobile residues, engaged in specific interactions with NEFs (e.g., N57, R258, R262, E283, D285). The observed interplay between these respective intrinsic (pre-existing, structure-encoded) and specific (co-evolved, sequence-dependent) interactions provides us with insights into the allosteric dynamics and functional evolution of the modular Hsp70 ATPase domain

Collective Dynamics Differentiates Functional Divergence in Protein Evolution

Protein evolution is most commonly studied by analyzing related protein sequences and generating ancestral sequences through Bayesian and Maximum Likelihood methods, and/or by resurrecting ancestral proteins in the lab and performing ligand binding studies to determine function. Structural and dynamic evolution have largely been left out of molecular evolution studies. Here we incorporate both structure and dynamics to elucidate the molecular principles behind the divergence in the evolutionary path of the steroid receptor proteins. We determine the likely structure of three evolutionarily diverged ancestral steroid receptor proteins using the Zipping and Assembly Method with FRODA (ZAMF). Our predictions are within ∼2.7 Å all-atom RMSD of the respective crystal structures of the ancestral steroid receptors. Beyond static structure prediction, a particular feature of ZAMF is that it generates protein dynamics information. We investigate the differences in conformational dynamics of diverged proteins by obtaining the most collective motion through essential dynamics. Strikingly, our analysis shows that evolutionarily diverged proteins of the same family do not share the same dynamic subspace, while those sharing the same function are simultaneously clustered together and distant from those, that have functionally diverged. Dynamic analysis also enables those mutations that most affect dynamics to be identified. It correctly predicts all mutations (functional and permissive) necessary to evolve new function and ∼60% of permissive mutations necessary to recover ancestral function

Allo-network drugs: Extension of the allosteric drug concept to protein-protein interaction and signaling networks

Allosteric drugs are usually more specific and have fewer side effects than orthosteric drugs targeting the same protein. Here, we overview the current knowledge on allosteric signal transmission from the network point of view, and show that most intra-protein conformational changes may be dynamically transmitted across protein-protein interaction and signaling networks of the cell. Allo-network drugs influence the pharmacological target protein indirectly using specific inter-protein network pathways. We show that allo-network drugs may have a higher efficiency to change the networks of human cells than those of other organisms, and can be designed to have specific effects on cells in a diseased state. Finally, we summarize possible methods to identify allo-network drug targets and sites, which may develop to a promising new area of systems-based drug design

ATPase Subdomain IA Is a Mediator of Interdomain Allostery in Hsp70 Molecular Chaperones

The versatile functions of the heat shock protein 70 (Hsp70) family of molecular chaperones rely on allosteric interactions between their nucleotide-binding and substrate-binding domains, NBD and SBD. Understanding the mechanism of interdomain allostery is essential to rational design of Hsp70 modulators. Yet, despite significant progress in recent years, how the two Hsp70 domains regulate each other's activity remains elusive. Covariance data from experiments and computations emerged in recent years as valuable sources of information towards gaining insights into the molecular events that mediate allostery. In the present study, conservation and covariance properties derived from both sequence and structural dynamics data are integrated with results from Perturbation Response Scanning and in vivo functional assays, so as to establish the dynamical basis of interdomain signal transduction in Hsp70s. Our study highlights the critical roles of SBD residues D481 and T417 in mediating the coupled motions of the two domains, as well as that of G506 in enabling the movements of the α-helical lid with respect to the β-sandwich. It also draws attention to the distinctive role of the NBD subdomains: Subdomain IA acts as a key mediator of signal transduction between the ATP- and substrate-binding sites, this function being achieved by a cascade of interactions predominantly involving conserved residues such as V139, D148, R167 and K155. Subdomain IIA, on the other hand, is distinguished by strong coevolutionary signals (with the SBD) exhibited by a series of residues (D211, E217, L219, T383) implicated in DnaJ recognition. The occurrence of coevolving residues at the DnaJ recognition region parallels the behavior recently observed at the nucleotide-exchange-factor recognition region of subdomain IIB. These findings suggest that Hsp70 tends to adapt to co-chaperone recognition and activity via coevolving residues, whereas interdomain allostery, critical to chaperoning, is robustly enabled by conserved interactions. © 2014 General et al

Combining Sequence and Structure Information to Model Biological Systems Dynamics

