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Perturbation Response Scanning Reveals Key Residues for Allosteric Control in Hsp70 David Penkler, Ozge Sensoy, Canan Atilgan, and Ozlem Tastan Bishop J. Chem. Inf. Model., Just Accepted Manuscript • Publication Date (Web): 15 May 2017 Downloaded from http://pubs.acs.org on May 16, 2017
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Perturbation Response Scanning Reveals Key
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Residues for Allosteric Control in Hsp70
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David Penkler1, Özge Sensoy2, Canan Atilgan3 and Özlem Tastan Bishop1
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Research Unit in Bioinformatics (RUBi), Department of Biochemistry and Microbiology,
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Rhodes University, Grahamstown, 6140, South Africa 2
School of Engineering and Natural Sciences, Istanbul Medipol University, Beykoz, 34810,
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Istanbul, Turkey 3
Faculty of Engineering and Natural Sciences, Sabanci University, Tuzla 34956, Istanbul, Turkey
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Abstract
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Hsp70 molecular chaperones play an important role in maintaining cellular homeostasis, and are
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implicated in a wide array of cellular processes including protein recovery from aggregates, cross
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membrane protein translocation, and protein biogenesis. Hsp70 consists of two domains, a
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nucleotide binding domain (NBD) and a substrate binding domain (SBD), each of which
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communicates via an allosteric mechanism such that the protein interconverts between two
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functional states, an ATP bound open conformation and an ADP bound closed conformation.
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The exact mechanism for interstate conversion is not as yet fully understood. However, the
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ligand bound state of the NBD and SBD as well as interactions with co-chaperones such as DnaJ
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and nucleotide exchange factor (NEF) are thought to play crucial regulatory roles. In this study,
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we apply Perturbation Response Scanning (PRS) method in combination with molecular
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dynamics simulations as a computational tool for the identification of allosteric hot residues in
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the large multi-domain Hsp70 protein. We find evidence in support of the hypothesis that
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substrate binding triggers ATP hydrolysis, and that the ADP-substrate complex favours interstate
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conversion to the closed state. Furthermore, our data is in agreement with the proposal of there
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being an allosterically active intermediate state between the open and closed states and vice
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versa, as we find evidence that ATP binding to the closed structure and peptide binding to the
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open structure allosterically “activates” the respective complexes. We conclude our analysis by
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showing how our PRS data fits the current opinion for Hsp70’s conformational cycle and present
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several allosteric hot residues that may provide a platform for further studies to gain additional
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insight into Hsp70 allostery.
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Introduction
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The 70-kDa heat shock proteins (Hsp70s) are highly conserved molecular chaperones that play
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an important role in maintaining the cellular homoeostasis in organisms ranging from bacteria to
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humans1. Hsp70s are able to rescue misfolded, partially denatured, or aggregated proteins by
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mediating their unfolding and refolding via a complicated ATP driven allosteric mechanism2–6.
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From a structural standpoint Hsp70 is made up of two main domains; a nucleotide binding
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domain (NBD) at the N-terminal, and a substrate binding domain (SBD) at the C-terminal
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(Figure 1A). The NBD and SBD are connected via a 10-12 residue-long, highly conserved
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hydrophobic linker7. The NBD is made up of lobes (lobe I and lobe II), each of which consist of
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two subdomains (IA, IIA, IB, and IIB). The SBD consists of a hydrophobic β-sandwich peptide
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binding cavity (SBDβ) and a helical lid (SBDα) that entraps bound peptide8.
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Hsp70's function relies on the nucleotide mediated allosteric interaction of its two domains1,6.
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Structural insights into Escherichia coli Hsp70, DnaK, have greatly contributed to the
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understanding of Hsp70's dynamics, leading to the description of two key functional nucleotide
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dependent conformations. X-ray crystallography of the ATP bound protein reveals the precise
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packing of the interdomain linker within a binding cleft located between subdomains IA and IIA,
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together with interdomain docking of the SBD onto the NBD via multiple interactions with the
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SBD, such that the substrate binding cavity is exposed for substrate binding9,10 (Figure 1A; PDB
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4B9Q). Solution NMR studies show that in the ADP bound state, subdomain rotation at the NBD
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releases the interdomain linker, leading to the detachment of the SBD from the NBD, such that
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the two domains are largely independent of one another, allowing the SBDα lid to firmly bind to
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the SBDβ and entrap bound substrate11,12 (Figure 1B; PDB 2KHO). We refer to these two
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structural conformations/states with respect to the exposure of the substrate binding cavity of the
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SBD, namely the ATP “open” and ADP “closed” states.
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Figure 1C provides a schematic representation of the series of events that take place during
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Hsp70's multi-conformation functional cycle1,3,4,7,9,13–16: (1) Client substrate binding at the SBD
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initiates an allosteric signal that is transmitted from the SBD to the NBD leading to an
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allosterically active intermediate state, in which the SBD partially detaches from the NBD7,
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activating ATPase activity. Bound ATP at the NBD results in low peptide binding affinity and
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high on-off transfer rates17–19, thus for efficient client binding, Hsp70 is facilitated by the co-
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chaperone DnaJ, interactions of which have also been shown to accelerate ATPase activity2–5. (2)
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ATP hydrolysis and destabilisation of the proline switch results in major conformational
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rearrangements in both the NBD and SBD, leading to the eventual transition to the closed state.
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(3) In the ADP bound closed state the NBD and SBD are largely independent of one another and
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the substrate binds with a higher affinity and slower transfer rates1,2,5. (4) Intervention by
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nucleotide exchange factor (NEF) and the exchange of ATP for ADP, initiates an allosteric
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signal at the NBD which is transmitted to the SBD resulting in the partial docking of the SBD to
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the NBD and an allosterically active intermediate7. (5) Structural rearrangements and
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conformational dynamics at the SBD leads to client substrate dissociation, returning the
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chaperone to the start of the cycle once again9.
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Figure 1. A) Experimental crystal structure of Hsp70 in its open conformation (PDB ID 4B9Q),
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coloured by subdomain. The hyphenated box describes the loop regions of the SBD showing; L12
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(res 404-406), L23 (res412-420), L34 (res428-434), L45 (res439-457), L56 (res458-476), L67 (res
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479-482), and L78 (res 490-496). B) Closed experimental NMR structure of Hsp70 in closed
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peptide bound conformation, showing ADP (sticks) bound to the NBD (red) and peptide
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substrate (black) bound to the SBD (blue). The linker region is coloured green. The red-green-
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blue colouring of the regions is also used in all following schematics. C) Hsp70's conformational
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cycle; DnaJ facilitates client substrate binding to the SBD of the open conformation (4B9Q) (1-
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2), triggering ATP hydrolysis and domain undocking and the eventual entrapment of client
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substrate (2-3). ADP is exchanged for ATP through NEF intervention, triggering domain
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docking and the opening of the SBD, allowing for the release of client substrate (3-5).
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It is clear that for efficient binding and release of client substrates, Hsp70s must undergo major
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conformational changes during their functional cycle, regulation of which is tightly controlled
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through interdomain allosteric communication in relation to the bound state of the NBD3,7,9–11,20–
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25
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thoroughly investigated through the efforts of several research groups (see ref.
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overview), and it is widely thought that a core set of residues are important for substrate-
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triggered ATPase activity and ATP-stimulated peptide release. This hypothesis however is
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difficult to rigorously test using in vitro techniques, and a number of research groups have turned
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to computational approaches for further insight. Coarse-grained molecular dynamics (MD)
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studies have demonstrated the collective motions involved in allosteric communication between
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the SBD and NBD28,29, while all-atom simulations in conjunction with several computational
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analysis techniques have been utilised to better understand the conformational dynamics of the
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two end-point states22,23,30–32. However, given the computational and hardware limitations of MD
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simulations, the complete transition from the open to the closed state and vice versa cannot be
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observed in all-atom studies, with complete protein conformational transitions typically
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occurring on the micro-millisecond time scale33. Despite this, several studies have demonstrated
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the elegant use of computational techniques to describe key residue networks essential for
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interdomain allostery in Hsp70: Smock and co-workers defined a structurally contiguous group
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of residues that form a physical linkage network between NBD and SBD of which the linker is
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centrally positioned15; General et al. (2014) combine computational techniques together with
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experimental validation to report several highly conserved residues in subdomain IA to be key
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allosteric communicators between the NBD and SBD34; a further study has demonstrated the use
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of free-energy landscapes to analyse the structural changes induced by ATP binding, reporting
and SBD13,16,26,27. To date, aspects of this complex allosteric control mechanism have been 6
for a detailed
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27 internal coordinates that correspond to a 91 residue network thought to be relevant for
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allosteric communication23. Molecular simulations combined with protein stability analysis and
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network modelling of residue interactions, suggest allostery to be primarily determined by
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nucleotide induced conformational redistributions in the NBD and SBD, and that several
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mediating residues with high network centrality are crucial for conformational stability and
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allosteric communication35. Allosteric inhibition of DnaK was recently investigated by Stetz et
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al. (2016), in which molecular simulations and binding free energy analysis were coupled with
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extensive network-based modelling of residue interactions, to compare and characterise the
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molecular signatures of the apo, substrate bound, and allosteric inhibitor bound closed
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conformation complexes. This study reports a mechanism by which the allosteric inhibitor PET-
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16 can stabilise the ADP bound closed conformation and inhibit interdomain allosteric control36.
