Allosteric Landscapes of Eukaryotic Cytoplasmic Hsp70s Are Shaped by Evolutionary Tuning of Key Interfaces

Allosteric landscapes of eukaryotic cytoplasmic Hsp70s are shaped by evolutionary tuning of key interfaces

Wenli Menga, Eugenia M. Clericoa, Natalie McArthura, and Lila M. Gierascha,b,1

aDepartment of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, MA 01003; and bDepartment of Chemistry, University of Massachusetts Amherst, Amherst, MA 01003

Edited by Susan Marqusee, University of California, Berkeley, CA, and approved October 10, 2018 (received for review June 27, 2018) The 70-kDa heat shock proteins (Hsp70s) are molecular chaperones result, the SBD adopts an open conformation with high substrate that perform a wide range of critical cellular functions. They assist on/off rates and low affinity for substrate. Our earlier studies of in the folding of newly synthesized proteins, facilitate assembly of DnaK showed that its bias toward different allosteric states results specific protein complexes, shepherd proteins across membranes, from an energetic tug-of-war between two orthogonal interfaces: and prevent protein misfolding and aggregation. Hsp70s perform the NBD–SBD interface and the β-SBD–α-lid interface (SI Ap- these functions by a conserved mechanism that relies on allosteric pendix,Fig.S1) (13). The NBD–SBD interface is fully formed in cycles of nucleotide-modulated binding and release of client the ATP-bound, docked conformation of the Hsp70, while the proteins. Current models for Hsp70 allostery have come from β-SBD–α-lid interface forms in the undocked state. extensive study of the bacterial Hsp70, DnaK. Extending our un- Thirteen isoforms of human Hsp70s have been identified derstanding to eukaryotic Hsp70s is extremely important not only and are present in all major cellular compartments, including in providing a likely common mechanistic framework but also be- two major cytoplasmic isoforms: the stress-inducible Hsp70, cause of their central roles in cellular physiology. In this study, we HspA1, and the constitutive cognate Hsp70, Hsc70. The ex- examined the allosteric behaviors of the eukaryotic cytoplasmic pression level of HspA1, which is implicated in the etiology of Hsp70s, HspA1 and Hsc70, and found significant differences from several cancers and neurodegenerative diseases (14–16), is low that of DnaK. We found that HspA1 and Hsc70 favor a state in under physiological conditions but greatly augmented under which the nucleotide-binding domain (NBD) and substrate-binding stress. Hsc70, which is 86% identical in sequence to HspA1, domain (SBD) are intimately docked significantly more as com- carries out distinct cellular functions. It plays a central role in BIOPHYSICS AND

