Dynamical Structures of Hsp70 and Hsp70-Hsp40 Complexes.

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Structure. Author manuscript; available in PMC 2017 July 06. Published in final edited form as: Structure. 2016 July 6; 24(7): 1014–1030. doi:10.1016/j.str.2016.05.011.

Dynamical structures of Hsp70 and Hsp70–Hsp40 complexes Thomas Reid Alderson1,2,*, Jin Hae Kim3, and John Lute Markley3 1Department

of Chemistry, University of Oxford, South Parks Road, Oxford OX1 5QY, United

Kingdom 2Laboratory

of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA

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3National

Magnetic Resonance Facility at Madison and Biochemistry Department, University of Wisconsin-Madison, Madison, WI 53706, USA

Abstract

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Protein misfolding and aggregation are pathological events that place a significant amount of stress on the maintenance of protein homeostasis (proteostasis). To prevent and repair protein misfolding and aggregation, cells are equipped with robust mechanisms that mainly rely on molecular chaperones. Two classes of molecular chaperones, heat shock protein 70 kDa (Hsp70) and Hsp40, recognize and bind to misfolded proteins, preventing their toxic biomolecular aggregation and enabling refolding or targeted degradation. Here, we review the current state of structural biology of Hsp70 and Hsp40-Hsp70 complexes and examine the link between their structures, dynamics, and functions. We highlight the power of nuclear magnetic resonance (NMR) spectroscopy to untangle complex relationships behind molecular chaperones and their mechanism(s) of action.

eTOC blurb

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*

Correspondence to [email protected], +447526760105. Author contributions T.R.A., J.H.K., and J.L.M. designed the research and wrote the article.

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Hsp70 and Hsp40 are molecular chaperones that prevent protein misfolding and aggregation. Here, Alderson et al. review the structures and internal dynamics of Hsp70 and its interaction with Hsp40.

Fire and Rain: protein misfolding and molecular chaperones


One of the fundamental principles of molecular biology is the idea that proteins need to assume the correct structure in order to function properly (Dobson, 2003; Fabrizio and Dobson, 2006). The view of what that correct structure entails has been evolving, and we now appreciate that all proteins are dynamic (Henzler-Wildman and Kern, 2007; McCammon et al. 1977) and that the ability to assume multiple conformations over the course of their lifetime is also essential (Baldwin and Kay 2009; Smock and Gierasch, 2009). Most proteins, on their own or with the assistance of folding catalysts, fold to a state of maximal stability. However, proteins can misfold and aggregate under conditions that destabilize their native structures (Hipp et al., 2014). Because protein misfolding and aggregation activate signaling pathways that repress vital cellular functions, prevention of erroneous inter-molecular interactions under native and stressful conditions constitutes a matter of biological significance (Hartl and Hayer-Hartl, 2002; Hipp et al., 2014; Kim et al., 2013). Organisms across all kingdoms have therefore heavily invested in cellular machinery to avert and counteract protein misfolding (Figure 1A), reflecting the evolutionary necessity for maintenance of internal protein homeostasis (proteostasis) (Hartl and Hayer-Hartl, 2002; Hipp et al., 2014; Kim et al., 2013). Cells commit sizeable amounts of energy to their protein quality control system, which consists of molecular chaperones, proteases, and folding catalysts. In humans, disruption of proteostasis can lead to protein misfolding diseases, including Parkinson’s, Alzheimer’s, and type II diabetes (Hipp et al., 2014). A class of molecular chaperones known as heat shock proteins (Hsps) prevent protein misfolding and recognize and degrade polypeptide chains

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that have misfolded. Hsps are generally categorized by their molecular masses: Hsp100s, Hsp90s, Hsp70s, Hsp60s, Hsp40s, and small Hsps. Except for sHsps, which function in a nucleotide-independent manner (Hilton et al., 2013), each family of Hsps interacts with nucleotides or other chaperones to mediate protein folding, refolding, holding, translocation, degradation, or oligomeric assembly (Hipp et al., 2014; Hartl and Hayer-Hartl, 2002). In these processes, molecular chaperones generally function in multi-chaperone complexes that consume ATP and utilize the resultant energy to facilitate the above-mentioned processes (Gao et al., 2015; Hartl and Hayer-Hartl, 2002; Kim et al., 2013; Nillegoda et al., 2015). Studying the structures and dynamics of multi-chaperone complexes at atomic resolution, however, has proven to be challenging, owing to the transient nature of interactions that Hsps engage in, driven by relatively weak binding affinities. Therefore, no single biophysical technique has provided a holistic view of the structure and dynamics of these molecular machines; rather, a multi-faceted approach has been needed to capture this information.

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In this review, we focus on the allosteric molecular chaperone Hsp70 (Figure 1B) and discuss advances in understanding its structure and dynamic interactions with its cochaperone Hsp40 (Figure 1C), co-chaperone and substrate binding, and ATPase cycle (Figure 1A. Although structural investigations have employed Hsp70 proteins from many different species, we limit this review to four model systems: T. thermophilus DnaK, E. coli DnaK, B. taurus Hsc70, and H. sapiens Hsp70. We offer a brief introduction to the structure of Hsp70 and its functional roles during its ATPase cycle, and we discuss what has been learned about nucleotide-dependent control of fast internal dynamics and their impact on global allosteric communication (Horwich, 2014; Mayer, 2013; Zuiderweg et al., 2013). We also review the structure of Hsp40 and the nucleotide-dependent interactions between Hsp70 and Hsp40, whose structural details have been subject to controversy (Jiang et al., 2007; Sousa et al., 2012; Zuiderweg and Ahmad, 2012) and have only recently been resolved (Ahmad et al., 2011; Kim et al., 2014). Throughout this review, we place special focus on insights obtained using nuclear magnetic resonance (NMR) spectroscopy, as this method is exceptionally suitable for studies of dynamics and transient interactions in complex systems like Hsp70 and Hsp40 (Burmann and Hiller, 2015; Rosenzweig and Kay, 2014).

Into the Mystic: Hidden conformational dynamics in the Hsp70 nucleotide binding domain regulate intradomain allostery

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In eukaryotes, Hsp70s reside in the cytosol and in membrane-bound organelles (e.g. mitochondria and endoplasmic reticulum) where they function to ensure that nascent polypeptides fold properly into their correct conformations, that endocytosed clathrin-coated vesicles are unassembled, and that organelle-targeted proteins arrive at their proper translocase machinery (Mayer, 2013). Misfolded and unfolded proteins are recognized by the co-chaperone Hsp40 and escorted to the substrate-binding domain (SBD) of Hsp70, which binds to solvent-exposed hydrophobic residues and allows abnormally folded proteins to re-fold into their native conformations. The binding of ATP to the nucleotide-binding domain (NBD) of Hsp70 and its hydrolysis to ADP allosterically regulate the substrate binding affinity of the SBD: ADP-bound Hsp70 (henceforth ADP-Hsp70) binds substrate


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peptides with 1–10 nM affinity, whereas ATP-Hsp70 has significantly lower affinity for substrates, Kd = 1–10 μM, and faster on/off rates. Reciprocally, substrate binding stimulates ATP hydrolysis through an allosteric mechanism by which both domains of Hsp70 communicate during its functional cycle (Figure 1B) (Mayer, 2013).

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Owing to their diverse and indispensable roles, Hsp70s are among the most highly conserved proteins, and many eukaryotic and prokaryotic genomes encode multiple Hsp70s (e.g. humans have 13 Hsp70s) (Mayer, 2013). However, given their ability to prevent cellular stress and prevent apoptosis, Hsp70s can be harmful: upregulation of Hsp70 expression and activity have been documented in numerous diseases, including various forms of cancer (Murphy, 2013). Given their biological importance, numerous groups have pursued structural studies of Hsp70s, leading structures of their isolated domains, but only recently have high-resolution structures been determined for the ADP- and ATP-bound forms of fulllength Hsp70 (FL Hsp70) (Bertelsen et al., 2009; Kityk et al., 2012; Qi et al., 2013)

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Structural studies revealed that ATP binding in the NBD of Hsp70 leads to large structural rearrangements throughout the protein (Mayer and Bukau, 2005; Zuiderweg et al., 2013). Yet, the mechanism by which allostery propagates between the two domains of Hsp70 remained largely unclear. NBDs of Hsp70s adopt a bi-lobed V-shaped structure, wherein nucleotide binding occurs at the central cleft (Figure 2A). Because nucleotide binding allosterically regulates substrate binding affinity, transmission of an allosteric signal from the NBD to the SBD could traverse any of the NBD sub-domains (IA, IIA, IB, IIB) that directly contact the adenosine and deoxyribose moieties. Crystal structures of the isolated NBD appeared to adopt identical structures in the presence of ATP, ADP, and other nucleotide analogues (Figure 2A) leaving the questions about allostery and motions within the Hsp70 NBD unanswered. Powerful methods to address solution behavior and dynamics, solution-state NMR spectroscopy and molecular dynamics simulations brought to light the “hidden” conformational dynamics within the Hsp70 NBD modulate intra- and interdomain allostery, and, therefore, the function of the chaperone.