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In this study, we demonstrate the use of extensive all-atom molecular dynamics simulations of
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Hsp70/DnaK, coupled with Perturbation Response Scanning (PRS) analysis, to investigate the
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relative effect different combinations of bound ligand at the NBD (ATP/ADP/apo) and SBD
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(peptide/apo) have on interdomain allostery, reporting the allosteric potential of each
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configuration as well as the residue contribution for conformational modulation. PRS analysis is
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a computational technique used to determine the allosteric influence each residue has on all other
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residues in a given protein when externally perturbed. Simply put, PRS measures the response of
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all residues in an initial state to the perturbation of a single residue k, and correlates this response
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to a known experimental target conformation37–41. PRS is an efficient approach for the
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identification of areas within a protein that moderate binding region motions in a mechanical
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manner. It is useful for locating functionally important residues in a given conformational
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change, and can therefore be used to determine candidate sites for mutational studies. Previously,
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the biological implications of PRS data has been extensively studied on a set of 25 different
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proteins that demonstrate various classified complex motions, including shear, allosteric, hinge
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and partial refolding38. Indeed, this same study demonstrated that residues whose perturbation
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lead to global displacements that correlate well with experimental values are also hot spots that
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have been previously shown through experimental and computational works to be of functional
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significance.
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The allosteric mechanism of the open ATP bound conformation of Hsp70 has been previously
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investigated with PRS34. Therein, the protein was modeled as an anisotropic network 42, and the
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relative influence and sensitivity of all residues analysed, reporting three distinct allosteric
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effector sites each located in a separate subdomain; the SBDß (F426, A435, I462, Q471, I472,
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and K491); subdomain IIB (Y239, L283, and M296); and subdomain IA (G6, A117, V139,
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K155, R167, and N170). Of these hot residues, the authors suggest those of the SBDß to be
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involved in the propagation of structural perturbations induced by client peptide binding, while
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the effector residues located in the IA subdomain may be integral for the efficient
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communication of allosteric signals from the SBD to the nucleotide binding site.
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The present study differs from the aforementioned work in that we make use of the
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experimental structures 4B9Q9 (open) and 2KHO11 (closed) as conformational templates, to
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prepare 12 configurations of DnaK (six open state and six closed state), by alternating the bound
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configuration of the NBD (ATP/ADP/apo) and SBD (peptide/apo) (Figure 2), all of which are
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subjected to all-atom molecular dynamics rather than a network model construction. Using these
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putative configurations as initial states in a series of PRS experiments, we compare and contrast
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the relative effect the bound ligand (ATP/peptide) at the NBD and SBD has on the allosteric
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potential for interstate conversion to the known experimental target structures 2KHO and 4B9Q.
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To the best of our knowledge there is no study to date that has investigated the residue
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contribution to modify the structure of Hsp70 from its open conformation to the closed
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conformation and vice versa, in conjunction with all combinations of bound ligand, as
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accomplished in the current PRS analysis. Furthermore, the external force perturbations used in
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PRS analysis allow us to not only identify allosteric mediators for future mutational studies, but
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also to predict residues that may be involved in protein-protein interactions such as those with
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Hsp70 co-chaperones. These interactions may elicit an allosteric response, and present putative
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drug target sites.
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Figure 2. Overview of PRS experiments (involving 12 unique configurations of DnaK) to
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investigate the potential for interstate conversion. The open to closed transition experiments are:
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A-F, and the closed to open transition experiments are: G-L.
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Our results show that bound ADP at the NBD of the open conformation promotes the closing
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transition, and that peptide binding to the ATP bound open state allosterically activates the
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chaperone complex. Likewise, for the closed conformation, ATP binding at the NBD promotes
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interconversion to the open state, via an allosterically active intermediate. Furthermore, bound
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ADP at the NBD stabilises the complex, affording it a resistance to interconvert to the open
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conformation. These findings are supported by the observation that allosteric effector residues
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identified in both transitions correlate well with previously published experimental and
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computational works. Additional key residues not previously reported are identified, providing a
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platform for future studies.
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Materials and Methods
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Preparation of ligand bound configurations
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A total of 12 unique configurations of DnaK were prepared, six open and six closed
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conformations in various combinations of bound/unbound ADP/ATP or peptide substrate.
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Experimental structures of DnaK in open and closed conformation were obtained from the
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Protein Data Bank; PDB codes 4BQ9 9 and 2KHO 11, respectively. PDB 4B9Q (residues 2-602)
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represents the open conformation of the protein, where the SBD is intimately docked to the NBD
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and SBDα is undocked from the SBDß exposing the substrate binding site. PBD 2KHO (residues
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4-603) is representative of the ADP bound closed conformation, in which each domain is
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independently positioned on either side of the interdomain linker, and the SBDα is bound to
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SBDß.
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Neither 4B9Q nor 2KHO contained a peptide substrate and as such the NBD co-crystallized
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with peptide NRLLLTG (PDB code 1DKZ) was superimposed over the NBD’s of 4B9Q and
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2KHO respectively and the coordinate data for the peptide incorporated into these structures. In
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the same manner, ADP coordinates were obtained from the PDB 3ATV, the NBD of human
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Hsp70 crystallized with ADP. ATP coordinates were obtained from 4B9Q.
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Perturbation-response scanning
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We analyse the conformational changes between various nucleotide and substrate bound
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conformations of DnaK by applying PRS to several configurations of the protein, thereby
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elucidating the propensity for interconversion between two states. The theory of PRS has been
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well described in previous studies
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(LRT), where a particular conformation of a protein can be described by the perturbation of the
37,38
. Briefly, this technique utilizes linear response theory
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Hamiltonian of an alternative conformation, and the shift in coordinates due to an external force
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such as ligand binding is approximated by 37,43,44;
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∆ = 〈〉 − 〈〉 ≅
〈∆R R∆ 〉 ∆ =
∆
(1)
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where subscripts 0 and 1 reflect unperturbed and perturbed protein configurations, respectively.
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The vector ∆ denotes the coordinates of the externally inserted force on a select residue. is the
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variance-covariance matrix which can be obtained by either imposing the approximation of
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harmonic springs between pairs of interacting residue atoms, or directly from the coordinate data
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of MD trajectories of suitable length. The PRS algorithm is thus the repetition of the above LRT
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calculation separately for each residue in the protein, identifying those residues whose
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perturbation results in a favourable overlap with the observed (targeted) conformational change
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∆ = 〈〉 − 〈〉 .
(2)
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For our purposes, the C matrix was constructed using the latter MD trajectory approach. The
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linear response theory that forms the basis of the PRS method relies on the assumption that the
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kernel used in connecting the inserted forces to the displacements comes from a single energy
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minimum that may be approximated by harmonic springs. While network models ensure this is
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the case, they are limited because (i) they require a PDB structure for network construction, and
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(ii) the selection of the cutoff distance is ambiguous45. Alternatively, one may directly use the
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variance-covariance matrix obtained from an MD simulations. In that case, one needs to use
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short enough MD simulation pieces to construct the Hessian so that the matrix represents
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information obtained from a single potential well. We have explored previously optimal lengths
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of MD simulations that will fit this requisite and using relaxation times of the backbone atom
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fluctuations, we have shown that simulations up to 40 ns will carry this information46,47.
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Moreover, using MD simulations frees one from the limitation of having alternative PDB
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structures, since one may generate alternative conformations using appropriate conditions, as
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long as one ensures that equilibrium or quasi-equilibrium has been reached in the portion of the
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MD trajectory that will be used to construct the variance-covariance matrix.
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The trajectory of each structure was reduced to Cα atoms and the deviation of each residue
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from an average structure over 20 ns time window w was calculated by ∆ = −
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〈 〉, and recorded in a 3N x w ∆ matrix. The covariance matrix C is then calculated
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by ∆∆ . In this manner a total of 12 separate C matrices were constructed, each pertaining to
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one of the 12 conformational states previously described (see Preparation of ligand bound
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configurations).
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For each PRS experiment, the coordinates of the initial and final states (states 0 and 1 in the LRT
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equation) were extracted from the initial frame in each state’s trajectory respectively,
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superimposing the final state on the initial state, and only then computing the targeted
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(experimental) residue displacement vectors.
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We sequentially perturb each residue in the initial state by applying random force vectors,
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recording the expected changes, ∆, as a result of the linear response of the protein. A total of
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250 forces were applied to each residue to resemble a sphere of randomly selected directions.