pared to DnaK. Past work established that the NBD–SBD interface cellular homeostasis and functions in specialized cellular pro- COMPUTATIONAL BIOLOGY and the helical lid–β-SBD interface govern the allosteric landscape cesses like clathrin-mediated endocytosis (17–19). Some of the of DnaK. Here, we identified sites on these interfaces that differ differences in cellular roles of HspA1 and Hsc70 relate to their between eukaryotic cytoplasmic Hsp70s and DnaK. Our mutational differential expression, but others may arise from differential analysis has revealed key evolutionary variations that account for substrate specificity and allosteric properties. Enhanced un- the population shifts between the docked and undocked confor- derstanding of the origin of allosteric tuning in eukaryotic mations. These results underline the tunability of Hsp70 functions Hsp70scanbeusedtobetterunderstandotherHsp70family by modulation of allosteric interfaces through evolutionary diver- members as well. sification and also suggest sites where the binding of small- Current models for the allosteric mechanism of eukaryotic molecule modulators could influence Hsp70 function. Hsp70s rely heavily on what has been learned from DnaK. However, HspA1 and Hsc70 are only 46% identical in sequence to DnaK. Importantly, sequence alignments reveal that many of Hsp70 | methyl-TROSY NMR | allostery | chaperone Significance he 70-kDa heat shock proteins (Hsp70s) are ubiquitous Tmolecular chaperones that play critical roles in a wide range of cellular processes, including protein folding/refolding, disag- Heat shock protein 70 (Hsp70) molecular chaperones act as a gregation, membrane translocation, and assembly of multiprotein central hub of cellular proteostasis network. The functions of complexes (1, 2). Hsp70s are composed of two domains: an N- Hsp70s rely on allosteric communication between nucleotide- terminal 45-kDa actinlike nucleotide-binding domain (NBD) and binding domain and substrate-binding domain. Current mechanistic models for Hsp70 allostery are based on extensive study a C-terminal 30-kDa substrate-binding domain (SBD), which are of the bacterial Hsp70, DnaK. Here, we report that the allosteric connected by a conserved, largely hydrophobic interdomain linker. landscapes of the eukaryotic cytoplasmic Hsp70s, HspA1 and The SBD consists of a β-sandwich subdomain (β-SBD), which Hsc70, diverge significantly from that of DnaK in that they contains a canonical substrate-binding pocket, an α-helical lid favor a domain-docked, low substrate-affinity state much more subdomain (α-lid), and a disordered C-terminal region. Hsp70s than DnaK does. Our mutational results illustrate how this perform their cellular functions by binding and releasing client evolutionary tunability of Hsp70s arises by modulation of key proteins in a nucleotide-dependent fashion (3–5). For example, in Escherichia coli allosteric interfaces. These insights will help in understanding the case of the Hsp70 DnaK, ATP binding facil- the mechanism of Hsp70 functional diversities and aid in the itates substrate release, and, in turn, substrate binding stimulates design of small-molecule modulators of Hsp70s. the ATP hydrolysis rate. In the ADP-bound state of DnaK, the NBD and SBD are undocked, the linker is relatively exposed and Author contributions: W.M., E.M.C., N.M., and L.M.G. designed research; W.M., E.M.C., dynamic, and the two-domain chaperone behaves as “beads on a and N.M. performed research; W.M., E.M.C., N.M., and L.M.G. analyzed data; and W.M., string” (Fig. 1A) (6, 7). The SBD adopts a closed conformation, E.M.C., and L.M.G. wrote the paper. and the two subdomains—the β-SBD and the α-lid—form a stable The authors declare no conflict of interest. β-SBD–α-lid interface, resulting in low substrate on/off rates and This article is a PNAS Direct Submission. high binding affinity. Upon ATP-binding, an allosteric signal is Published under the PNAS license. transmitted from the NBD to the SBD through the interdomain 1To whom correspondence should be addressed. Email: [email protected]. linker (8, 9), and the protein undergoes a major conformational This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. change (Fig. 1B). The α-lid detaches from the β-SBD and both the 1073/pnas.1811105115/-/DCSupplemental. α-lid and the β-SBD become docked to the NBD (10–12). As a