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To probe backbone mobility of the NBD in solution, Zhang and Zuiderweg measured residual dipolar couplings (RDCs) (Bax and Grishaev, 2005) in the isolated NDB of B. taurus Hsc70 (Zhang and Zuiderweg, 2004). The authors included equimolar ADP and inorganic phosphate (Pi) to mimic the nucleotide state directly following ATP hydrolysis (ADP-Pi- NBD). The RDC results indicated the presence of conformational flexibility within the isolated NBD that had not been detected in static crystal structures (Figure 1B) (Zhang and Zuiderweg, 2004). The relative orientation of sub-domains IA and IIA in solution were found to deviate significantly from their positions in the crystal structure of ADP-bound Hsc70 NBD (Zhang and Zuiderweg, 2004). Because residues between subdomains IA and IIA are known to interact with the Hsp70 interdomain linker, (Ahmad et al., 2011; Swain et al., 2007) conformational flexibility in this region could provide a conduit for interactions between the NBD and SBD during the Hsp70 ATPase cycle.


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Two subsequent solution-state NMR spectroscopic investigations provided further insight regarding dynamic structural rearrangements in the NBD induced by nucleotide binding (Bhattacharya et al., 2009; Revington et al., 2004). NMR chemical shifts are exceptionally sensitive to local chemical environments, and chemical shift perturbations (CSPs) are routinely used to monitors changes in protein structure. ADP-Pi binding to the isolated NBD of T. thermophilus resulted in significant CSPs to residues in all four subdomains (Figure 2C) (Zhang and Zuiderweg, 2004). Some of the affected residues are distant from the ADPPi binding site, which suggested the presence of allosteric networks and interactions (Zhang and Zuiderweg, 2004). Moreover, analysis of NMR signals from residues in the nucleotidebinding cleft of ADP-Pi-NBD indicated that a subset of these residues exists in two similarly populated conformations: one that resembles ATP-NBD and one that corresponds to ADPPi-NBD (Figure 2C) (Zhang and Zuiderweg, 2004). Since some residues in the active site of ADP-Pi-NBD already sample the ATP-NBD conformation, binding of ATP could occur through a conformational selection mechanism in which the “ATP-like” state of ADP-PiNBD selectively recognizes and binds to ATP. The authors posited that ADP-Pi binding could serve to facilitate sub-domain reorientation and open and close the nucleotide-binding cleft during nucleotide exchange (Zhang and Zuiderweg, 2004).

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Bhattacharya et al. used CSPs and RDCs to monitor allosteric changes in the NBD of DnaK, a member of the Hsp70 family, from T. thermophilus between the ADP- and ATP-bound states (Figure 2B) (Bhattacharya et al., 2009). To model the ATP-bound state, a nonhydrolyzable ATP mimic (AMPPNP) was employed. AMPPNP binding affected residues throughout the NBD, including sites in sub-domains IA and IIA near the interdomain linker and Hsp40 binding sites (Bhattacharya et al., 2009). Bhattacharya et al. detected a large difference between the orientation of sub-domain IIB in ADP-NBD and AMPPNP-NBD, which offered insight into the mechanism by which nucleotide-exchange factors (NEFs) exchange ADP for ATP. Crystallographic analyses of NEF-bound NBDs had revealed outward rotation of sub-domain IIB, suggesting that NEFs actively mediate structural rearrangement in the NBD to facilitate nucleotide exchange (Harrison et al. 1997). However, because ADP-NBD and AMPPNP-NBD exhibit different orientation of sub-domain IIB, it seems more likely that NEFs recognize the already-rotated conformation of ADP-NBD rather than induce structural changes (Bhattacharya et al., 2009). Indeed, recent work has verified that the BAG family of NEFs bind tighter to ADP- and apo-Hsp70 (Rauch and Gestwicki, 2014) than to the ATP-bound form, verifying the predictions put forth by (Bhattacharya et al., 2009). Moreover, interruption of the BAG-Hsp70 interaction has proved to be a potential target for cancer therapeutics (Li et al., 2015).

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These studies also revealed that Hsp70 NBD undergoes dynamical changes in response to nucleotide binding (Bhattacharya et al., 2009; Revington et al., 2004; Zhang and Zuiderweg, 2004). NMR-detected enhanced shearing motions within the sub-domains of ADP-NBD are indicative of interconversion between two (and likely more) conformations (Figure 2B and 2C) (Bhattacharya et al., 2009; Revington et al., 2004) Such conformational dynamics could serve functional purposes, such as facilitating the opening of the nucleotide-binding cleft for nucleotide exchange or remodeling the energy landscape of sub-domain orientations in response to co-chaperone or substrate interactions (Bhattacharya et al., 2009;, Revington et al., 2004). Structure. Author manuscript; available in PMC 2017 July 06.

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Nucleotide-dependent structural changes in the isolated Hsc70 NBD were also investigated using all-atom molecular dynamics (MD) simulations (Woo et al., 2009). In agreement with previous NMR results, the sub-domain IA/IIA interface underwent relatively large structural rearrangements upon ATP binding (Bhattacharya et al., 2009; Revington et al., 2004). Moreover, ATP binding diminished sub-domain motions and overall flexibility with respect to the fluctuating, dynamic apo state (Woo et al., 2009). Building upon this work, recent allatom MD simulations of apo, ADP-, and ATP-bound Hsc70 NBD revealed nucleotidemediated modulation of NBD dynamics at specific localized sites coinciding with those identified by solution-state NMR spectroscopy. (Gołaś et al., 2015). By utilizing a graph theory-based algorithm that models protein structures as networks and their residues as nodes, Liu et al. identified “central residues,” or residues with high probabilities of comprising the shortest possible allosteric pathway, in the NBD of open (NEF-bound and apo) and closed (ATP-bound) Hsc70 (Liu et al., 2010). These results were in general agreement with NMR spectroscopic data and demonstrated the ability of computational methods to identify protein allosteric networks from structural information.

Stuck in the Middle with You: structure and dynamics of Hsp70’s substrate-binding domain

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In terms of its structure, the Hsp70 SBD is remarkable: it has a unique topology when compared to the more than 100,000 protein structures been described to date (Zuiderweg et al., 2013). A crystal structure of the isolated SBD of DnaK (E. coli) bound to a substrate peptide (NRLLLTG; “NR”) was solved in 1996,(Zhu et al., 1996) and Zuiderweg and coworkers determined a solution-state NMR spectroscopy-based structural ensemble of the self-bound SBD in 1998.(Wang et al., 1998) Two additional structural ensembles of the DnaK (E. coli) apo-SBD determined by solution-state NMR followed shortly thereafter (Pellecchia et al., 2000; Stevens et al., 2003). Because Gierasch and coworkers recently reviewed the topic of substrate binding to Hsp70 (Clerico et al., 2015), we include only a brief description of the structure of its SBD as the framework for our detailed discussion of its dynamics.

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The 25 kDa SBD comprises two sub-domains (Figure 1B): a 10 kDa β-domain (SBDβ), composed of two antiparallel β-sheets of four strands each (β1–β8; Figure 3A), and a 15 kDa α-helical lid domain (SBDα) that contains four α-helices (helix A–D) and a disordered Cterminal tail. Substrate binding occurs between β-sheets in a hydrophobic cleft, and the substrate peptide interacts primarily with hydrophobic groups in strands β1, β3, and β4 (Figure 3A) (Clerico et al., 2015; Pellecchia et al., 2000; Zhu et al., 1996). The substratebiding cleft is capable of enclosing a seven-residue peptide, and thus Hsp70 is believed to interact with exposed patches of ca. seven residues in non-natively folded proteins. Largescale structural rearrangements occur throughout the SBD upon the binding of substrate and/or ATP to Hsp70 (Clerico et al., 2015; Kityk et al., 2012; Pellecchia et al., 2000; Stevens et al., 2003; Wang et al., 1998). 15N

NMR spin relaxation experiments have demonstrated that both the isolated DnaK and Hsc70 SBDs are quite rigid on the ps-ns timescale, with residues in the β3 strand and in


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loops at the substrate binding cleft displaying evidence of conformational exchange on the μs-ms timescale (Pellecchia et al., 2000; Stevens et al., 2003; Wang et al., 1998). These μsms motions in the β3 strand and substrate binding cleft loops are abolished upon substrate binding (Pellecchia et al., 2000; Stevens et al., 2003), leading to the suggestion that they serve as a “conformational switch” between apo and substrate-bound SBD.