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The quality of the predicted displacements ∆R k for each residue i in response to an applied
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force was assessed using the Pearson correlation coefficient between the predicted and
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experimental displacements averaged over all affected residues k,
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Ci =
i ! ∑N k=1 [∆R k - ∆ ]∆Sk $∆%
&$'( ')
(3)
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The overbar represents the average, ∆Sk are the experimental displacements, calculated
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between the initial MD frame and the target conformation (PDB structures 4B9Q and 2KHO for
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the open and closed conformations respectively), *% and *+ are the corresponding root mean
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squared values. Thus ∆R k is compared with ∆Sk and the goodness of fit assessed with the
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Pearson correlation coefficient to calculate Ci for each residue i (it is important to note that Ci is
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calculated for each of the 250 perturbation, but that only the maximum Ci is retained). In this
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assessment, a Ci value close to 1 implies good agreement with the experimental changes, and
247
values close to zero, no agreement or lack of correlation with the experimental findings.
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The residue-based displacements analysed in PRS are critically dependent on the alignment
249
between the two conformational structures. Given Hsp70’s large conformational changes that
250
involve both rotational and translational transitions, it is important to note that a whole protein
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alignment approach was followed in this study, using the Kabsch algorithm48 (see Figure S1 A-F
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for schematics illustrating this point). Thus, residue overlaps are not used as a measure of the
253
conformational change, because for large scale transitions, the overlap, which is calculated as the
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dot product ∆ ∙ ∆-/|∆ ||∆-|, is not expected to be close to 1 because the direction of the
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initial motion may be entirely different from the overall displacements that characterise the end
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points of the conformational change. Indeed this very problem has been previously discussed,
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and alternative approaches offered49,50. Here, we found the correlation between the relative
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residue displacements to be adequate to classify residues whose perturbation leads to satisfactory
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and unsatisfactory induced conformational change. The reproducibility of this approach is
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demonstrated in the supporting documentation (Figure S1 G), where PRS calculations are
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reproduced using independent MD trajectories for the closed NBD(ATP)-SBD(pep) complex.
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Furthermore, the selection of residues using equation 1 as the criterion is also illustrated in the
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supporting documentation (Figure S2), where we graphically exemplify the difference in the
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displacements by perturbing residues that provide a high and a low Ci value, respectively.
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Molecular dynamics simulations
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All MD simulations were performed with the GROMACS 5.1.2 54–56
51–53
employing the
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CHARMM 22 force-field (with CMAP corrections)
. Systems were subjected to energy
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minimization using a conjugate gradient algorithm prior to MD runs. All systems were energy
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relaxed with 1000 steps of steepest-descent energy minimization. MD simulation on the
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minimized systems were then carried out in the NVT ensemble at 310 K using the Berendsen
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temperature coupling algorithm. The final structures were then run in the NPT ensemble at 1 atm
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and 310 K until volumetric fluctuations stabilized and the desired average pressure maintained,
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allowing for adequate data points to be collected for further analysis. MD simulations were run
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for a minimum of 100 ns and maximum of 200 ns until the backbone RMSD of the protein
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equilibrated with a fluctuation of no more than 3 Å for at least 20 ns. All coordinates were saved
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at 2 ps intervals for analysis. All MD simulations were carried out using the LINCS algorithm to
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constrain bond lengths, while the Long-range electrostatic forces were handled using the Fast
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Particle-Mesh Ewald method (PME), and Van der Waals forces treated with a 0.9 nm cut-off.
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Periodic boundary conditions were applied in all directions and the simulation time-step was set
280
to 2 fs. Water molecules were modelled using the TIP3P model. NH3+ and COO- groups were
281
used to achieve the zwitterionic form of the protein whereas the N- and the C- termini of the
282
peptide substrate were capped using methyl-carbonyl and methyl-amine, respectively, to mimic
283
as if the peptide were part of a protein.
284
Prediction of intra-protein contact residues
285
Inter-subdomain contact residues were determined using the Protein-Interaction Calculator 57
286
(PIC)
. The equilibrated perturbed structures of the open and closed NBD(ATP)-SBD(pep)
287
configurations were submitted as input and all default parameter settings selected. In house
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288
Python scripts were used to parse the interaction data to determine the inter-subdomain
289
interactions.
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Results and Discussion
291
Molecular dynamics simulations
292
PRS relies on linear response theory (LRT) to relate external forces to perturbed positions on a
293
protein, and requires the construction of the Hessian (covariance) matrix (C). Here we follow
294
previously published methodology whereby C is constructed using a MD simulation trajectory of
295
the initial state, a method which is advantageous in that it takes into consideration the
296
interactions of all atoms in the protein over time40,46,58.
297
A total of 12 Hsp70 configurations, six in the closed conformation (NBD-SBD undocked) and
298
six in the open conformation (NBD-SBD docked), were prepared using the experimental NMR
299
and X-ray crystal structures 2KHO11 and 4B9Q9 respectively (Figure 2 A-L). Each conformation
300
has a unique combination of bound nucleotide and/or peptide substrate (see methodology). We
301
refer to each of these configurations using the following nomenclature to describe the bound
302
state of the NBD and SBD respectively; NBD(ATP/ADP/apo)-SBD(pep/apo). Each
303
configuration was submitted to MD simulation for a minimum of 100 ns and a maximum of 200
304
ns, to obtain a 20 ns equilibrated trajectory segment (where RMSD fluctuation of backbone is
305
maintained around 2 Å), required for the construction of the Hessian matrix (see methods and
306
Table 1 for more details). The initial frame for each equilibrated segment was used as the initial
307
state (perturbed) structure in the respective PRS calculations, and it was thus important to
308
consider the structural arrangement of each configuration post MD simulation, to investigate the
309
immediate, short-range effect of bound nucleotide and substrate on the conformation and
310
interdomain rearrangement of each configuration. Table 1 summarizes each of these simulations
311
in terms of simulation length, equilibrated region and RMSD fluctuation in the equilibrated
312
region.
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Configuration
Simulation length (ns)
Equilibrated region (ns)
RMSD deviation (Å)
NBD(ATP)-SBD(apo)
100
80-100
2
NBD(ATP)-SBD(pep)1
200
180-200
2
NBD(ATP)-SBD(pep)2
150
130-150
2
NBD(ADP)-SBD(apo)
100
80-100
2
NBD(ADP)-SBD(pep)1
100
61-81
2
NBD(ADP)-SBD(pep)2
100
61-81
2
NBD(apo)-SBD(apo)
150
120-140
2.3
NBD(apo)-SBD(pep)
150
68-88
3
NBD(ATP)-SBD(apo)
100
56-76
2.3
NBD(ATP)-SBD(pep)
200
116-136
2.3
NBD(ADP)-SBD(apo)
100
53-73
2.3
NBD(ADP)-SBD(pep)
100
80-100
2
NBD(apo)-SBD(apo)
100
62-82
2.3
NBD(apo)-SBD(pep)
100
66-86
2.3
313 314
Table 1. Summary of MD trajectories indicating the simulation length and the backbone RMSD
315
fluctuation used to obtain the respective 20 ns equilibrated region used in the PRS calculations.
316
Note that the NBD(ATP)-SBD(pep) and NBD(ADP)-SBD(pep) simulations were repeated twice
317
to test the reproducibility of the results.
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Interdomain arrangement of closed complexes
319
Substrate free complexes. First we compare the relative effect of bound ATP and ADP on the
320
conformational dynamics of the closed state complexes. Over the course of the MD simulation
321
for NBD(ADP)-SBD(apo), the SBD rotates 180° relative to the NBD and forms stabilising
322
contacts between the SBDβ loops L23, L67 and the linker (Figure S3), and between the linker and
323
subdomain IA. This stabilisation between both functional domains is evident in a tightly
324
equilibrating RMSD plot after 50 ns (Figure S4). In comparison, NBD(ATP)-SBD(apo) only
325
stabilised after 70 ns, courtesy of interactions between the SBDβ and loop 210 of subdomain IIA,
326
in addition to interactions between loops L23, L67, and the linker (Figure S3). The NBD(apo)-
327
SBD(apo) complex established contacts between the linker and SBDβ loops L23, L67, and
328
between loop Asp20-Thr23 and the linker, in addition to a contact between Ser505 and loop
329
Gly132-Glu137 (Figure S3). In comparison to the nucleotide bound complexes, the NBD(apo)-
330
SBD(apo) resulted in a far less stable complex requiring a simulation time of 150 ns to yield a
331
stable segment between 120-140 ns, with a maximum backbone RMSD fluctuation of 2.3Å
332
(Figure S4).