www.pnas.org/cgi/doi/10.1073/pnas.1811105115 PNAS Latest Articles | 1of6 Downloaded by guest on September 27, 2021 on DnaK in the apo state or when bound to ADP or ADP plus peptide NR and when bound to ATP or ATP plus peptide (Fig. 2). Note that the band corresponding to full-length 70-kDa DnaK disappears at low proteinase K concentrations and that two bands at ∼45 and 25 kDa, corresponding to individual NBD and SBD, emerge. These observations are consistent with the fact that the NBD and SBD are undocked (in the apo, ADP- bound, and ADP/peptide-bound states) or largely undocked (ATP/peptide-bound state), which exposes the proteolytically labile interdomain linker. In ATP-bound DnaK, a band corresponding to the two-domain construct (appearing at 60 kDa due to a partial cleavage of the SBD α-lid) persists after protease treatment, arguing that the linker region is more resistant to proteolysis, in agreement with the fact that ATP-bound DnaK adopts a domain-docked conformation in which the linker is intimately packed between the NBD and SBD (10, 11). By contrast, limited proteolysis experiments on HspA1 show that the full-length protein is protease-resistant to a substantial extent in the ADP/peptide-, ADP-, ATP/peptide-, and ATP- bound states (Fig. 2). We quantified the SDS/PAGE gels by Fig. 1. Conformational features of Hsp70 allostery. (A and B) Structures of densitometry and used portions of the two-domain bands to DnaK showing the canonical ADP-bound undocked state [Protein Data Bank evaluate the resistance of HspA1 to proteinase K in different (PDB) ID code 2kho] (A) and the canonical ATP-bound docked state (PDB ID SI Appendix B C β –α ligand-bound states ( , Fig. S4). Only in the apo state code 4b9q) ( ). ( ) Residue conservation on the undocked state -SBD -lid is the full-length protein seen to be rapidly digested into indi- interface. Residues are mapped on the SBD structure of HspA1 (PDB ID code vidual NBD and SBD fragments. All other ligand-bound states 4po2). (D) Residue conservation on the docked state NBD–SBD interface among HspA1, Hsc70, and DnaK. Residues are mapped on the homology are resistant to proteolysis. Specifically, the ATP-, ATP/peptide-, model of HspA1 based on ATP–BiP structure (PDB ID code: 5e84). (C and D) and ADP-bound states have a similar degree of resistance, Residues in blue are conserved among HspA1, Hsc70, and DnaK; residues in whereas peptide binding makes the ADP-bound state of HspA1 pink are conserved between HspA1 and Hsc70, but different in DnaK; resi- become increasingly labile to proteolysis. These results argue dues in orange are not conserved between HspA1 and Hsc70. (E) HspA1 and that the docked conformation of HspA1 is favored under a much Hsc70 constructs used in the current study. All NBD and two-domain con- wider array of ligand-binding conditions than that of DnaK. The structs contain a T204A mutation; A1(residues 1 to 555)y and A1 SBD(resi- behavior of Hsc70 falls between those of DnaK and HspA1, with dues 386 to 555)y constructs contain an L542Y mutation. ADP-, ATP/peptide-, and ATP-bound states protease resistant and the apo and ADP/peptide-bound states labile to proteolysis, suggesting that its domain docking preference is higher than the evolutionary residue changes among Hsp70s occur on the DnaK, but lower than HspA1. More subtle distinctions upon interdomain interfaces that are critical to the allosteric mechanism limited proteolysis can be seen in the band patterns and are (Fig. 1 C and D and SI Appendix,Figs.S1–S3). Thus, we hy- discussed in SI Appendix, SI Discussion. pothesized that the differences in sequence between eukaryotic Hsp70s and DnaK modulate their allosteric landscapes, giving rise The ATP Hydrolysis Rates of Eukaryotic Hsp70s Are Less Stimulated by to significant functional diversification. Therefore, it is critical to Model Peptide Substrates As Compared with DnaK. A classic hall- assess the mechanistic impact of the evolutionary sequence di- mark of allostery in Hsp70s is the two-way communication be- versification between DnaK and different eukaryotic Hsp70s. tween the SBD and NBD, as indicated by ATP modulation of substrate-binding affinity and substrate activation of the ATP Results hydrolysis rate. In the case of DnaK, binding of peptide sub- Design of Eukaryotic Hsp70 Constructs for Study of Allostery. The strates can stimulate the ATP hydrolysis rate significantly; the allosteric transitions of Hsp70 molecular chaperones involve extent of stimulation is qualitatively correlated with the mea- docking and undocking of the NBD and SBD as modulated by sured affinity of binding, consistent with an energetic coupling ligand binding (Fig. 1). Studies of DnaK and other Hsp70s have between ligand binding and the population of an NBD state allowed us to dissect the allostery of Hsp70s based on different characterized by high ATPase activity (23). We performed allosteric units (6, 8, 13). In the current study, we designed two- ATPase assay for wild-type DnaK, Hsc70, and HspA1 using both domain constructs as well as individual NBD and SBD constructs peptide p5 and peptide NR. Either model peptide stimulated the E to probe the allosteric landscape of HspA1 and Hsc70 (Fig. 1 ). DnaK ATPase approximately sixfold (Fig. 3A). Strikingly, we The T204A mutation is incorporated to decrease the ATP hydrolysis rate in all NBD and two-domain constructs (20). In this work, HspA1 T204A is referred to as HspA1, and Hsc70 T204A is referred to as Hsc70. We characterized the conformational properties of HspA1 and Hsc70 in five different ligand-bound states: the apo state (no nucleotide and no peptide), the ADP- bound state, the ADP/peptide-bound state, the ATP-bound state and the ATP/peptide-bound state. Two classic Hsp70 model substrate peptides, NR (NRLLLTG) and p5 (CLLLSAPRR), are used as substrates in the current study to provide parallel comparisons with DnaK.