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Since 1989, it has been known that short, hydrophobic peptides bind to Hsp70s and stimulate ATPase activity (Flynn et al., 1989; Gragerov and Gottesman, 1994). Initial studies of the mechanism of substrate binding employed phage display arrays and peptide libraries to screen for amino acid preferences among peptides that bind to Hsp70. No consensus amino acid recognition sequence arose, yet a strong preference was evident for aliphatic (Leu, Ile, and Val), aromatic (Phe and Tyr), and basic (Arg and Lys) amino acids (Fourie et al., 1994; Rudiger et al., 1997). Aliphatic residues were favored in the core of the peptide sequence, and aromatic and basic residues were favored in the flanking regions (Rudiger et al., 1997). The crystal structure of DnaK (E. coli) bound to NRLLLTG (NR) indicated that the central leucine (termed the 0-site) binds to a deep, hydrophobic pocket in the SBD.(Zhu et al., 1996) The three sites on either side (−3, −2, −1, 0, +1, +2, +3) form hydrogen bonds via their backbone atoms. The positions of basic residues with respect to the central leucine were found not to be critical (Clerico et al., 2015). Moreover, the bound peptide can also exist in a register-shifted conformation, with the central Leu in site -1 (Zahn et al., 2013).

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In order to determine if Hsp70s recognize small regions of unfolded or misfolded proteins in a manner similar to the binding and recognition of peptides, two protein substrates σ32 proteins, and apo-myoglobin, were employed. DnaK interacted with the region of each intact protein corresponding to its preferential small peptide substrate (Rodriguez et al., 2008; Vega et al., 2006). Additionally, two recent studies investigated DnaK binding to SRC homology 3 domain (SH3) and human telomere repeat binding factor 1 (hTRF1) (Lee et al., 2015; Sekhar et al., 2015). While both of these substrate proteins are natively folded, they also contain a population of unfolded conformations (e.g. hTRF1 is 4% unfolded at 308 K) under native conditions, thereby enabling a unique assessment of conformational selection by DnaK. DnaK was found to reversibly and selectively bind to the unfolded state of these substrates, and the conformation of the bound substrates was not affected by the presence or absence of nucleotide (Lee et al., 2015; Sekhar et al., 2015). The conformation of DnaKbound hTRF1 resembled that of the natively unfolded state, whereas DnaK-bound SH3 adopted multiple conformations that were different from the unfolded state (Lee et al., 2015; Sekhar et al., 2015).

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Interestingly, in a follow-up study of DnaK binding to hTRF1, Sekhar et al. probed residual tertiary structure in the bound substrate protein. The authors used NMR to measure transient long-range contacts in natively unfolded, urea unfolded, and DnaK-bound hTRF1 (Sekhar et al., 2016). Long-range contacts were observed in the natively unfolded state, but these contacts were quenched upon urea unfolding and DnaK binding, suggesting that ureaunfolded and DnaK-bound hTRF1 adopt similarly extended conformations. In contrast, the residual secondary structure of DnaK-bound hTRF1 was more similar to the natively unfolded state. Therefore, their results indicate that DnaK binding to substrate proteins disrupts long-range contacts and promotes extended conformations, but enables formation of


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local secondary structure that could bias the folding pathways of bound targets upon release of DnaK (Sekhar et al., 2016). NMR data for DnaK bound to hTRF1 was similar to that of the peptide-bound form, suggesting that the chaperone itself adopts similar conformations when bound to either small peptides or full-length proteins (Sekhar et al., 2015). However, a disulfide fixation- and EPR spectroscopy-based investigation of DnaK (E. coli) revealed that the conformation of the SBD in the presence of an unfolded protein was different from that in the presence of a short substrate peptide (Schlecht et al., 2011). Helix B was shown to adopt at least three conformations (Figure 3B), none of which closed over the bound proteins in the manner observed with SBD bound to heptapeptides. Therefore, it appears that more investigation is needed to define the mechanistic details of Hsp70 binding to peptides and native substrates (Clerico et al., 2015; Lee et al., 2015; Sekhar et al., 2015).

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To investigate the potential link between allostery and internal SBD dynamics, Zhuravleva and Gierasch used NMR spectroscopy and MD simulations to study the coupling between intra- and interdomain allostery (Figure 3C) (Zhuravleva and Gierasch, 2015). The authors employed a suite of DnaK SBDβ single point-mutants and provided mechanistic insight regarding the role of internal dynamics in the SBD of DnaK. The hydrophobic core around β-strands β5, β7, and β8 was found to control intradomain allostery and the resultant ability to dock onto the NBD and to bind substrate. Loops L3,4 and L5,6 were identified as key regulators of interdomain allostery: truncation of the L3,4 loop resulted in structural changes to the entire SBD, whereas mutations to residues in L5,6 led to changes in the μs–ms dynamics (Figure 3C) (Zhuravleva and Gierasch, 2015).

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In summary, this study demonstrated that the conformational dynamics within the SBDβ domain of Hsp70 are under thermodynamic and kinetic control by motions in the L3,4 and L5,6 loops. Fluctuations in these loop regions create a transient population of the SBD that allows for substrate binding and propagation of intra- and interdomain allostery, which is crucial to the function of Hsp70s. Moreover, the entropically-driven conformational flexibility of SBDβ likely enables interaction with a wide variety of substrate shapes and sizes (Zhuravleva and Gierasch, 2015).

Can’t You Hear Me Docking? Role of the interdomain linker

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Despite elegant structural studies of isolated domains, structural and mechanistic details of interdomain communication and allostery in FL Hsp70 remained elusive. Combined results from various biophysical techniques demonstrated that ATP binding stimulates a large structural rearrangement in Hsp70 leading to a more compact structure (Buchberger et al., 1995b; Han and Christen, 2003; Moro et al., 2006; Liberek et al., 1991; Palleross et al., 1992; Shi et al., 1996; Wilbanks et al., 1995)., In X-ray diffraction maps of Hsp70s, the lack of electron density arising from the interdomain linker hindered elucidation of its structural role. Results from hydrogen-deuterium exchange experiments detected by mass spectrometry (HDX-MS) and limited proteolysis indicated, respectively, that ATP-binding to DnaK protects interdomain linker residues from exchange and cleavage (Buchberger et al., 1995b; Liberek et al., 1991; Rist et al., 2006). Therefore, it was suggested that the linker is


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solvent exposed and flexible in both apo and ADP states, yet structured or stabilized in the ATP state (Buchberger et al., 1995b; Liberek et al., 1991; Rist et al., 2006). Clear structural evidence for such a structural transition, however, is still lacking.

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In order to examine the functional role of the Hsp70 interdomain linker, Vogel et al. examined truncated variants of E. coli DnaK containing the NBD and a partial interdomain linker (Vogel et al., 2006a). This variant displayed a 40-fold increase in basal ATPase activity in comparison to either FL DnaK or the isolated NBD. Similarly, HscA truncated at the end of the linker had much higher ATPase activity than the isolated NBD; in addition, ATPase activity of the isolated NBD was stimulated by adding a peptide with the linker sequence (Alderson et al., 2014). Mutagenesis studies of E. coli DnaK have identified two surface-exposed, positively charged residues (K155 and R167) in sub-domain IA that appear to be involved in interdomain communication via interaction with the negatively charged aspartate residues that flank hydrophobic leucine residues within the linker (388DLLLD393) (Vogel et al., 2006a). Protein variants of FL DnaK, designed from analysis of crystal structures of Hsc70 NBDs, led to the identification of another subset of residues in subdomain IA that mediate interdomain allostery (P143, R151, and E171). In both of these studies, control experiments demonstrated that the DnaK variants (P143G, R151A, K155A, R167A, E171Q) were structurally sound and fully functional in nucleotide binding and exchange assays. However, this group of sub-domain IA variants failed to refold substrate proteins and was deficient in substrate- and Hsp40-mediated enhancement of ATPase activity. These results collectively support a mechanism by which the interdomain linker interacts with sub-domain IA in an allosteric manner (Vogel et al., 2006a).

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In detailed NMR spectroscopic investigation of the functional role of the Hsp70 interdomain linker, the Gierasch group found that the linker interacts with the NBD in a nucleotidedependent manner (Swain et al., 2007; Zhuravleva and Gierasch, 2011). ATP binding to an NBD variant with a partial interdomain linker (DnaK392) resulted in structural rearrangement throughout the NBD (Swain et al., 2007), whereas structural changes in ADP-DnaK392 were localized specifically to the interdomain linker binding site. A hydrophobic patch between sub-domains IA and IIA was determined to comprise the interdomain linker binding site, which had previously been shown to be sensitive to nucleotide binding (Zhang and Zuiderweg, 2004; Zuiderweg et al., 2013).