333
Substrate bound complexes. NBD(ADP)-SBD(pep) stabilised after 50 ns (Figure S4), once
334
again forming contacts between the linker and loops L23, L67 in the SBDβ, as well contacts
335
between L67 and L210 of subdomain IIA were also observed (Figure S3). Interestingly
336
NBD(ATP)-SBD(pep) only stabilised with a RMSD fluctuation of 2 Å after 150 ns (Figure S4).
337
In this complex, the linker appears to form extensive contacts with several residues that form a
338
cleft between subdomains IA and IIA, in addition to contacts between loops L23, and L67 in the
339
SBD. The resultant conformation being notably distinct from the NBD(ATP)-SBD(apo) complex
340
(Figure S3 & S5A). NBD(apo)-SBD(pep) was simulated for 150 ns before stabilising briefly
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341
between 68-88 ns at a maximum fluctuation of 3.0 Å (Table 1; Figure S4). Stabilising contacts
342
for this complex were observed between the linker and L23, and between the linker and loop
343
Asp20-Thr23 (Figure S3).
344
Our observations of conformational flexibility and rearrangement of NBD and SBD for the
345
closed configurations are largely in line with previous findings. Solution NMR studies have
346
shown that for the ADP bound complex, the SBD and linker collide randomly with the IA and
347
IIA subdomains11, but that the IA/IIA binding cleft remains closed. Similar behavior in our MD
348
simulations is observed, and 20 ns stable segments invariably involve some form of stabilising
349
contact between the linker and subdomain IIA. The only evidence of direct contact between the
350
SBDβ and NBD was observed for the NDB(ATP)-SBD(pep) complex via contacts between L23
351
and subdomain IA, contacts previously suggested to be a direct result of ATP binding4.
352
Furthermore, the linker in this complex is positioned within the IA/IIA cleft (Figure S5A) as seen
353
in the experimental crystal structures of the open conformation 9,10. This structural rearrangement
354
is also in agreement with similar MD studies by Chiappori and colleagues
355
authors use extensive trajectory analysis techniques and conclude that the orientation of the
356
linker within the IA/IIA binding cleft to be in line with the proposed allosteric intermediate state
357
previously reported by Zhuravleva7. Given the significant SBD rearrangement and reorientation
358
over the course of the MD simulations, replicate simulation runs of the NBD(ADP)-SBD(pep)
359
and NBD(ATP)-SBD(pep) complexes were produced to validate the findings of an intermediate
360
state in which the linker of the ATP complex binds the NBD IA/IIA cleft. The NBD(ADP)-
361
SBD(pep) complex once again reached a stable equilibrium within 100 ns (Figure S4, red plot),
362
while the NBD(ATP)-SBD(pep) complex stabilised within 150 ns (Figure S4, red plot).
363
Comparing the equilibrated 3D structures from the respective MD runs, for NBD(ATP)-
22
. In that study, the
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SBD(pep) the SBDs assume slightly different orientations with respect to the NBDs, but notably,
365
the linker is almost identically oriented within the NBD IA/IIA binding cleft (Figure S5 B,
366
spheres). For the NBD(ADP)-SBD(pep) complexes however, the linker appears to assume
367
variable orientations, and is not bound to the IA/IIA cleft in either replica (Figure S5 C, spheres).
368
These data and the findings of previous studies support the hypothesis that an intermediate
369
conformational structure may exist between the closed ADP bound configuration and the ATP
370
bound open configuration7,22,23.
371
Interdomain arrangement of open complexes
372
In this case, unlike the closed conformation MD simulations, neither bound nucleotide, nor
373
peptide substrate have significant effects on the interdomain structural arrangement of the SBD
374
in relation to the NBD. However, we do note a high degree of flexibility for the SBDα, in each
375
configuration (Figure S6, green helix). Despite the flexibility of the SBDα, all configurations
376
stabilised within 100 ns except for NBD(ATP)-SBD(pep), which stabilised after 130 ns (Figure
377
S7). The ATP bound complexes and apo complex had the largest RMSD displacements (>10Å).
378
Details of the stable segments used for PRS analysis are summarised in Table 1. The maximum
379
RMSD fluctuation of 2.3 Å for these segments accounts for the flexibility observed in the SBDα.
380
Previous studies have reported several key contacts between the SBD and NBD in the
381
NBD(ATP)-SBD(pep) complex (Lys414-Asp326; Asn415-Thr221; Gln442-Asp148; Asp481-
382
Arg151/Arg167/Ile168; Leu484-Asp148)9,22. These contacts form part of an important stabilising
383
hydrogen bond network, thought to be essential for interdomain allostery2,12,14,59. Asp481 is
384
central to this network, and for the NBD(ATP)-SBD(pep) and ADP complexes, contacts with
385
Arg151 and Ile168 are absent in our stable segments (Table 2). Furthermore, there is a complete
386
loss of contact between Leu484 and Asp148 in these complexes, an interaction that has
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387
previously been suggested to play a crucial role in sensing bound peptide at the SBD2,3,12–15,59,60,
388
this interaction only being observed in the NBD(ATP)-SBD(apo) and apo NBD complexes.
389
Indeed, NBD(ATP)-SBD(apo) was the only configuration that maintained all of the
390
aforementioned hydrogen bond contacts with the exception of Asp148 and Gln442 (Table 2). 414
415
442
326
221
147
NBD(ATP)-SBD(apo)
x
x
x
NBD(ATP)-SBD(pep)
x
NBD(ADP)-SBD(apo)
x
NBD(ADP)-SBD(pep)
x
NBD(apo)-SBD(apo)
x
NBD(apo)-SBD(pep)
x
481
484
Contact pairs
x
x
148
151
167
168
148
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
391 392
Table 2: Summary of interdomain hydrogen bond contacts observed for the open conformation
393
simulations, showing how peptide binding reduces the number of contact pairs.
394
PRS analysis
395
In this study PRS was carried out following previously published methodology 40,41,58. The 12
396
Hsp70 configurations were divided into two groups; the open (Figure 2 A-F) and closed (Figure
397
2 G-L) conformations. PRS analysis was completed for each of these configurations, where the
398
initial state was the first frame from the respective stable 20 ns MD segment, and the final state
399
the experimental structures of the opposite conformation (2KHO and 4B9Q respectively) (Figure
400
2). In this manner, the PRS analyses in this study can be broken down into 12 unique
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401
experiments, six representing the closing transition (Figure 2 A-F) and six representing the
402
opening transition (Figure 2 G-L) of Hsp70.
403
For each set of experiments (closing/opening), we identify all residues that are accentuated by
404
PRS analysis, as being residues whose perturbation selects a coordinate change towards the
405
target conformation. There are several approaches for the selection of these residues depending
406
on the distribution of the observed correlation coefficient values (Ci): (i) First the residues are
407
ranked in descending order of Ci and plotted on the same set of axes. This allows for the
408
identification of the maximum Ci value (Cimax) for the given transition, and affords the
409
opportunity for quantitative comparison between each configuration. (ii) Next, two threshold
410
correlation values are set; a minimum threshold of 0.641, and an upper threshold which is equal to
411
the maximum Ci value observed over all experiments less 0.1, (Cimax–0.1). (iii) If the majority of
412
residues have Ci > upper threshold, we do not report any residues to be accentuated by PRS, and
413
interpret this behavior as a natural propensity for the protein to interconvert to the target
414
conformation. (iv) If the majority of residues have Ci < lower threshold, this behavior is
415
interpreted as resistance of the protein to interconvert to the target conformation. (v) Finally, the
416
Ci profiles of only those experiments with select residues that are accentuated by PRS are
417
analysed in more detail. For the ease of reading, the following discussions have been separated
418
into the closing transitions and opening transitions, in which we discuss relevant findings and
419
observations that pertain to the transition of interest.
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420 421
Figure 3: Descending Ci profiles for the closing transition experiments, graphically indicating
422
the propensity for allosteric interconvertion for each configuration. The upper correlation
423
threshold value of 0.79 is shown as a black line.
424
Closing transitions
425
In the following section we present the PRS analysis data for the closing transition
426
experiments (Figure 2 A-F) by plotting the Ci values for all six experiments as descending curves
427
on the same set of axes (Figure 3). We note that the shape of the ranked Ci curve which shows a
428
sharp drop for the first ranked residues and for the last ranked residues, with a quasi-linear
429
behavior between these two extreme values is not Universal. For a protein that displays simple
430
hinge bending motions there will be many residues that will accomplish this change so that the
431
Ci curves will gradually decrease having many residues with high Ci. For proteins with complex
432
motions; e.g. a mix of shearing and hinge motions typical of the multi-domain structure, only a
433
few residues might provide higher correlations, leading to a sudden drop in the initial part of the
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434
curve. In the low correlation extreme, there will always be residues that resist forcing since they
435
are too connected, too central to the structure that their motion does not accomplish any
436
productive change (e.g. residues located in the core of the protein) and a sudden decrease in the
437
Ci values at this end is expected for all proteins.
