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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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Journal of Chemical Information and Modeling

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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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ACS Paragon Plus Environment

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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

193

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

195

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;

199

∆ = 〈〉 − 〈〉 ≅

〈∆R R∆ 〉 ∆ =

∆

(1)

200

where subscripts 0 and 1 reflect unperturbed and perturbed protein configurations, respectively.

201

The vector ∆ denotes the coordinates of the externally inserted force on a select residue. is the

202

variance-covariance matrix which can be obtained by either imposing the approximation of

203

harmonic springs between pairs of interacting residue atoms, or directly from the coordinate data

204

of MD trajectories of suitable length. The PRS algorithm is thus the repetition of the above LRT

205

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

207

∆ = 〈〉 − 〈〉 .

(2)

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For our purposes, the C matrix was constructed using the latter MD trajectory approach. The

209

linear response theory that forms the basis of the PRS method relies on the assumption that the

210

kernel used in connecting the inserted forces to the displacements comes from a single energy

211

minimum that may be approximated by harmonic springs. While network models ensure this is

212

the case, they are limited because (i) they require a PDB structure for network construction, and

213

(ii) the selection of the cutoff distance is ambiguous45. Alternatively, one may directly use the

214

variance-covariance matrix obtained from an MD simulations. In that case, one needs to use

215

short enough MD simulation pieces to construct the Hessian so that the matrix represents

216

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

218

fluctuations, we have shown that simulations up to 40 ns will carry this information46,47.

219

Moreover, using MD simulations frees one from the limitation of having alternative PDB


12

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220

structures, since one may generate alternative conformations using appropriate conditions, as

221

long as one ensures that equilibrium or quasi-equilibrium has been reached in the portion of the

222

MD trajectory that will be used to construct the variance-covariance matrix.

223

The trajectory of each structure was reduced to Cα atoms and the deviation of each residue

224

from an average structure over 20 ns time window w was calculated by ∆ = −

225

〈 〉, and recorded in a 3N x w ∆ matrix. The covariance matrix C is then calculated

226

by ∆∆ . In this manner a total of 12 separate C matrices were constructed, each pertaining to

227

one of the 12 conformational states previously described (see Preparation of ligand bound

228

configurations).

229

For each PRS experiment, the coordinates of the initial and final states (states 0 and 1 in the LRT

230

equation) were extracted from the initial frame in each state’s trajectory respectively,

231

superimposing the final state on the initial state, and only then computing the targeted

232

(experimental) residue displacement vectors.

233

We sequentially perturb each residue in the initial state by applying random force vectors,

234

recording the expected changes, ∆, as a result of the linear response of the protein. A total of

235

250 forces were applied to each residue to resemble a sphere of randomly selected directions.

236

The quality of the predicted displacements ∆R k for each residue i in response to an applied

237

force was assessed using the Pearson correlation coefficient between the predicted and

238

experimental displacements averaged over all affected residues k,

239

Ci =

i ! ∑N k=1 [∆R k - ∆ ]∆Sk $∆%

&$'( ')

(3)

240

The overbar represents the average, ∆Sk are the experimental displacements, calculated

241

between the initial MD frame and the target conformation (PDB structures 4B9Q and 2KHO for


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Page 14 of 59

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the open and closed conformations respectively), *% and *+ are the corresponding root mean

243

squared values. Thus ∆R k is compared with ∆Sk and the goodness of fit assessed with the

244

Pearson correlation coefficient to calculate Ci for each residue i (it is important to note that Ci is

245

calculated for each of the 250 perturbation, but that only the maximum Ci is retained). In this

246

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.

248

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

251

alignment approach was followed in this study, using the Kabsch algorithm48 (see Figure S1 A-F

252

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

254

dot product ∆ ∙ ∆-/|∆ ||∆-|, is not expected to be close to 1 because the direction of the

255

initial motion may be entirely different from the overall displacements that characterise the end

256

points of the conformational change. Indeed this very problem has been previously discussed,

257

and alternative approaches offered49,50. Here, we found the correlation between the relative

258

residue displacements to be adequate to classify residues whose perturbation leads to satisfactory

259

and unsatisfactory induced conformational change. The reproducibility of this approach is

260

demonstrated in the supporting documentation (Figure S1 G), where PRS calculations are

261

reproduced using independent MD trajectories for the closed NBD(ATP)-SBD(pep) complex.

262

Furthermore, the selection of residues using equation 1 as the criterion is also illustrated in the

263

supporting documentation (Figure S2), where we graphically exemplify the difference in the

264

displacements by perturbing residues that provide a high and a low Ci value, respectively.


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265

Molecular dynamics simulations

266

All MD simulations were performed with the GROMACS 5.1.2 54–56

51–53

employing the

267

CHARMM 22 force-field (with CMAP corrections)

. Systems were subjected to energy

268

minimization using a conjugate gradient algorithm prior to MD runs. All systems were energy

269

relaxed with 1000 steps of steepest-descent energy minimization. MD simulation on the

270

minimized systems were then carried out in the NVT ensemble at 310 K using the Berendsen

271

temperature coupling algorithm. The final structures were then run in the NPT ensemble at 1 atm

272

and 310 K until volumetric fluctuations stabilized and the desired average pressure maintained,

273

allowing for adequate data points to be collected for further analysis. MD simulations were run

274

for a minimum of 100 ns and maximum of 200 ns until the backbone RMSD of the protein

275

equilibrated with a fluctuation of no more than 3 Å for at least 20 ns. All coordinates were saved

276

at 2 ps intervals for analysis. All MD simulations were carried out using the LINCS algorithm to

277

constrain bond lengths, while the Long-range electrostatic forces were handled using the Fast

278

Particle-Mesh Ewald method (PME), and Van der Waals forces treated with a 0.9 nm cut-off.

279

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


15


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Page 16 of 59

288

Python scripts were used to parse the interaction data to determine the inter-subdomain

289

interactions.


16

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290

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.


17

Open

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Closed


Page 18 of 59

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.


18

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318

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


19


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Page 20 of 59

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


20

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364

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


21


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Page 22 of 59

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


22

Page 23 of 59

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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.


23


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Page 24 of 59

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


24

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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.


25


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Page 26 of 59

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-


26

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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


27


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Page 28 of 59

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


28

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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


29


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Page 30 of 59

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


30

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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


31


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Page 32 of 59

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


32

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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


33


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Page 34 of 59

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


34

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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


35


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Page 36 of 59

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


36

Page 37 of 59

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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


37


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Page 38 of 59

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


38

Page 39 of 59

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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


39


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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


40

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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.


41


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Page 42 of 59

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.


42

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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).


43


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Page 44 of 59

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


44

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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


45


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Page 46 of 59

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


46

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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.


47


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825

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: [email protected]. Note: The authors declare no competing financial interest.


48

Page 49 of 59

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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


49


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

859 860

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Hilser, V. J.; Wrabl, J. O.; Motlagh, H. N. Structural and Energetic Basis of Allostery. Annu. Rev. Biophys. 2012, 41, 585–609.


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Perturbation Response Scanning Reveals Key Residues for Allosteric Control in Hsp70

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