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The conformation of the interdomain linker was shown to depend on the presence and nature of the bound nucleotide. Linker residues in apo-DnaK392 were flexible and disordered, in the presence of ADP, the linker was only partially disordered, and in the presence of ATP these residues formed a β-strand (Zhuravleva and Gierasch, 2011). Therefore, residues in the interdomain linker are exquisitely sensitive to ADP or ATP binding, propagate allosteric signals throughout the NBD in the presence of ATP, and enable direct communication between the NBD and SBD in a nucleotide-dependent fashion (Swain et al., 2007). The combination of all of these insights formed the basis for a proposed allosteric cycle. The linker remains flexible and solvent-exposed in apo-DnaK, and only transiently interacts with the NBD in ADP-DnaK so as to minimize contact between the NBD and SBD (Swain et al., 2007; Zhuravleva and Gierasch, 2011). ATP binding, however, induces a global structural


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rearrangement in DnaK, in which the linker forms a β-strand and docks at the hydrophobic sub-domain IA/IIA interface (Figure 1B) (Swain et al., 2007; Zhuravleva and Gierasch, 2011). Interestingly, an analysis of co-evolving amino acid residues in Hsp70 indicated that residues in sub-domains IA and IIA modulate interdomain signal transduction and binding of Hsp40 (General et al., 2014). Such exquisite coupling of linker and ATP binding locally remodels the orientations of NBD sub-domains and alters their internal dynamics. Global communication between the NBD and SBD is, in turn, regulated by nucleotide-induced binding and dissociation of the interdomain linker binding (Swain et al., 2007; Zhuravleva and Gierasch, 2011).

At Last: structures of two-domain Hsp70 constructs

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Previously solved structures of isolated Hsp70 domains in their apo, substrate-, and nucleotide-bound states offered mechanistic insight into their functions. Moreover, these isolated domains provided numerous avenues for structure-function experiments, including creation of the frequently used ATPase deficient DnaK variant, DnaK(T199A). However, elucidation of structural details regarding interdomain communication was hindered by failures of FL Hsp70 to crystallize.

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A crystal structure of a truncated, two-domain apo-Hsc70 construct (residues 1–554, E213A and D214A) delivered the first high-resolution, structural evidence for this chaperone’s allosteric cycle (Jiang et al., 2005). Both the NBD and the SBD were included in this truncated Hsc70 construct, which excluded the 10 kDa helical lid sub-domain in the SBD known to promote Hsc70 aggregation. The NBD was more open in this two-domain structure than in structures of isolated ADP- and ATP-NBDs (Jiang et al., 2005) and, instead, resembled structures of NEF-bound NBDs (Harrison et al., 1997). Surprisingly, although the Hsc70 construct was in the apo-form, electron density was observed for the solvent-exposed interdomain linker, and residues in the SBD were docked at the linker binding site between NBD sub-domains IA and IIA (Jiang et al., 2005). The observation that docking occurred between the NBD and SBD contradicted previous biophysical assays indicating that apo-Hsc70 adopts an extended conformation in the apo and ADP-bound states (Wilbanks et al., 1995). Thus, either the truncation of Hsc70 or the two dramatic mutations in the NBD-SBD interface, may have led to the observed interactions. NBD-SBD contacts observed in this X-ray structure were not found in solution studies of WT apoHsc70 (Zuiderweg et al., 2013).

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A solution-state NMR study focused on the ADP-Pi-bound form of a truncated variant of T. thermophilus DnaK (residues 1–501, ΔT422, A423E) (Revington et al., 2005). NMR signals arising from residues in the nucleotide-binding site could not be observed, likely due to conformational dynamics on the μs-ms timescale. Residues in the interdomain linker were found to adopt β-strand conformations (Revington et al., 2005). Peak width and RDC measurements from residues in the NBD and SBD indicated that these domains were relatively rigid, and it was observed the NBD was in contact with either the SBD or the interdomain linker (Revington et al., 2005). It should be noted, however, that the NMR data were collected at 55 °C, approximately 25 °C below the optimal physiological temperature


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of 80 °C. Thus, the diminished temperature could have influenced interdomain contacts and internal dynamics in this thermophilic chaperone.

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By combining X-ray crystallography and solution-state NMR spectroscopy, Bertelsen et al. determined high-resolution structures of peptide-bound WT ADP-DnaK and ADP- DnaK (1-605) (Figure 1B, top) (Bertelsen et al., 2009). These structures excluded the C-terminal 33 residues that were shown to be dynamic and disordered. Analyses of NMR spectra from peptide-bound WT ADP-DnaK (1-605) indicated that the NBD and SBD move independently of one another and that interdomain linker residues are highly flexible and adopt random coil conformations. RDC and paramagnetic relaxation enhancement (PRE) measurements reported on the relative orientation of the NBD and SBD, which were loosely coupled and capable of moving in cones of ± 35° opening angles, yet adopting a timeaveraged structure compatible with that presented at the top of Figure 1B (Bertelsen et al., 2009).

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Serving as initial points in the structure calculations, high-resolution crystal structures of GrpE-bound NBD and NR-bound SBD were combined with RDC and PRE NMR data to define the relative orientations of NBD sub-domains (Bertelsen et al., 2009). In support of the previous NMR studies of T. thermophilus and E. coli DnaK, (Bhattacharya et al., 2009; Revington et al., 2004) the RDC data collected by Bertelsen et al. indicated that the orientation of ADP-NBD sub-domain IIB differs by 20° or more from those of crystallized NBD sub-domains (Bertelsen et al., 2009). Long-range distance information from PRE data indicated the presence of transient contacts between sub-domain IIA of the NBD and the β2 and β3 strands of the SBD. Thus, from the NMR data presented by Bertelsen et al. it was clear that ADP-peptide-bound DnaK adopts an extended conformation wherein each domain, while relatively independently mobile, senses the presence of the other, thus limiting motion to cones of ± 35° opening angles (Bertelsen et al., 2009). In 2012, Mayer and coworkers successfully crystallized ATP-bound DnaK(T199A) from E. coli (Kityk et al., 2012). The final 32 disordered residues of the protein were removed, and cysteine residues were introduced in the NBD (E47C) and helix B of the SBD (D526C), resulting in a fully functional triple mutant, DnaK T199A/E47C/D526C. In the presence of ATP, DnaK E47C/D526C formed a stable, intramolecular disulfide bond, which suggested that the NBD and helix B of the SBD contact one another in this state; however, this disulfide bond also formed in nucleotide-free and ADP-bound DnaK E47C/D526C. This result, while surprising, indicated that apo- and ADP-DnaK sample ATP-DnaK-like conformations that place the NBD and helix B of the SBD in relatively close proximity, although these conformations were sampled less frequently than in ATP-DnaK.

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Striking differences were observed between the structure of ATP-DnaK T199A/E47C/ D526C and those of ADP-bound DnaK (Kityk et al., 2012). Whereas such structural rearrangements had been predicted on the basis of lower-resolution biophysical methods, only now were they detected at high resolution. Notably, as described above, in ADP-bound DnaK, the NBD and SBD remain relatively independent of one another, whereas ATP binding promotes significant conformational changes and domain-domain interactions. In ATP-DnaK, SBDβ establishes contacts with NBD sub-domains IA, IB, and IIA, while


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SBDα, which comprises four helices (A, B, C, and D), docks onto NBD sub-domain IB via helix A (Kityk et al., 2012). Numerous electrostatic and polar interactions between NBD, interdomain linker, and SBD residues facilitate NBD-SBD docking. Whereas the structures of isolated Hsp70 NBD bound to various nucleotides (including ATP) were nearly identical (Figure 2A), the NBD in the context of ATP-bound FL Hsp70 displayed large structural rearrangements (Kityk et al., 2012). As indicated previously, (Mayer and Bukau, 2005; Zuiderweg et al., 2013) all four sub-domains of the NBD are involved in coordinating Mg2+ and ATP; however, ATP binding in the full-length protein induces sub-domain rotations to place sub-domain IA closer to SBDβ, move sub-domain IIB to position it above the ATP-binding cleft, and create a hydrophobic cleft for interdomain linker binding between sub-domains IA and IIA (Kityk et al., 2012).


As with the NBD, the SBD of ATP-bound FL Hsp70 exhibited extensive structural differences from that of the isolated domain (Kityk et al., 2012). Alterations to β-strand orientations and inter-strand hydrogen bonding patterns of the upper and lower β-sheets culminated in closure of the SBDβ hydrophobic binding pocket and an apparent increase in conformational flexibility of the substrate-enclosing loops (L3,4 and L5,6). These structural features were consistent with biochemical data showing that ATP-Hsp70 binds to substrate with lower affinity and faster on/off rates than apo- or ADP-Hsp70. Additionally, kinetic measurements of fluorescently labeled, double cysteine DnaK variants resolved the order in which ATP-induced structural rearrangements occur (Kityk et al., 2012). First, the interdomain linker inserts into the hydrophobic groove between sub-domains IA and IIA; second, SBDβ docks onto the NBD; and third, helix B relocates and binds to the NBD. Fluorescence measurements in the presence of substrate peptide revealed that, by the time helix B contacts the NBD, the peptide has already been released (Kityk et al., 2012). In 2013, Qi and coworkers solved the crystal structure of E. coli ATP-bound DnaK (Figure 1B, bottom) by introducing two stabilizing mutations: the above-mentioned T199A and truncation of loop 3,4 (L’3,4) (Qi et al., 2013). Although the structure of this variant was very similar to that of Kityk et al. (Kityk et al., 2012) (backbone RMSD = 0.44 Å), it exhibited upward protrusions of L’3,4 and L5,6 indicative of conformational flexibility in this region. Although these differences could have been the result of removing residues T428 and Q433, whose presence stabilizes hydrogen bonds within L3,4 and between L3,4 and L5,6, (Qi et al., 2013) prior solution-state NMR spectroscopic studies of the SBD showed that L3,4 and L5,6 are dynamic on the millisecond timescale (Pellecchia et al., 2000; Wang et al., 1998) Thus, the crystal structures of Qi et al. and Kityk et al. may have captured these loops in one of their multiple accessible conformations (Kityk et al., 2012; Qi et al., 2013).