438
In figure 3, we observe the maximum Ci = 0.89 for the NBD(ADP)-SBD(pep) complex. As such
439
the upper threshold is 0.79. For the ADP bound complexes, 86% and 43% of residues have Ci >
440
0.79, for the NBD(ADP)-SBD(apo) NBD(ADP)-SBD(pep) complexes respectively (Figure 3
441
yellow & green respectively). Given the elevated number of accentuated residues, this suggests
442
that when bound by ADP, Hsp70 has a natural tendency to interconvert to the closed
443
conformation. Conversely, the NBD(ATP)-SBD(apo) complex (Figure 4 dark blue) has the
444
lowest number of Ci > 0.79 (6%), while the peptide bound equivalent, NBD(ATP)-SBD(pep)
445
(Figure 4 red), has 31%. Interestingly, this complex shares a similar descending correlation
446
curve, with the NBD(apo)-SBD(apo/pep) complexes (Figure 3 brown and light blue), with 32%
447
and 30% Ci > 0.79 respectively.
448
ADP drives the closing transition
449
NMR studies have shown that the nucleotide free and ADP bound states of Hsp70 appear to be
450
more dynamic than the ATP bound state, particularly with respect to NBD subdomain tilting and
451
shearing movements and SBDα-SBDβ and NBD-SBDβ distances1,25. MD studies have indicated
452
that bound ADP promotes spontaneous closing of a DnaK homology model to a semi-closed
453
intermediate state32, and that the SBD does not interact with the NBD23. Our PRS data is in
454
agreement with these findings; the ADP bound configurations recorded 86% (peptide free) and
455
43% (peptide bound) of residues to be accentuated by PRS, indicating that under ADP bound
456
conditions, Hsp70 has a natural propensity to interconvert to the closed conformation.
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457 458
Figure 4: Correlation (Ci) profiles for the closing transition experiments showing the distribution
459
of residues with Ci > 0.79. A – 6%, B – 31%, C – 86%, D – 43%, E – 32%, F – 30%. Each
460
configuration is colour coded according to the respective curves in Figure 3. The upper threshold
461
value of 0.79 is shown as a black line.
462 463
Peptide binding allosterically activates the ATP bound complex
464
Previous studies on mitochondrial Hsp70 conformational dynamics have indicated that the
465
NBD(ATP)-SBD(apo) configuration is more stable than the ADP or apo NBD configurations,
466
although undocking of the otherwise bound NBD and SBD is transiently observed on the time-
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467
scale of seconds61. MD of an ATP bound DnaK homology model indicated the NBD to have
468
decreased mobility, with the SBD remaining stably docked to the NBD23,32. Hydrogen exchange
469
mass spectrometry as well as NMR experiments show that the addition of a peptide to the ATP
470
complex leads to a semi-closed conformational state in-between the open and closed
471
conformations, a state in which the SBDβ detaches from the NBD and is thought to be
472
allosterically active7,62. Our PRS data shows that for the ATP bound complexes, only 6% of
473
residues for the peptide deficient configuration are capable of selecting the closed conformation
474
when externally perturbed, while 31% of residues were accentuated in the peptide bound
475
configuration (Figure 4 A-B). The increased number of allosterically active residues in the
476
peptide bound configuration suggests this complex to have a higher propensity to interconvert to
477
the closed state. These data are in line with the abovementioned experimental work, and supports
478
the theory that the open NBD(ATP)-SBD(apo) conformation is relatively stable, and that peptide
479
binding may allosterically activate the protein favoring a transition towards an allosterically
480
active semi-closed intermediate state 1,7. Indeed, in a previous study statistical coupling analysis
481
was used to define a contiguous group of 115 co-evolving residues implicated in mediating
482
allosteric coupling in Hsp7015. Of these 115 residues, 64 are accentuated in our PRS analysis of
483
the NBD(ATP)-SBD(pep) configuration, while only 12 residues are observed for NBD(ATP)-
484
SBD(apo), again pointing to the allosteric natures of the peptide bound complex.
485
Location
IA
NBD(ATP)-SBD(apo)
NBD(ATP)-SBD(pep) 7, 113, 120, 127, 139, 140, 141, 143, 144*-146, 148, 149-150, 151, 152 153, 156, 164, 166, 167, 170, 171, 381
References
2–4,14,15,24
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IB
IIA
IIB
226
192, 193, 195, 198-200*, 201, 205, 207*, 216*, 217*, 218, 224, 225, 226, 227*, 310-314, 316, 338, 339, 340, 344-347, 349-351, 354, 361, 362
228-229, 232
228, 229, 230, 231, 232-234, 235*, 236, 237*, 238-240, 243, 267, 270, 271, 273, 274, 275-281, 297*, 298, 299*, 303*-304*-308*-309*
linker
392-393
SBDβ
398*, 400*-409*, 410*, 413, 414*, 419*, 425*-431*, 432, 433*, 434436*-438*-440*, 446*, 447*, 449*, 450 451*, 453*, 454, 455-456, 457*, 458*, 459, 460-461, 462467*, 468*, 469-471*, 472, 473, 474, 475-479-482, 483, 484, 485*, 486, 487, 488 489-490*, 493, 494 497*, 498*, 499*, 500*-502*
399, 418-419,438-440442, 444, 446, 450, 451-453-461 , 485, 487, 489, 494, 495498, 499, 500
503, 504, 505, 515, 520, 539*, 540, 543, 550*-551, 554, 582
SBDα
14,15,20, 23,49,50
14,20,24
2,14,15
9,10,15,23, 37,49,57
4,15,23,71
486 487
Table 3: Summary of all accentuated residues for the ATP bound complexes. Emboldened
488
residues indicate previously reported functional or allosterically relevant residues. (*) Residues
489
also observed in the apo NBD complexes.
490
Visual comparison of the Ci profiles for the ATP complexes (Figure 4 A-B) shows that
491
residues accentuated by PRS tend to be grouped by residue number, forming distinct peaks of
492
contiguous residues. Comparing the location of Ci for these complexes, we find that the
493
distribution of Ci for the peptide bound configuration spans both the NBD and SBD, with the
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494
exception of subdomain IB (Table 3). For the peptide free configuration Ci are predominantly
495
restrained to the SBDβ, with only four observations in the NBD (subdomains IIA and IIB, Table
496
3). We analyse the residue composition of each peak for functional and allosteric importance by
497
drawing comparisons with experimental studies.
498 499
Functional relevance of accentuated residues in NBD(ATP)-SBD(apo/pep)
500
Starting with the NBD of the NBD(ATP)-SBD(pep) configuration; residues P143-F146, R151,
501
and E171, have been previously shown to contribute to the functioning of the proline switch3. Of
502
these residues Y145 and F146 play a crucial role in stabilizing the open conformation through
503
aromatic interactions with P1433,4. Residues E171, G198, T199, K270, S274, T344-R355 are
504
reported to be either implicated in direct interactions with nucleotide, or the coordination of ATP
505
hydrolysis, their mutation leading to a loss of allosteric control3,20. Also in the NBD are the hinge
506
residues L227, G228, and G229 which are intricately involved in coordinating shearing motions
507
between subdomain IA and IIA. Alanine point mutations of these residues caused DnaK to have
508
reduced in vitro and in vivo chaperone activities24. All of these residues were found to be
509
accentuated by PRS.
510
ATP hydrolysis has been previously shown to be synergistically stimulated >1000-fold by
511
peptide substrate and DnaJ binding2–5, highlighting the functional role and importance of DnaJ
512
interactions in Hsp70's ATPase cycle. To date only residues; Y145, N147, D148, R167, T170,
513
E217, V218, G400, D526, and G539 have been implicated in DnaJ binding2,4,63,64. Of these
514
residues, only N147 and D526 are not accentuated by PRS in the NBD(ATP)-SBD(pep)
515
configuration, however none were observed to be accentuated in the NBD(ATP)-SBD(apo),
516
suggesting DnaJ binding to be a putative allosteric activator for the closing transition only when
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517
substrate is simultaneously bound. A further study by Gao and co-workers shows the SBDα to be
518
essential for J-domain binding and ATPase stimulation65. In line with these results, we found
519
several accentuated residues the SBDα of the NBD(ATP)-SBD(pep) complex alone; M515,
520
E520, G539, L543, V550, E551 G554, A582, all of which could be naturally stimulated by J-
521
protein binding at the C-terminal of Hsp70.