Biochemical activity and core stability are essential properties of proteins, maintained usually by conserved amino acids. Structural dynamics emerged in recent years as another essential aspect of protein functionality, which enables the adaptation of the protein to substrate binding. It also underlies its ability to undergo allosteric transitions, while maintaining its fold. Key residues that mediate structural dynamics would thus be expected to be conserved, or exhibit co-evolutionary patterns at least. Yet, the correlation between sequence evolution and structural dynamics is yet to be established. To this end, we have performed in-depth analyses of a number of representative proteins, using a combined approach of sequence analyses and coarse-grained physics-based models. For the Hsp70 family, we studied the interactions of Hsp70 ATPase domains with four different nucleotide exchange factors (NEFs) and revealed two classes of key residues: (i) those highly conserved residues involved in nucleotide binding, which mediate the ATPase domain opening via a global hinge-bending, and (ii) those co-evolving and highly mobile residues engaged in specific interactions with NEFs. The observed interplay between these respective intrinsic (pre-existing, structure-encoded) and specific (co-evolved, sequence-dependent) interactions provides us with insights into the allosteric dynamics and functional evolution of the modular Hsp70 ATPase domain, and inspired a follow-up study that identified a group of key residues mediating the Hsp70 allosteric pathways using perturbation analysis. Along the same lines, a systematic study has been performed on a set of 34 enzymes representing various folds and functional classes, which generalizes the previous findings and unravels a unique correlation between sequence evolutionary properties and conformational dynamics. Our findings suggest that there is a balance between physical adaptability (enabled by structure-encoded motions) and chemical specificity (conferred by correlated amino acid substitutions), and this balance underlies the selection of a relatively small set of versatile folds by proteins. In another study, HIV-1 protease was investigated as a special case in which short-term evolutionary pressure plays a significant role. With advanced clustering techniques, we differentiated multi-drug resistant mutations from those arising from phylogenetic variations; correspondingly, these mutations exhibit distinctive structural/dynamical features, underlying the role of protein dynamics in conferring drug resistance

ANALYSIS OF THE BIOCHEMICAL AND CELLULAR ACTIVITIES OF SUBSTRATE BINDING BY THE MOLECULAR CHAPERONE HSP110/SSE1

Molecular chaperones ensure protein quality during protein synthesis, delivery, damage repair, and degradation. The ubiquitous and highly conserved molecular chaperone 70-kDa heat-shock proteins (Hsp70s) are essential in maintaining protein homeostasis by cycling through high and low affinity binding of unfolded protein clients to facilitate folding. The Hsp110 class of chaperones are divergent relatives of Hsp70 that are extremely effective in preventing protein aggregation but lack the hallmark folding activity seen in Hsp70s. Hsp110s serve as Hsp70 nucleotide exchange factors (NEF) that facilitate the Hsp70 folding cycle by inducing release of protein substrate from Hsp70, thus recycling the chaperone for a sequential round of folding and allowing successfully folded substrates to exit the folding cycle. In the model organism Saccharomyces cerevisiae, Hsp110 is represented by the proteins Sse1 and Sse2, which possess an Hsp70-like substrate binding domain (SBD), making them unique among other functionally similar, but structurally distinct, NEFs. Studies of Hsp110 and Sse1 have demonstrated that this chaperone/NEF family can bind polypeptides and prevent proteins from aggregating in vitro and that this ability is conferred by the SBD. However, attempts to study Hsp110 protein binding in vivo have not been successful. To date, the impact of peptide binding by Hsp110 is unknown. This study elucidates and defines substrate binding by the yeast Hsp110 and addresses the contributions of this activity toward protein and cellular homeostasis as well as begins inquiries into substrate binding by the Drosophila melanogaster Hsp110, Hsc70cb. As a major partner of Hsp70, determining cellular Hsp110 activities is a prerequisite to a full understanding of chaperone-mediated protein homeostasis. By studying chaperone functions and activities in yeast and animal models, we can understand human cellular protein quality control systems which can then be pharmacologically targeted to combat protein conformational disorders, including Alzheimer’s, Huntington’s, and Parkinson’s diseases

Structural Dynamics and Allosteric Signaling in Ionotropic Glutamate Receptors

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate excitatory neurotransmission events in the central nervous system. All distinct classes of iGluRs (AMPA, NMDA, Kainate) are composed of an N-terminal domain (NTD) and a ligand-binding domain (LBD) in their extracellular domain, a transmembrane domain (TMD) and an intracellular carboxy-terminal domain (CTD). Ligand binding to the LBD facilitates ion channel activation. The NTDs modulate channel gating allosterically in NMDA receptors (NMDARs). A similar function of the NTD in AMPA receptors (AMPARs) is still a matter of debate. Taking advantage of recently resolved structures of the NTD and the intact AMPAR, the main focus of this dissertation is a comprehensive examination of iGluR NTD structural dynamics, ligand binding and allosteric potential of AMPARs. We use a multiscale, multi-dimensional approach using coarse-grained network models and all-atom simulations for structural analyses and information theoretic approaches for examination of evolutionary correlations. Our major contribution has been the characterization of the global motions favored by iGluR NTD architecture. These intrinsic motions favor ligand binding in NMDAR NTDs and are also shared by other iGluR NTDs. We also identified structural determinants of flexibility in AMPARs and confirmed their role through in silico mutants. The overall similarity in collective dynamics among iGluRs hints at a putative allosteric capacity of non-NMDARs and has propelled the elucidation of interdomain and intersubunit coupling in the intact AMPAR. To this end, we identified “effector” and “sensor” regions in AMPARs using a perturbation-response technique. We identified potentially functional residues that enable information propagation between effector regions and proposed an efficient mechanism of allosteric communication based on a combination of tools including network models, graph theoretical methods and sequence analyses. Finally, we assessed the “druggability” of iGluR NTDs using molecular dynamics simulations in the presence of probe molecules containing fragments shared by drug-like molecules. Based on our study, we offer key insights into the ligand-binding landscape of iGluR NTD monomers and dimers, and we also identify a novel ligand-binding site in AMPAR dimers. These findings open an avenue of searching for molecules able to bind to iGluR NTDs and allosterically modulate receptor activity