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You Ain’t Seen Nothing Yet: insight into interdomain allostery and the mechanism of Hsp70 action Given that the accumulated structural insights into Hsp70 have documented structural flexibility and large conformational changes that accompany the Hsp70 functional cycle, the major question in the field revolves around allostery and what orchestrates these changes. Molecular simulations of FL Hsp70s (human and E. coli) played an important role in Structure. Author manuscript; available in PMC 2017 July 06.

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modeling the allosteric network involved in propagating nucleotide-induced structural transitions (Chiappori et al., 2012; General et al., 2014; Gołaś et al., 2012; KA, 2010; Nicolaï et al., 2010, 2013). Here, we discuss only recent simulations of the bacterial chaperone, as previous studies have been summarized (Gołaś et al., 2015; Gołaś et al., 2012)

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Building on work by Gołaś and coworkers that reported so-called “initiating” structural transitions in the presence of ADP and substrate peptide, which promoted dissociation of SBDα from SBDβ and exposure of the hydrophogic interdomain linker site, Chiappori et al. identified DnaK residues that initiate structural rearrangements between the ATP- and ADPbound states (Chiappori et al., 2012). In all simulations, they observed that when ATP was modeled into the structure of ADP-DnaK, SBDβ docked onto the NBD. This interdomain docking accompanied or followed local unfolding of the SBD helical turn (Chiappori et al., 2012), consistent with the formation of a uniform SBD helix as described above. In agreement with solution-state NMR results, the dynamics of ADP-NBD subdomain IIB were quenched in the ATP complex, and sub-domain IIB formed a stable contact with subdomain IB. Additionally, Chiappori et al. modeled ADP into the structure of ATP-bound DnaK, which was based on an ATP-Hsp110 homology model (Chiappori et al., 2012; Gołaś et al., 2012). Both the ADP- and ATP-DnaK MD trajectories produced a similarly structured “intermediate” state, in which SBDα had dissociated from SBDβ and SBDα–NBD contacts were absent (Chiappori et al., 2012).

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The allosteric pathway describing the mechanism of ATP hydrolysis in E. coli DnaK was recently analyzed using mutagenesis and functional assays (Kityk et al., 2015). ATP binding was determined to rotate lobe I of the NBD with respect to lobe II, which allows for exposure of the hydrophobic patch at the sub-domain IA/IIA interface and concomitant binding of the interdomain linker (Kityk et al., 2015). Contacts between SBDβ and I168 and D326 in the NBD provide means to open the substrate-binding cleft and eject bound substrate. Incoming substrate, in turn, interacts with the SBD, which facilitates enhancement of ATP hydrolysis via signal propagation to D148 and subsequent rotation of lobe I (Kityk et al., 2015).

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Zhuravleva et al. applied methyl-TROSY NMR experiments (Kay, 2011; Rosenzweig and Kay, 2014) to investigate interdomain allostery in DnaK (Zhuravleva et al., 2012). This study probed allostery using multiple two-domain constructs in which distinct portions of the SBD were removed to dissect the roles of these regions (Zhuravleva et al., 2012). In corroboration with prior NMR results (Swain et al., 2007; Zhuravleva and Gierasch, 2011), both apo and ADP-bound states closely recapitulated NMR spectra from the isolated domains (Figure 4C) (Zhuravleva et al., 2012), indicating that individual domains within the two-domain construct behave like “beads on a string.” The addition of ATP led to massive structural rearrangements and dynamical changes on the μs-ms timescale (Zhuravleva et al., 2012). Mapping these structural and dynamical changes onto a homology model of DnaK demonstrated that the affected residues were diffuse throughout the NBD and at the NBDSBD interface. Analysis of the structural changes led the authors to conclude that the two “end-points” in DnaK’s allosteric cycle rely upon a distinct pair of interactions: NBD-SBDβ contacts in the domain-docked (ATP-bound) state and helix B-SBDβ contacts in the domainundocked (ADP-bound) state (Zhuravleva et al., 2012).


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The ATP-substrate-bound state of DnaK yielded NMR signals at positions not observed in the ATP-bound state, suggesting that substrate binding leads to formation of a distinct conformation (Figure 4A) (Zhuravleva et al., 2012). In this allosterically-active state, the interdomain linker was found to oscillate rapidly between the linker-bound and linkerunbound conformations (Figure 4B, C, and D) (Zhuravleva et al., 2012). The SBD remained relatively unperturbed from its isolated substrate-bound state, which suggested that the SBD dissociates from the NBD upon the binding of both ATP and substrate. Furthermore, this work indicated that stabilization or destabilization of the NBD–SBDβ or helix A-SBDβ interfaces modulates substrate-binding-induced docking or undocking of the NBD and SBD (Zhuravleva et al., 2012). In ATP- and substrate-bound DnaK, therefore, a “tug-of-war” between orthogonal interfaces determines the allosterically-active state of the molecule (Figure 4D).


Conformational heterogeneity in Hsp70s has been addressed by Förster resonance energy transfer (FRET) measurements on BiP (Marcinowski et al., 2011) (ER-based Hsp70 from Mus musculus), Ssc1 (Mapa et al., 2010; Sikor et al., 2013) (mitochondrial Hsp70 from S. cerevisiae), and DnaK (Mapa et al., 2010). FRET data demonstrated surprising conformational heterogeneity within the SBD during the ATPase cycle of these Hsp70s. Fluorophores were placed at sites in sub-domain IIA, SBDα, and SBDβ, such that interdomain and intra-SBD distances could be measured. Notably, at least three distinct SBD conformations were observed for ADP-bound Ssc1 (Mapa et al., 2010; Sikor et al., 2013) and BiP (Marcinowski et al., 2011). As expected, ATP binding led to higher ensemble FRET efficiency between fluorophores in the Ssc1 NBD and the SBD. Collectively, these results dissected the Hsp70 allosteric pathway in unprecedented detail, and a relationship was established between internal dynamics, nucleotide and substrate binding, and interdomain contacts.

Two Princes: Interactions between bacterial Hsp70s and Hsp40 cochaperones

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Hsp40 co-chaperones function together with Hsp70 chaperones to maintain cellular proteostasis (Figures 1A and 1C) (Cyr and Ramos, 2015; Hartl and Hayer-Hartl, 2002; Kampinga and Craig, 2010). By binding to misfolded polypeptides and delivering them to Hsp70 SBDs, Hsp40s mediate the re-folding process while also stimulating ATPase activity in the NBDs of Hsp70s. This mechanism, which allows for dual binding of Hsp40 and substrate peptide, creates a synergistic enhancement in the rate of Hsp70-mediated ATP hydrolysis, with stimulation over basal levels in some cases by 1,000-fold (Figure 1A) (Cyr and Ramos, 2015; Hartl and Hayer-Hartl, 2002; Kampinga and Craig, 2010; Vickery and Cupp-Vickery, 2007). Family members of the Hsp40 class of proteins are categorized by their domain organization (Figure 1C). All members contain a J-domain of ~approximately 70-residues; in type I and type II Hsp40s this domain is located at the N-terminus, whereas in type III Hsp40s, the Jdomain can be found anywhere in the protein (Kampinga and Craig, 2010). J-domains comprise four helices (helix I–IV) and contain a highly conserved His-Pro-Asp (HPD)


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tripeptide motif. Mutation of the HPD motif has been shown to abolish the ability of Hsp40s to stimulate the ATPase activity of their cognate Hsp70s (Kampinga and Craig, 2010). At their C-termini, type I and type II Hsp40s have peptide-binding domains that are separated from the J-domain by a linker rich in glycine and phenylalanine residues (GF linker). Type I Hsp40s contain zinc-finger (ZF) motifs between the GF linker and peptide-binding domain that distinguish them from type II and type III Hsp40s, which lack ZF motifs.