522
ATP binding has been shown to result in the opening of a distinct cleft between subdomains
523
IA and IIA. This cleft provides a suitable binding groove for the interdomain linker, a structural
524
arrangement that has been shown to be essential for both ATP-stimulated peptide release as well
525
as polypeptide-stimulated ATP hydrolysis2,12,14,59,60. We find that the allosteric mediator linker
526
residues L392 and D393 to be accentuated by PRS for the NBD(ATP)-SBD(pep) configuration;
527
in vitro mutation of these residues impairs interdomain allostery2,14. Furthermore, PRS also
528
accentuates the cleft lining residues in this configuration; Y145, D148, K155, E217 and V218,
529
which have also been shown to perturb interdomain allostery when mutated14,63, along with the
530
linker coordinating reside P419 in the SBDß, which has been shown to be crucial for maintaining
531
allosteric communication9,10,15. A previous study suggests that peptide binding and interactions
532
with the co-chaperone DnaJ may be necessary to reposition the of the linker within the binding
533
cleft in such a ways as to conformationally stimulate residues R151, K155, and R167, which in
534
turn act as a trigger for the proline switch and eventually ATP hydrolysis3,14. It was thus
535
interesting to note that these linker coordinating residues along with the DnaJ and proline switch
536
residues were all accentuated in the NBD(ATP)-SBD(pep) configuration but not the NBD(ATP)-
537
SBD(apo).
538
A further effect of ATP binding is the apparent rotation of the NBD lobe I relative to lobe
539
II66,67. Premature back rotation of lobe I and therefore ATPase activity may be prevented through
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540
stabilising contacts with the SBDß4. Several residues have since been shown to facilitate SBD
541
docking to the NBD; D148, K414, Q442, D481, L484, S505, and G5064,9,10, all of which are
542
accentuated by PRS for NBD(ATP)-SBD(pep).
543
Next the SBDβ is investigated, and in particular residues that are known to be involved in the
544
recognition and binding of peptide substrates. A recent study characterised the residue
545
composition of the peptide binding cavity of DnaK, to include residues; I401-T409, Q424-H439,
546
N458, R467-M469, I472, and V47468. We observe several distinct peaks in the SBDβ of the
547
NBD(ATP)-SBD(pep) complex that have significant overlaps with these binding cavity residues;
548
G400-T410, V425-V440, and S453-E473. Interestingly, residues S398, T403, G405, T428,
549
D431, I438, F457, L459, and G468 experience large NMR chemical shift changes when a
550
peptide substrate is bound12, further pointing to their relative importance of peptide binding.
551
Despite there being several peaks in the SBDβ of the NBD(ATP)-SBD(apo) configuration, there
552
is a complete absence of these cavity residues (except N458), further highlighting the allosteric
553
impact of bound peptide.
554
A previous in silico alanine mutation scan study on the full length ATP bound open structure
555
(4B9Q), found the highly conserved SBDß residues; S427, T428, M469-I472 to be particularly
556
intolerant to mutation69. Here, we find these residues to also be accentuated by PRS in the
557
NBD(ATP)-SBD(pep) configuration, but not the NBD(ATP)-SBD(apo) complex (Table 3).
558
Since these are also sites directly involved in binding the peptide, their perturbation in the open
559
state (as would happen when the peptide first binds) may trigger the conformational change
560
towards the closed state. In the same study69, mutations of the SBDß residues G405 and V407
561
severely disrupted the intra-domain communication pathways between residues of the SBDα and
562
SBDß. SBDα residues V533 and R536 are bridged over to G405 and V407 positioned around the
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563
peptide binding site via a hydrophobic patch, and G405S and M408I mutants have been
564
experimentally shown to diminish peptide binding26. Given their location and contribution to
565
peptide binding, it is not surprising that these residues are accentuated by PRS for the
566
NBD(ATP)-SBD(pep)configuration, but not the NBD(ATP)SBD(apo) complex. The PRS results
567
in the current study suggest that perturbations arriving at these residues may be central in
568
controlling the grip of DnaK over the substrate and that this role may be perfectly fulfilled by
569
DnaJ in the initial substrate binding step.
570
As mentioned in the introduction, PRS has previously been used to investigate the interdomain
571
allostery of Hsp70 in a previous study34. In this study several groups of effector residues were
572
reported in the IA, IIB, and SBDß subdomains of the open NBD(ATP)-SBD(apo) configuration.
573
Interestingly we find that the Ci profile for this configuration (Figure 4A) closely resembles that
574
of General et al., but note that only the SBDß residues reported therein (F426, A435, I462,
575
Q471-I472, and K491) overlap with the hot residues reported here (Table 3). In the
576
aforementioned study, ANM is used to construct the Hessian matrix, and the quality of predicted
577
effector residues is evaluated by the magnitude of the resulting displacements, rather than a
578
correlation with a known experimental difference. While this generates a similar profile to the
579
one seen in this study, the definition of hot residues is not the same. Rather note, that all effector
580
residues reported by General and co-workers overlap with the hot residues observed for our
581
allosterically active NBD(ATP)-SBD(pep) configuration.
582 583
Peptide binding residues may be allosteric mediators of the closing transition
584
We have reported a number of residues accentuated by PRS for the six open conformation
585
configurations, and find that the bound state of the NBD and SBD affects the number and
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Journal of Chemical Information and Modeling
586
distribution of accentuated residues. This prompts the question, are there any residues that are
587
accentuated regardless of bound nucleotide or peptide substrate? The Ci profiles illustrated in
588
figure 4, show the SBDß to be consistently accentuated in all six configurations, and analysis of
589
these peaks reveals 10 residues in the peptide binding cavity to be accentuated in all six
590
configurations (I438-V440, K446, D450, N451, Q456, H485, Q497, and T500). Of these, special
591
mention must be made of resides I438-V440 and H485. Peptide substrate has been shown to
592
interact directly with residues I438-V44016,68,70, and a recent study has shown that alanine
593
mutations to V440 and L484 abrogate substrate simulated ATPase activity4. Indeed the authors
594
of that study suggest a plausible mechanism for ATPase activation, whereby peptide binding
595
exerts a force through V440 and L484 onto the residue D148, which acts as a release trigger for
596
lobe I back rotation and thus activation of ATP hydrolysis4. While it is as yet unclear what the
597
functional relevance of the remaining seven residues is, their conserved accentuation along with
598
the key substrate recognition residues I438-V440, and H485 (neighboring L484) in all
599
configurations, suggests these residues may be potential key allosteric hotspots for initiating the
600
closing transition.
601 602
The nucleotide free complexes favour the closing transition
603
Previous work has shown that for the nucleotide free configuration, the SBD and NBD are
604
mostly separated, but that docking of the two domains can occur on a 5-ms timescale23,71. MD
605
studies show that for the apo state the SBDα occasionally explores partially closed and open
606
conformations32. For our NBD(apo)-SBD(apo/pep) configurations, approximately 30% of
607
residues are accentuated in each, and of these residues 53% are observed in both configurations
608
(Table S1, bold). Furthermore, 64 of these residues overlap with accentuated residues in the
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609
NBD(ATP)-SBD(pep) (Table 3*), suggesting these configurations to have a similar tendency to
610
interconvert to the closed conformation. Indeed the functional residues; E217, G400, G539
611
(DnaJ interactions); D398 (linker); G400-T410, Q424-V440, N458, R467-M469, and I472
612
(substrate binding cavity), are conserved in all three of these complexes. From these data we
613
conclude that the presence of bound peptide has little impact on the allosteric nature of the
614
nucleotide free complexes, and that the number of accentuated residues is conserved and
615
distributed similarly to that of the NBD(ATP)-SBD(pep). This suggests the nucleotide free
616
complex to be allosterically active or to have a natural tendency to interconvert toward the closed
617
conformation.
618 619
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620
Opening transitions
621
Here we report the PRS analysis data obtained for the six opening transition experiments
622
(Figure 2G-L). The descending Ci curves indicate the Cimax for the six configurations to be 0.80,
623
for NBD(ATP)-SBD(pep), and the upper Ci threshold value is set to 0.70. Comparing the
624
descending curves, there is distinct pairing of experiments according to their bound NBD
625
configuration. Interestingly the ATP configurations (Figure 5 blue & red), have the highest Ci
626
values; with 44% and 34% of residues having Ci > 0.7 for the NBD(ATP)-SBD(apo) and
627
NBD(ATP)-(SBD(pep) configurations respectively. For the ADP configurations; only 3% of
628
residues have Ci > 0.7 for the NBD(ADP)-SBD(apo) complex, and a mere 1% in the
629
NBD(ADP)-SBD(pep) complex (Figure 5 yellow and green). In contrast, no residues in the apo
630
NBD configurations have Ci > 0.7 (Figure 5 light blue and brown), behavior that indicates a
631
natural resistance for the apo NBD configurations to interconvert to the open ATP bound
632
conformation.
633
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634
Figure 5: Descending Ci profiles for the open transition experiments, graphically comparing the
635
propensity for allosteric interconvertion for each configuration. The upper correlation threshold
636
value of 0.70 is shown as a black line.