A Comparison of Mitochondrial Heat Shock Protein 70 and Hsp70 Escort Protein 1 Orthologues from Trypanosoma brucei and Homo sapiens

The causative agent of African trypanosomiasis, Trypanosoma brucei (T. brucei), has an expanded retinue of specialized heat shock proteins, which have been identified as crucial to the progression of the disease. These play a central role in disease progression and transmission through their involvement in cell-cycle pathways which bring about cell-cycle arrest and differentiation. Hsp70 proteins are essential for the maintenance of proteostasis in the cell. Mitochondrial Hsp70 (mtHsp70) is a highly conserved molecular chaperone required for both the translocation of nuclear encoded proteins across the two mitochondrial membranes and the subsequent folding of proteins in the matrix. The T. brucei genome encodes three copies of mtHsp70 which are 100% identical. MtHsp70 self-aggregates, a property unique to this isoform, and an Hsp70 escort protein (Hep1) is required to maintain the molecular chaperone in a soluble, functional state. This study aimed to compare the solubilizing interaction of Hep1 from T. brucei and Homo sapiens (H. sapien). The recently introduced Alphafold program was used to analyze the structures of mtHsp70 and Hep1 proteins and allowed observations of structures unavailable to other modelling techniques. The GVFEV motif found in the ATPase domain of mtHsp70s interacted with the linker region, resulting in aggregation, the Alphafold models produced indicated that the replacement of the lysine (K) residue within the KTFEV motif of DnaK (prokaryotic Hsp70) with Glycine (G), may abrogate bond formation between the motif and a region between lobe I and II of the ATPase domain. This may facilitate the aggregation reaction of mtHsp70 orthologues and provides a residue of interest for future studies. Both TbHep1 and HsHep1 reduced the thermal aggregation of TbmtHsp70 and mortalin (H. sapien mtHsp70) respectively, however, TbHep1 was ~ 15 % less effective than HsHep1 at higher concentrations (4 uM). TbHep1 itself appeared to be aggregation-prone when under conditions of thermal stress, Alphafold models suggest this may be due to an N-terminal α- helical structure not present in HsHep1. These results indicate that TbHep1 is functionally similar to HsHep1, however, the orthologue may operate in a unique manner which requires further investigation.Thesis (MSc) -- Faculty of Science, Biotechnology Innovation Centre, 202

CONFORMATIONAL DYNAMICS OF PROTEINS: INSIGHTS FROM STRUCTURAL AND COMPUTATIONAL STUDIES

Proteins are not static; they undergo both random thermal fluctuations near a given equilibrium state, and transitions between different sub-states. These motions are usually intricately connected to the function of the protein. Therefore, understanding the dynamics of proteins is important to gain insights into the mechanisms of many biological phenomena. Only the combination of structure and dynamics does allow for describing a functional protein (or biological molecule) properly. Therefore, this thesis is centered on computational and structural studies of protein dynamics. I carried out full atomic simulations and coarse-grained analyses(using elastic network models) as computational approaches, and used NMR as well as X-ray crystallography on the experimental side. With regard to the understanding of the fluctuations accessible under equilibrium conditions, a detailed analysis of high-resolution structural data and computationally predicted dynamics was carried out for a designed sugar-binding protein. The mean-square deviations in the positions of residues derived from NMR models and those inferred from X-ray crystallographic B-factors for two different crystal forms were compared with the predictions based on the Gaussian network model (GNM) and the results from molecular dynamics (MD) simulations. The results highlighted the significance of considering ensembles of structures (or structural models) from experiments, in order to make an accurate assessment of the fluctuation dynamics of proteins under equilibrium conditions. Moreover, we analyzed the amplitudes, correlation times, and directions of residue motions in multiple MD runs of durations varying in the range 1 ns – 400 ns. Our data show that the distribution of residue fluctuations is insensitive to the simulation length, while the amplitudes increase with simulation time with a power law. Another area of interest concerned the phenomenon of “domain swapping”. We investigated the molecular basis of this unusual multimerization, using a broad range of approaches. A systematic analysis of a large set of domain-swapped structures was performed to this aim. Results suggest that almost any protein may be capable of undergoing domain swapping, and that domain swapping is solely a specialized form of oligomer assembly but is closely associated with the unfolding/folding process of proteins. We also use experimental 19F-NMR to study the thermodynamic and kinetic properties in CV-N domain swapping. The activation energy barrier for the passage between monomeric and domain swapped dimeric form is of similar magnitude to that for complete unfolding of the protein, indicating that the overall unfolding of the polypeptide is required for domain swapping. Crystal structures of a domain-swapped trimer and a tetramer of CV-N provide further insights into the potential mechanics of CV-N domain swapping