Many Hsp40s, including DnaJ, are known to exist as dimers (Figure 1C) (Barends et al., 2013; Kampinga and Craig, 2010; Shi et al., 2005). Numerous crystal and solution structures of Hsp40 J-domains and other fragments have been published (Huang et al., 1999a; Pellecchia et al., 1996; Stark et al., 2014), including a model of full-length E. coli DnaJ (Barends et al., 2013). Yet, despite the existence of these Hsp40 structures, structural information regarding Hsp40-Hsp70 complexes lagged for many years. The crystal structure of a disulfide-linked auxilin J-domain:apo-Hsc70-NBD complex was published in 2007 (Jiang et al., 2007). This disulfide-linked complex, was active and included direct contacts between the auxilin HPD motif and the Hsc70 NBD (Jiang et al., 2007). Closer examination of this structure noted the existence of an alternative auxilin-NBD interface that was formed by crystal packing. This alternative interface contained a larger buried surface area (582 Å2) than the covalently-linked J-domain:NBD complex (372 Å2) (Jiang et al., 2007; Sousa et al., 2012; Zuiderweg and Ahmad, 2012; Zuiderweg et al., 2013), which suggested that the covalent auxilin-NBD complex could have trapped an otherwise sparsely populated state that is influenced by non-native contacts (Jiang et al., 2007; Sousa et al., 2012; Zuiderweg and Ahmad, 2012; Zuiderweg et al., 2013). As discussed below, the bacterial Hsp40-Hsp70 interaction in the absence of nucleotide and in the presence of ADP turned out to be highly dynamic, thus supporting the view of multiple states and contact points (Figure 5) (Ahmad et al., 2011; Zuiderweg et al., 2013).

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Landry and coworkers used NMR CSP mapping and line broadening analyses to probe the interaction between the J-domain of DnaJ and FL DnaK in solution (Greene et al., 1998). The results indicated that the binding site for both ADP- and ATP-DnaK comprised helix II and the HPD motif of DnaJ75 (Greene et al., 1998). This conclusion was supported by mutagenesis and functional assays conducted by Gross and coworkers, which collectively demonstrated that DnaJ(D35N) does not interact with ATP-DnaK (Suh et al., 1998). Furthermore, mutations in the HPD motif and nearby residues of DnaJ were shown to abolish its ability to stimulate the ATPase activity of Hsp70 (Suh et al., 1998). These studies led to the prevailing model for the interaction between DnaJ and DnaK, which consists of HPD-motif mediated docking of the negatively charged DnaJ helix II onto the positively charged region of the DnaK NBD near R169 (Greene et al., 1998; Suh et al., 1998). The direct role of the GF linker in this interaction remained unclear, although NMR relaxation studies had indicated this region could be involved in regulating the internal dynamics of the J-domain (Huang et al., 1999b). From an NMR investigation of the non-covalent complex of E. coli DnaJ70 and ADPpeptide-DnaK, Zuiderweg and coworkers concluded that the HPD motif of DnaJ does not directly contact ADP-peptide-DnaK (Figure 5A, B, and C) (Ahmad et al., 2011). This claim was supported by PRE measurements, which revealed that DnaK was more than 15 Å distant


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from residues in the HPD motif of DnaJ. Ahmad et al. instead determined that helix II of DnaJ70 scans the surface of ADP-peptide-DnaK in a highly dynamic manner (Ahmad et al., 2011). NMR data were used in a restrained MD simulation to visualize the mobile complex, the results of which are shown in Figure 5B (Ahmad et al., 2011).

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Direct evidence of a native Hsp40–Hsp70:ATP interaction with full-length proteins in solution came from a study by Markley and coworkers (Kim et al., 2014). The authors employed NMR to study binding between a specialized Hsp40 (HscB) and an ATP-bound Hsp70 (HscA). In order to model the ATP-bound state of HscA, Kim et al. included the well-known Hsp70 mutation (T212V in HscA, T199A in DnaK) that minimizes ATP hydrolysis but still enables conformational changes upon ATP binding (Kim et al., 2014). NMR data indicated that ATP-HscA(T212V) binds directly to the HPD motif of HscB (Figure 5D, bottom), and a second binding site with lower affinity was identified in the Cterminus. The interaction between HscB and HscA was nucleotide-dependent, as the observed effects did not occur or were severely diminished in both the apo- and ADP-bound state (Figure 5D, top and middle). In addition, mutation of HPD to AAA abolished the functional interaction between ATP-HscA and HscB, which demonstrated that the HPD motif was directly responsible for binding to ATP-HscA and stimulating ATPase activity (Kim et al., 2014). Interestingly, titrations with HscB and the NBD of HscA(T212V) demonstrated that the isolated NBD – in the absence of nucleotide or in the presence of excess ADP or ATP – did not bind to the J-domain. Therefore, both the SBD and NBD of HscA are required to elicit functional interactions with HscB, and the J-domain and HPD motif of HscB underlie its ability to sense the nucleotide-bound state of HscA (Kim et al., 2014).

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With a Little Help from My Friends: Hsp70 and its complexes with cochaperones

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Early investigations of Hsp70 revealed that the protein was prone to form high molecular weight species (Benaroudj et al., 1995; Carlino et al., 1992; Kim et al., 1992; Osipiuk et al., 1993; Schönfeld et al., 1995; Schröder et al., 1993). This oligomerization was subsequently determined to originate from binding of the SBD of one molecule to the interdomain linker of another (Aprile et al., 2013). E. coli DnaK dimers and multimers were found to efficiently bind and hydrolyze ATP, but were deficient in their interaction with DnaJ and ability to refold denatured substrate (Thompson et al., 2012). Heat shock promoted oligomerization of Hsp70 in cell lysates (Thompson et al., 2012), whereas binding of substrate to apo- or ADPbound Hsp70 reversed the oligomerization process, resulting in a large population of monomeric protein (Aprile et al., 2013). These and other investigations concluded that the distinct formation of oligomeric and monomeric Hsp70 is part of the chaperone’s cycle. Based on its ability to reactivate denatured substrate, the ATP-bound chaperone was termed the “active” species. This view, however, is currently undergoing some reassessment, because recent observations of dimeric bacterial and human Hsp70s have led to a different model for the quaternary assembly of Hsp70 chaperones (Morgner et al., 2015; Sarbeng et al., 2015). For example,


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recombinantly expressed human Hsp70 from E. coli was found to homo-dimerize via its 80 C-terminal residues (Marcion et al., 2015). Additionally, Liu and coworkers (Sarbeng et al., 2015) used mutagenesis to dissect the putative dimeric interface observed between two ATPHsp70 molecules in the asymmetric unit of their crystal structure (Figure 6A) (Qi et al., 2013). Intriguingly, variants that prevented dimerization exhibited normal interdomain allostery and chaperone function, but were less sensitive to stimulation by DnaJ (Sarbeng et al., 2015). The authors suggested that dimerization of E. coli DnaK was transient, yet constituted an important step in the chaperone’s cycle, such that catalytic interactions with dimeric DnaJ could occur efficiently (Sarbeng et al., 2015).

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Robinson and coworkers used native mass spectrometry (native MS), which preserves noncovalent interactions in the gas phase, to reveal that post-translational modifications (PTMs) modulate the dimerization of human Hsp70 (Morgner et al., 2015). They demonstrated that human Hsp70 expressed in E. coli (i.e. with no PTMs, Hsp70Ec) formed dimers at relatively high concentrations, but dissociated into monomers at lower concentrations (Morgner et al., 2015). On the other hand, human Hsp70 expressed in Sf9 insect cells (Hsp70Sf9) maintained a population of dimers throughout the concentration range, and the ability to form dimers at low concentrations was abolished by phosphatase treatment, suggesting that the phosphate group at T504 strengthens inter-subunit affinity. The same study also employed comparative chemical crosslinking (XL) (Schmidt and Robinson, 2014), which determined the antiparallel orientation of the Hsp70Sf9 dimer by revealing XLs between NBDs and SBDs in different subunits (Morgner et al., 2015). The Hsp70Sf9 PTMs localized mainly to the dimeric interface (acetylated lysines) or at the SBDβ/ α hinge (T504). The authors also probed Hsp70 dimerization in the presence and absence of nucleotides, of known cochaperones (Hsp40, Hsp70/Hsp90 organizing protein (Hop), and Hsp90), and of a substrate (a fragment of glucocorticoid receptor, GR) (Morgner et al., 2015). These results are summarized in Figure 6B and 6C. Catalytic amounts of Hsp40 significantly increased the proportion of Hsp70Ec and Hsp70Sf9 dimers, with Hsp70Sf9 again exhibiting a larger population of dimers than Hsp70Ec (Morgner et al., 2015). In this case, comparative XLs in the presence of nucleotides mapped contacts between the Hsp40 J-domain and the acidic groove between subunits in the NBD, and between the C-terminal domain of Hsp40 and the SBD of Hsp70Ec. A comparison of the formation of hetero-complexes with Hsp70Ec or Hsp70Sf9 and Hsp90 and Hop revealed that Hsp70Ec almost exclusively formed Hsp902Hop complexes with low incorporation of Hsp70Ec, whereas Hsp70Sf9 readily formed Hsp902Hsp702Hop complexes (Figure 6C) (Morgner et al., 2015), which formed the basis for engaging substrate, as illustrated by the native and XL-MS view of Hsp902Hsp702HopGR complex (Figure 6, central panel). However, acquiring atomic-level information about this large, and presumably dynamic, hetero-complex remains a significant challenge for the field (Morgner et al., 2015) Progress toward this was made in a recent cryo-EM study of human and yeast Hsp90:Hop:Hsp70 complexes, in which the structures of these ternary complexes were determined at ca. 25 Å resolution (Alvira et al., 2014). Both structures were heterogeneous and dynamic, and cycling between multiple conformations was observed (Alvira et al., 2014). Similar results were observed in another cryo-EM and biochemical study of substrate transfer between Hsp70 and Hsp90 using the glucocorticoid receptor (GR) (Kirschke et al., Structure. Author manuscript; available in PMC 2017 July 06.