637
ATP binding may promote an allosterically active state
638
The elevated number of residues with Ci > 0.7 for the ATP complexes indicates that ATP
639
binding may promote an allosterically “sensitive” or active state. This observation is in
640
agreement with previous work which indicates the notion of there being an allosterically active
641
intermediate state between the NBD(ADP)-SBD(pep) closed state, and the NBD(ATP)-
642
SBD(apo) open state7,11,22,23. Indeed, our equilibrated structure obtain from MD simulations of
643
the closed NBD(ATP)-SBD(pep) closely resembles this proposed state (Figure S3 & S5 A), a
644
structure that is also observed in other MD simulation studies22,23. Interestingly the Ci profiles of
645
the NBD(ATP)-SBD(pep) and NBD(ADP)-SBD(pep) complexes share very similar distributions
646
(Figure 6 B), the major difference being higher Ci values (~ 0.1) for the ATP configuration
647
(Figure 6B red), supporting the hypothesis that ATP binding favours allosteric signal
648
propagation.
649 650
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651 652
Figure 6: Ci Profiles for the opening transition experiments showing the distribution of residues
653
with Ci > 0.70. A – 44%, B – 34%, C – 3%, D – 1%, E – 0%, F – 0%. Each configuration is
654
colour coded according to the respective curves in Figure 5. The upper threshold value of 0.70 is
655
shown as a black line. In B, the data for D is superposed on the NBD(ATP)-SBD(pep) complex
656
to show the clearly similar profiles with lowered Ci values when ADP is bound instead of ATP.
657
Bound peptide decreases allosteric potential
658
Previous works have shown that peptide interactions with the SBD influence the
659
conformational dynamics of Hsp70, such that the presence of bound peptide dramatically
660
reduces the frequency of spontaneous α-lid opening9,61. Comparing the number of accentuated
661
residues between the peptide free and peptide bound configurations, the elevated 44% in the
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662
peptide free configuration is indicative of a natural propensity for this complex to interconvert to
663
the open conformation, and suggests that the decreased 34% in the peptide bound configuration
664
may be a direct result of bound peptide, as suggested in the aforementioned studies.
665
It is interesting to note that the highest Ci values for NBD(ATP)-SBD(pep) were recorded in
666
the SBDß. An observation that may be significant in that peptide disassociation must occur
667
before the protein can fully resume the open conformation4,9,61,72. Previous biochemical studies
668
have shown that residues R536-Q538 constitute an important hinge site for substrate release, and
669
that “latch” residues D431, R467, D540, H544, and K548 form crucial hydrogen bonds between
670
the SBDα and SBDß, and may be important for maintaining the stability of substrate bound
671
closed conformation73–75. These findings have been further supported by a computational study
672
which found allosteric inhibition of DnaK by PET-16 to disrupt this stabilising network, leading
673
to the loss of substrate affinity36. With the exception of H544, these residues recorded the highest
674
Ci values in the NBD(ATP)-SBD(pep) complex; indeed much higher than those recorded for the
675
NBD(ATP)-SBD(apo) complex, in which there is no client peptide present.
676
Furthermore, PRS also accentuate residues
S398, G400, K414, G443, and E444 in the
677
NBD(ATP)-SBD(pep) configuration, mutation has been shown to significantly decrease
678
substrate specificity26. Similarly, residues F146 and D148 have been implicated in an intricate
679
mechanism for the modulation of substrate release4, both of which are also accentuated in this
680
configuration. Taken together, these findings indicate that disassociation of substrate together
681
with ADP/ATP exchange is necessary for the successful transition to the open conformation9,61.
682 683
Functional relevance of NBD(ATP)-SBD(pep) accentuated residues
684
Given the hypothesis that the NBD(ATP)-SBD(pep) configuration may be an allosterically
685
active intermediate, we explore the functional relevance of some key resides within the
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686
accentuated peaks of this complex (Table 4 bold). Residues; R71, P143, A144, and F146 are
687
implicated in the functioning of the proline switch3; G196-T199 are involved in direct
688
interactions with the nucleotide20; while residues G223, L227-G229 form important hinge sites
689
in the NBD24. K414, whose mutation leads to a loss of interdomain allosteric communication13.
690
Residues N98-G9923, F45715, and T437-H43915,68 have been reported to be involved in allosteric
691
signal propagation. In addition to these findings, several residues throughout this configuration
692
correspond with residues that have also been suggested to be involved in allosteric signal
693
propagation in two previous computational works15,23 (Table 4 bold).
694 Location
NBD(ATP)-SBD(pep)
References
IA
11, 12, 17-19, 113, 116, 117, 142, 143, 144, 146, 148, 149-150, 156-157, 167, 370, 373-374, 376-377, 380
3,4,15,23,50
IB
59-60, 66, 68, 70, 71, 73, 96-99
IIA
195-201, 202-203, 219, 220, 221, 222, 223, 225, 226, 227, 311-315, 316, 317-321, 324-325, 329-330, 333, 353, 356,
IIB
228-229, 231, 232-233-235, 252, 255, 256, 261, 264265, 281, 283-284,
linker
392
SBDβ
396-410, 411, 412, 413, 414, 417, 429-440, 441, 442444, 448, 450, 451-453, 454, 455, 457, 464-467, 478, 479, 485, 486, 498, 500-502
SBDα
503-508, 511, 512, 514, 515, 516-517, 520, 530, 532533-534-536-538, 540, 546-548-568, 571, 581-602
3,20,23,24
14,15,20,23,49,50
14,20,24 2,14,15
9,10,15,23,37,49,57,59
4,15,23,60,71,72
695
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Page 40 of 59
696
Table 4: Summary of all accentuated residues for the ATP peptide bound complex. Emboldened
697
residues have previously been reported to have functional or allosteric relevance.
698
Comparison of closing and opening allosteric hot residues
699
Our data supports the assumption that Hsp70 may adopt an allosterically active intermediate
700
state when bound by both ATP and peptide, regardless of the transition direction. A comparison
701
of the location of hot residues for these respective complexes (Tables 3 and 4) found 78 residues
702
accentuated in both configurations including the previously validated residues: the proline
703
switch, the NBD hinge residues, multiple residues from the substrate binding cavity including
704
the highly conserved I438, the substrate trigger residues D148, V440 and H485, and the
705
allosteric modulator K414 to name a few (see previous sections for details).
706 707
PRS indicates inter-subdomain interfaces as hot residues
708
By filtering out all residues previously implicated in protein function or allosteric modulation,
709
we select only those residues unique to this study (Table S2) and map them to their respective
710
perturbed structures (Figure S8). For the closing transition (Figure S8A), hot residues appear to
711
be clustered around subdomain and interdomain interfaces (SBDβ-IA; IIA-IIB; linker-IA), with
712
no observations in subdomain IB, while several residues appear at the extreme C-terminal end of
713
the SBDα. For the opening transition (Figure S8B), we note the extensive accentuation of the C-
714
terminal helices, as well as the interface between the N-terminal of SBDα and loops of the
715
SBDβ. Furthermore, there are subdomain interface hotspots between IA-IB; and IB-IIB
716
subdomains, as well as several isolated residues in subdomain IIA.
717
Previous studies have shown that allosteric signal propagation from the NBD to SBD and vice
718
versa involves the intricate structural rearrangement of its respective subdomains, highlighting
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Journal of Chemical Information and Modeling
719
the relative importance of key interdomain contact residues4,16,20. Interestingly the present study
720
shows PRS accentuation of several key interdomain contact residues previously validated (D148,
721
K414, Q442, D481, L484, S505, and G506)4,9,10. Based on these findings, we analysed the
722
perturbed structures of the two NBD(ATP)-SBD(pep) (open and closed) complexes with the
723
Protein Interaction Calculator (PIC)57, to determine all inter-subdomain interacting residues.
724
Correlating these residues with the hot residues unique to this study, shows several overlaps
725
(Table S2) which are mapped to their respective perturbed structures (Figure 7). For the open
726
configuration (Figure 7A), the accentuation of several inter-subdomain interacting residues
727
(P113, A149, Q150, A153, I207, H226, E267, L305, E306, V309, D311, L312, V313, I338,
728
V381, A413, I501, and S504) is observed, with their locations spanning all seven subdomains
729
(Table S3). In particular, we note the accentuation of the SBDα residue S504, which we find to
730
be in hydrogen bond contact with the NBD through residue R76. For the closed configuration
731
(Figure 7B) we find residues; I73, V142, Q150, F232, L312, P396, L397, A448, K452, L454,
732
I478, K502-S504, I512, D530, V533, Q534, Q549, E585, A592, Q593, S595, Q596, and I601 to
733
be accentuated, and particularly note that several in the SBDα are in either hydrogen bond or
734
hydrophobic contact with several SBDß loop residues (Table S4). Furthermore, for several
735
interacting pairs (P396-I512, L397-A503, L454-A503, I478-A503, and F232-L312), both
736
interacting residues are accentuated by PRS. Comparing the data from both structures, only
737
residues Q150, L312 and S504 are accentuated in both configurations. Table S5 presents the PRS
738
accentuated residues for the closed counterparts of those in Table S4 for the sake of
739
completeness.