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2014). The cryo-EM reconstruction of the GR:Hop:Hsp70:Hsp90 complex revealed contacts between the NBDs of both Hsp90 and Hsp70, which, combined with extensive biochemical assays, provided evidence of coupling between the two ATPase cycles that promote substrate transfer (Kirschke et al., 2014). Complete Hsp90 activity required ATP hydrolysis and the presence of co-chaperones Hop and p23 (Kirschke et al., 2014). Elucidation of the functional role of conformational rearrangements and internal dynamics in the Hsp90:Hop:Hsp70 complex will prove critical toward understanding the molecular mechanisms of this chaperone complex and its proteostatic functions. Moreover, understanding the interactions between other multi-chaperone complexes such as Hsp70:Hsp104, Hsp70:Hsp90, and Hsp70:GrpE will provide a long-sought atomic-level description of the processes involved in substrate recognition, transfer, and refolding (Doyle et al., 2015; Genest et al., 2015).

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It’s a Small (and Dynamic) World After All: conclusions and outlook

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The biological and chemical investigations of Hsp70 carried out over a period of more than three decades (Ellis, 1996; Hartl and Hayer-Hartl, 2002; Kim et al., 2013; Liberek et al., 1991; Martin and Hartl, 1994; Mayer, 2013; Mayer and Bukau, 2005; Yamamori et al., 1978; Zuiderweg et al., 2013) have laid the groundwork for our current understanding of the molecular mechanism of this dynamic enzyme. Elegant biochemical experiments have demonstrated that inter-domain allosteric communication regulate the function of this molecular chaperone (Figure 1A and B) (Mayer, 2013; Zuiderweg et al., 2013). The majority of this biochemical information, however, was gathered in a limited structural context, in which structures of the isolated Hsp70 domains were employed to interpret functional data (Figures 2A and 3A). Determination of the relationships between internal dynamics, nucleotide and substrate binding, and global interdomain communication provided a breakthrough in understanding the mechanism of Hsp70. The dynamical biology of Hsp70 has now come to light, and a picture has emerged wherein internal dynamics in both domains regulate allostery and function (Mayer, 2013; Zuiderweg et al., 2013). However, remaining questions about Hsp70 internal dynamics persist. For example, it will be of interest to examine the μs-ms dynamics in ATP-bound Hsp70 in more detail to ascertain what conformations are sampled in this exchange-broadened state. NMR measurements are capable of determining the conformation of and rate of exchange with such “invisible states” in which NMR peaks have become significantly broadened (Baldwin and Kay, 2009).

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Transient and dynamic interactions between chaperones have long proved challenging for structural biology, yet NMR spectroscopy has now begun to offer detailed insights regarding these elusive molecular chaperone complexes (Burmann and Hiller, 2015; Rosenzweig and Kay, 2014). For example, structural information on the complex of Hsp70 and Hsp40 was long sought in order to describe the molecular details that mediate how Hsp40 stimulates both ATP hydrolysis in Hsp70 and the process of substrate binding, refolding, and transfer. Atomic-level information about the formation and dynamics of multi-chaperone, substratebound complexes comprises another challenge in the field. Recent NMR work from the Kay group characterized the interaction with client proteins and the 580 kDa Hsp100 (ClpB) hexamer (Rosenzweig et al. 2015), but many interesting multi-chaperone or chaperonesubstrate complexes remain to be characterized. Structure. Author manuscript; available in PMC 2017 July 06.

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Pioneering studies with cryo-EM and native MS demonstrated the ability to dissect the stoichiometries and assemblies of multi-chaperone complexes (Alvira et al., 2014; Kirschke et al., 2014; Morgner et al., 2015). Moving forward, native MS, solid-state (ss) and methylTROSY NMR spectroscopy, and cryo-EM appear poised to expand on these studies, offering the potential to elucidate atomic-resolution dynamical information about these large chaperone complexes. ssNMR spectroscopy, in particular, is not limited by size, and can provide site-specific details about protein structure and dynamics (Lewandowski et al., 2015; Sinnige et al., 2015). Such information will be critical to understanding the proteostatic roles of these chaperones and to devising ways to prevent their hyperactivity in human diseases, including many cancers (Lianos et al., 2015; Murphy, 2013). Indeed, promising results using small-molecule inhibitors of Hsp70 and Hsp70-containing chaperone complexes have recently appeared in the literature (Assimon et al., 2013; Cesa et al., 2013; Colvin et al., 2014; Li et al., 2013, 2015; Miyata et al., 2013; Rousaki et al., 2011). Other molecular chaperones have been identified as candidate drug targets, and understanding their complex structures, dynamics, and interactions at atomic-resolution will aid in elucidating their mechanisms of action (Blair et al., 2014; Jego et al., 2013).

Acknowledgments The authors thank William Ford Freyberg, Justin L. P. Benesch, and Iva Pritišanac for critically reading this manuscript and providing helpful comments and suggestions. Supported in part by the NIH Oxford-Cambridge Scholars Program and US National Institutes of Health grant 5U01GM094584.

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Highlights •

Hsp70 is a dynamic molecular machine that undergoes allosteric regulation

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Internal dynamics in Hsp70 regulate inter-domain communication

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Dynamic interactions between Hsp40 and Hsp70 mediate complex formation

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Transiently formed, multi-chaperone complexes can be captured and studied indetail

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FIGURE 1. The Hsp70 ATPase-substrate interaction cycle and the structures of Hsp70 and Hsp40

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(A) (1) A J-protein (Hsp40) binds to a non-natively folded client protein via its C-terminal domain. The N-terminal J-domain of Hsp40 interacts with ATP-bound Hsp70 (2) and both the substrate and Hsp40 catalyze ATP hydrolysis, which leads to release of inorganic phosphate (Pi) and dissociation of Hsp40 from ADP-bound Hsp70. The client protein is transferred from Hsp40 to ADP-Hsp70 (3), which binds to substrate with a higher affinity than ATP-Hsp70. A nucleotide-exchange factor (NEF) binds to the nucleotide-binding domain (NBD) of ADP-Hsp70 (4) and promotes the exchange of ADP (5) for ATP (6). The NEF dissociates from ATP-Hsp70, and the client protein is released from ATP-Hsp70. If native-like contacts have not been established in the client protein, the J-protein will bind to exposed hydrophobic residues and the cycle will repeat. Image taken from (Kampinga and Craig, 2010) with permission from the Nature Publishing Group. (B) (Top) NMR-based structural model (PDB 2kho) of E. coli DnaK (1-605) bound to ADP and substrate peptide. The domains of DnaK are colored as follows: NBD (black), SBD (teal), interdomain linker (red). Specific regions of the NBD and SBD are annotated. (Bottom) X-ray crystal structure (PDB 4jne) of ATP-bound DnaK (T199A) containing truncation of loop3,4 and removal of the disordered C-terminus. The domains are colored as above. Upon ATP binding, the interdomain linker adopted a β-strand conformation and docked onto a region of the NBD between sub-domains IA and IIA (inset). Note that text indicating the SBD sub-domains has been removed for clarity. (C) Structures of various Hsp40 proteins and their J-domains. (i) A combined EPR and X-ray crystallographic structural model of FL, dimeric DnaJ from T. Structure. Author manuscript; available in PMC 2017 July 06.

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thermophilus (PDB 4j80). The respective domains within DnaJ are colored as indicated and one monomer within the dimer is colored black for clarity. Helices I and IV have been colored orange to highlight their presence or absence in the ensuing J-domain structures. (ii) The solution structural ensemble of DnaJ103 from E. coli (PDB 1bq0). This variant included helices I-IV in the J-domain, the proline-rich region, and a portion of the GF-linker; however, only the structure of the J-domain was solved by NMR. An inset displays the HPD motif as red sticks. (iii) A crystal structure of the auxilin J-domain from B. taurus, which comprised residues 810–910 (PDB 1nz6). Note that helix I is much shorter and helix IV is absent. (iv) A crystal structure of full-length HscB from E. coli (PDB 1fpo). The additional helices comprise the C-terminal domain, which binds to substrate protein.


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Author Manuscript Author Manuscript FIGURE 2. Conformational dynamics of the Hsp70 nucleotide-binding domain


(A) Alignment of Hsc70 nucleotide-binding domain (NBD) crystal structures in the presence or absence of various nucleotides. Here, the structures of isolated Hsc70 NBDs (residues 4– 381) have been superimposed by alignment of their backbone atoms. Five structures were used in this alignment, comprising the apo (PDB ID 2qw9; 0.47 Å), ATP (PDB ID 1kax), ADP (PDB ID 2qwl; 0.32 Å), ADP-Pi (PDP ID 3hsc; 0.34 Å), and ADP-Vi (PDP ID 2qwm; 0.30 Å) states of Hsc70 NBD. (E. coli DnaK residues 1–37, 112–184, 363–383 IA; (38–111) IB; (185–227, 310–362) IIA; (228–309) IIB). (B) Refined structural models of the DnaK (T. thermophilus) NBD residues 1–381 with HN RDCs in both AMPPNP- and ADP-bound states. Note the structural rearrangements as compared to Figure 2A. Figure adapted from (Bhattacharya et al., 2009) with permission from Elsevier. (C) Solution-state NMR spectra of the 40 kDa NBD of DnaK (T. thermophilus). Overlay of 1H-15N TROSY-HSQC spectra of 400 μM apo- (blue) and ADP- NBD (black). Overlay of 1H-15N TROSY-HSQC spectra of ADP- and AlFx-NBD (green). AlFx, which is an ATP analogue, suppressed peak doubling that was otherwise observed in the spectrum of ADP-NBD. Figure adapted from (Revington et al., 2004) with permission from the American Society for Biochemistry and Molecular Biology. See text for more details. (D) Interdomain linker-induced chemical shift perturbations (CSPs) mapped onto the ATP-bound NBD homology model of DnaK. Red spheres indicate residues with large CSPs. Green residues are those that displayed sensitivity to nucleotide, as measured by the observation of large differences in chemical shifts between ADP- and ATP-bound NBD. Space-filling model of the results depicted adjacently, with red, yellow, cyan, and grey residues indicating large, medium, or insignificant CSPs and no data, respectively. Figure adapted from (Zhuravleva and Gierasch, 2011) with permission from the National Academy of Sciences.


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FIGURE 3. Conformational dynamics of the Hsp70 substrate-binding domain

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(A) Solution-state NMR spectroscopy-derived structural ensembles of the isolated SBD of DnaK (E. coli) in the presence (top) and absence (bottom) of substrate peptide NRLLLTG. The β-strands are colored as follows: β1 red, β2 orange, β3 yellow, β4 green, β5 teal, β6 blue, β7 pink, and β8 purple. Functionally relevant loops 3,4 (L3,4) and L5,6 are indicated. See text for details regarding these loops. (B) Summary of EPR spectroscopy results that measured the accessible distances to the isolated SBD of DnaK (E. coli) in the presence of substrate. The cartoon depicts three models that are consistent with the distance restraints. Figure adapted from (Schlecht et al., 2011) with permission from the Nature Publishing Group. (C) A structural model of the DnaK (E. coli) SBD displaying significant CSPs (red) upon mutation of loop 3,4 (L3,4) is shown. Note the large number of residues affected by this mutation. A region of a 1H-15N TROSY-HSQC spectrum of ATP-bound DnaK (1-552) T199A/L542Y/L543E (black) overlaid upon the same protein with mutations in the loop 5,6 (L5,6) region (red). Note that many of the resonances in the SBDβ either broaden into the noise or are reduced in intensity, indicative of enhanced μs–ms motions. A cartoon below depicts such motions in the L5,6 mutant. Figure adapted from (Zhuravleva and Gierasch, 2015) with permission from the National Academy of Sciences, USA.


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Author Manuscript Author Manuscript FIGURE 4. Detection of the allosterically active intermediate in the chaperone cycle of Hsp70


(A) Methyl-TROSY spectra from ATP-bound DnaK552 in the apo (grey) and substratebound (black) states. Overlaid upon these spectra are those recorded on the isolated NBD (red) and linker-bound NBD (blue). Substrate binding to the ATP-bound full-length protein modulates the conformational ensemble of the interdomain linker, as both linker-bound and unbound states are populated, with chemical shifts between these states found in the isolated NBDs. (B) Residues with significant CSPs (yellow) between the NBDs of ATPγS-bound DnaK552 and DnaK552 (L390V) are mapped onto a homology model of ATP- and substratebound DnaK. (C) The methyl-TROSY spectrum of Ile-labeled DnaK601(389DDD391) bound to ATP and substrate (black) overlays nearly perfectly with the isolated ADP-bound NBD (red). This variant of DnaK601 is allosterically defective. Note that additional resonances that are absent in the NBD spectrum have arisen from isoleucines in the SBD. (D) A schematic that depicts conformational transitions in DnaK. (1) The transition between domain-docked (ATP) and domain-undocked (ATP and substrate) ensembles, which corresponds to the grey and black resonances in Figure 3A. (2) The transition between linker-bound (ATP) and (3) linker-unbound (ATP and substrate) ensembles, which corresponds to the blue and red resonances in Figure 3A. In ATP- and substrate-bound FL DnaK, this transition occurs on a fast timescale, as only one resonance is observed. This resonance corresponds to the population-weighted average of the two linker conformations. Residues stabilized by binding of substrate, ATP, or both are colored red, blue, or yellow, respectively. Figure adapted from (Zhuravleva et al., 2012) with permission from Elsevier.


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FIGURE 5. Conformational dynamics of Hsp40-Hsp70 complexes

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(A) DnaJ70 is shown in white, and its average position with respect to DnaK601 (NBD in yellow, SBD in cyan) determined from MD simulations and PRE restraints with MTSL tags at the indicated residues in both DnaJ70 and DnaK601. Green residues were not in contact with either DnaJ70 or DnaK601 when an MTSL spin-label was placed at those positions in DnaK601 or DnaJ70, respectively. DnaJ70 residues in blue were found to contact the red and orange residues in DnaK601. (B) Overlaid snapshots from the 30 ns MD simulation of DnaJ70 (white) bound to DnaK NBD (yellow). (C) A zoomed in region of the non-covalent complex indicating functional residues that were discussed in the text. The HPD motif is shown in green. Other residues have been colored as in A. Figure adapted from (Ahmad et al., 2011) with permission from the National Academy of Sciences, USA. (D) NMR spectroscopic results acquired from the interaction between unlabeled, ATPase-deficient HscA(T212V) and 15N-labeled HscB. HscB residues that were broadened upon addition of HscA(T212V) are colored red; residue that showed CSPs are colored blue. The nucleotide state of HscA(T212V) is indicated in the central boxes. Note that ATP-HscA(T212V) caused significant broadening in the both the J-domain and C-terminal domain (CTD) Figure adapted from (Kim et al., 2014) with permission from the American Chemical Society.


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FIGURE 6. Evidence from X-ray crystallography and native mass spectrometry for dimeric Hsp70 and its multi-chaperone complexes

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(A) The asymmetric unit of E. coli DnaK bound to ATP (PDB 4JNE) contains two molecules. Right, inter-domain contacts are indicated in circles. Figure adapted from (Sarbeng et al., 2015). (B) Native mass spectra of Hsp90 and Hop with Hsp70 from either E. coli (left) or Sf9 cells (right). The y-axis displays relative peak intensity and the x-axis is m/z; each protein and protein complex with a given mass m yields a Gaussian-shaped charge series. The authors assigned the peaks within each charge series to the indicated chaperone complexes. Note that Hsp70 from Sf9 cells yielded dimeric Hsp70 and Hsp902Hsp702Hop complexes. (C) A catalytic amount of Hsp40 mixed with E. coli Hsp70, Hsp90, Hop, and GR substrate protein led to the formation of a hexameric complex (indicated by rectangles). Other stoichiometries of chaperone-chaperone and chaperone-substrate complexes were observed as well. (D) Increasing the relative amount of Hsp70 from (C) led to the stable formation of the hexameric chaperone-substrate complex. (Cartoon) Comparative XLs were identified in the hexameric complex formed in (C) and (D). The lysine residues that formed


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XLs between each protein are annotated. Figure adapted from (Morgner et al., 2015) with permission from Elsevier.


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Nonintegrable semidiscrete Hirota equation: gauge-equivalent structures and dynamical properties.

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Comparison of the dynamical structures of lipoamide dehydrogenase and glutathione reductase by time-resolved polarized flavin fluorescence.

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Combining NMR and EPR to Determine Structures of Large RNAs and Protein-RNA Complexes in Solution.

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Synthesis, characterization and modeling structures of isatin-3-Girard T (IGT) and P (IGP) hydrazone complexes.

Prediction of three-dimensional structures of enzyme-substrate and enzyme-inhibitor complexes of lysozyme.