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740 741
Figure 7: Structural representation of allosteric hot residues identified in this study alone that are
742
specifically involved in inter-subdomain interactions, for the closing (A) and opening (B)
743
NBD(ATP)-SBD(pep) transition configurations. Hot residues are displayed as spheres and
744
substrate in stick representation.
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745
Conclusions
746
This study has utilized the computational technique of PRS analysis to gain insight on the
747
allosteric potential of large multi-domain proteins such as Hsp70. The data generated can be
748
analysed in terms of: (i) the propensity for the system to interconvert to an alternate state, and (ii)
749
the quantitative contribution of individual residues to select such a conformational transition.
750
PRS was applied to the molecular chaperone Hsp70, and the results found to be largely in
751
agreement with the current opinion regarding the allosteric control mechanism for this protein.
752
Looking once again at the conformational stages illustrated in Figure 1C, we summarize our
753
results in terms of Hsp70’s functional cycle as follows:
754
Phase 1 and 2 – substrate binding and ATP hydrolysis. Our PRS data suggests that bound ATP
755
alone is insufficient to trigger the closing transition, this configuration recording the lowest
756
allosteric potential out of all tested configurations. Conversely, the addition of a client peptide at
757
the SBD appears to increase the allosteric potential across both domains of the protein, in a
758
manner suggestive of a putative allosteric intermediate state. PRS accentuates many key
759
functional residues in this configuration (Figure 8A). Most notable are residues D148, R151,
760
K414, V440, D481, and L484 (Figure 8-black), which have recently been heavily implicated in
761
a complex mechanism for peptide stimulated ATPase activation4. The accentuation of several
762
known DnaJ interaction residues supports the theory that J-protein interaction may be necessary
763
to overcome the energetic barrier present between the open and closed states. PRS also
764
accentuates several groups of residues that have be implicated in the coordination of the bound
765
nucleotide (Figure 8, red); as well as the known proline switch3 (Figure 8-yellow) and NBD
766
hinge residues24 (Figure 8-cyan).
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767
Phase 3 – ADP bound functional state. PRS analysis indicates that bound ADP favours a
768
transition from the open to closed state, supporting previous findings that ADP drives the
769
transition to the closed state23,32. Our data also shows that ADP provides the closed form of the
770
protein with a natural resistance to interconvert back to open state, stabilising the chaperone in a
771
functionally closed state.
772
Phase 4 – nucleotide exchange and conformational rearrangement: Replacing ADP with ATP
773
at the NBD appears to allosterically activate the closed complex, drastically increasing the
774
number of accentuated residues. Our MD simulations of this complex shows complex
775
rearrangement of SBD with respect the NBD, suggestive of a previously reported intermediate
776
state. Furthermore, PRS once again accentuates several key functional residues for this complex
777
(Figure 8B), including the nucleotide coordinating residues, the Proline switch, and the NBD
778
hinge residues. Nucleotide exchange is heavily dependent on the functionality NEF, and we
779
report several residues (with no known function) located on the SBDα lid that may indicate the
780
location of a putative NEF binding site, which was previously suggested by Melero and co-
781
workers76. Indeed these hot residues may suggest that external force perturbations by other
782
binding partners such as NEF may be required to drive the opening transition.
783
Phase 5 – client peptide release stabilises return to the open state. The SBD residues were
784
among the highest correlating residues for the ATP and peptide bound closed complex. PRS
785
accentuates multiple residues involved in peptide binding interactions, including several SBDα
786
residues in direct contact with loops of the SBDß, including the known latch (D431, R467,
787
D540, and K548) and hinge residues (R536-Q538)73 (Figure 8B). These data suggest that
788
conformational dynamics of the SBD together with peptide release may be important
789
determinants for the opening transition, and supports the theory that completion of opening
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790
transition is dependent on complete dissociation of the client substrate to stabilise the open
791
conformation9,16.
792
793 794
Figure 8: Graphical representation of residues accentuated by PRS for (A) Closing and (B)
795
Opening NBD(ATP)-SBD(pep) transitions. Residues unique to this study are shown as gray
796
spheres, residues that overlap with experimentally determined functional residues are coloured
797
according to known function (inset legend), or coloured orange.
798
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799
Overall, our data demonstrate that the simultaneous binding of ATP and client peptide, may
800
lead to allosterically active intermediate states, which are highly sensitive to external force
801
residue perturbations. In nature, these force perturbations could be linked to functional events,
802
such as peptide binding and release, nucleotide exchange, co-factor binding, and even
803
interdomain allosteric communication. The details of Hsp70’s allosteric mechanism has slowly
804
come to light through the efforts of numerous laboratories over the past decade, however the
805
recent study by Kityk and co-workers presents a comprehensive analysis on Hsp70’s
806
communication pathways and functional sites. In that study, the authors report several residues
807
central to Hsp70’s mechanism; P143, Y145, F164, D148, I168, D326, K414, V440, D481, and
808
L484 (Figure 8-black). Our PRS analysis is in agreement with these findings, our hot residues
809
overlapping with all these residues except I168 and D326. Both of these residues are thought be
810
matching interaction pairs with K414 and D481, and involved in stabilising lobe I back rotation;
811
both residues are accentuated by PRS. Given the significant overlap of our hot residues with
812
numerous findings reported in previous works, it stands to reason that our hot residues with no
813
known function (Figure 8-gray and Table S2), may in fact point to functionally important
814
regions, such as co-factor binding sites or inter-subdomain communication interfaces (Tables S3
815
and S4). These data may provide further insight into Hsp70’s allosteric mechanism and a
816
platform for future studies.
817
Finally, we note that PRS determines the propensity of single residues to modify the structure
818
of the protein from one known endpoint to another. Therefore, population shift allostery will be
819
detected by the method while it will be insensitive to entropy driven allostery77. PRS will also
820
pinpoint residues that cause local conformational changes, but it will not be sensitive to, e.g.
821
binding hot spots that do not cause conformational change. The latter would require free energy
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Journal of Chemical Information and Modeling
822
difference calculations to determine the effect of function altering mutations36. Thus, a
823
combination of computational tools is necessary to fully understand the many-faceted dynamics
824
of Hsp70.
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Page 48 of 59
Associated content
826
Figure S1: Further information pertaining to PRS methodology. Figure S2: Illustration of the
827
output from PRS in terms of predicted displacements of all residues in response to single
828
perturbations. Figure S3: Structural arrangements of the six closed configurations after MD
829
simulation. Figure S4: Backbone RMSD plots for the six closed configurations, indicating the
830
stable regions. Figure S5: Comparison of replica MD simulations, showing linker orientations.
831
Figure S6: Structural arrangements of the six open configurations after MD simulation. Figure
832
S7: Backbone RMSD plots for the six open configurations, indicating the stable regions. Figure
833
S8: Hot residues for the open and closed NBD(ATP)-SBD(pep) configurations mapped to their
834
respective perturbed structures. Table S1: Residues accentuated by PRS for the open NBD(apo)-
835
SBD(apo) and NBD(apo)-SBD(pep) configurations. Table S2: Accentuated residues for the open
836
and closed NBD(ATP)-SBD(pep) configurations that are unique to the present study. Table S3:
837
List of hot residues involved in inter-subdomain interactions of the open NBD(ATP)-SBD(pep)
838
configuration, indicating the interacting pair and their subdomain location. Table S4: List of hot
839
residues involved in inter-subdomain interactions of the closed NBD(ATP)-SBD(pep)
840
configuration, indicating the interacting pair and their subdomain location. Table S5: Residues
841
accentuated by PRS for the closed NBD(ATP)-SBD(apo) and NBD(ADP)-SBD(apo)
842
configurations. Residue in bold formatting are also accentuated in the peptide bound
843
configuration.This information is available free of charge via the Internet at http://pubs.acs.org
844 845
Author information
846
Correspondence to Özlem Tastan Bishop; Research Unit in Bioinformatics (RUBi), Department
847
of Biochemistry and Microbiology, Rhodes University, Grahamstown, 6140, South Africa; E-
848
mail: O.TastanBishop@ru.ac.za. Note: The authors declare no competing financial interest.
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Journal of Chemical Information and Modeling
849
Acknowledgements
850
Authors thank Centre for High Performance Computing (CHPC), South Africa for computing
851
time. This work is partially supported by the National Institutes of Health Common Fund under
852
grant number U41HG006941 to H3ABioNet; National Research Foundation (NRF), South
853
Africa (grant number 93690) and Scientific and Technological Research Council of Turkey
854
(grant number 110T624). The content of this publication is solely the responsibility of the
855
authors and does not necessarily represent the official views of the funders.
856
Abbreviations
857
Hsp70, 70-kilodalton heat shock protein; NEF, Nucleotide exchange factor; PRS, Perturbation
858
response scanning; LRT, Linear response theory; MD, Molecular dynamics
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