The Response of Eukaryotic Hsp70s to Nucleotide Binding Differs from That of DnaK. Limited proteolysis experiments are a useful ap- Fig. 2. Limited proteolysis experiment suggests a major allosteric landscape proach to investigate the conformation properties of Hsp70s (21, difference among HspA1, Hsc70, and DnaK. Limited proteolysis experiments 22). We interrogated the domain–domain docking of HspA1 in on HspA1, Hsc70, and DnaK at 15 min reaction time. The full-length protein different ligand-bound states using limited proteolysis with pro- (FL), full-length protein with lid truncated (FLΔlid), NBD, and SBD bands are teinase K. As a reference, we carried out the proteolysis reaction indicated. Peptide NR is used as a model substrate. pep, peptide.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1811105115 Meng et al. Downloaded by guest on September 27, 2021 compared chemical shifts for the Ile methyl groups in the full- length eukaryotic Hsp70s in the apo state and in the ADP- or ATP-bound states, plus or minus NR peptide as model substrate, to those of the corresponding NBD (Fig. 4 and SI Appendix, Fig. S6). Note first that in the apo state, the NBD peaks from the full- length eukaryotic Hsp70s overlay exactly on those of their re- spective NBDs(residues 1 to 390), arguing that the NBD and SBD are predominantly noninteracting. The full-length SBD construct of HspA1 encompassing residues 386 to 641 is very prone to self-association and thus cannot be directly used in this analysis. Therefore, we compared spectra of a truncated two- domain construct, A1(residues 1 to 555)y, with isolated A1 NBD(residues 1 to 390) and A1 SBD(residues 386 to 555)y spectra, and we observed exact overlaps between peak positions for the two-domain construct and those for the isolated domain constructs (SI Appendix, Fig. S7). Together, these data argue that the apo state of eukaryotic Hsp70s adopts predominantly an undocked conformation, which is the same behavior as that of DnaK in the apo state. Addition of ATP to either full-length eukaryotic Hsp70 caused significant chemical shift changes such that the observed resonances no longer overlap those of the ATP–NBD(residues 1 to Fig. 3. The eukaryotic Hsp70s exhibit different ATPase stimulation and 390) spectrum (Fig. 4 and SI Appendix, Figs. S6 and S7). In substrate binding properties compared with DnaK. (A) ATPase activity of – μ particular, the residues on NBD SBD interface exhibit signifi- HspA1, Hsc70, and DnaK (at 2 M) in the absence and presence of 1 mM cant chemical shift changes (SI Appendix, Fig. S8). These ob- model peptide substrate p5 or NR. Error bars represent the SDs from three independent experiments. (B–D) Binding affinities of FITC-labeled p5 to full- servations support the conclusion that the NBD and SBD dock on one another, which is also the same behavior displayed by length HspA1, Hsc70, and DnaK in apo (black), ADP-bound (red), and ATP- – bound (blue) states. Error bars represent the SDs from three independent ATP DnaK (10, 11). Striking differences emerge when the BIOPHYSICS AND experiments. The KD values are listed in SI Appendix, Table S1. ADP-bound eukaryotic Hsp70s are examined. Both ADP-bound COMPUTATIONAL BIOLOGY

found that the ATPase activity of wild-type HspA1 cannot be stimulated by either peptide NR or peptide p5, even in 500-fold molar excess, while that of wild-type Hsc70 can be stimulated to a moderate extent—approximately twofold (Fig. 3A). We measured the binding affinities of peptide p5 to HspA1 and Hsc70 and compared them with its binding to DnaK. HspA1 has significantly lower substrate-binding affinity than DnaK does in corresponding ligand-bound states, while the binding affinity of Hsc70 falls between those of HspA1 and DnaK (Fig. 3 B–D and SI Appendix, Table S1): The apparent KD of ADP-bound HspA1 to peptide p5 is 23 ± 2 μM, which is 120-fold higher than that of ADP-bound DnaK (KD = 0.19 ± 0.01 μM) and 10- fold higher than that of ADP-bound Hsc70 (KD = 2.3 ± 0.2 μM). The KD of ATP-bound DnaK/p5 is 8.2 ± 0.2 μM, whereas the KD of ATP-bound HspA1/p5 is 130 ± 10 μM. The decreased peptide-binding affinities in eukaryotic Hsp70s compared with DnaK may be due, in part, to sequence changes in the substrate- binding pocket of their SBDs. However, the preference for NBD–SBD docking and the consequent population of the low substrate-affinity state certainly account, in large measure, for the decreased peptide-binding affinity in eukaryotic Hsp70s. The lower peptide-binding affinity of HspA1 relative to Hsc70 sup- ports this interpretation, as their substrate-binding pockets are highly conserved (SI Appendix, Fig. S5). Presumably, the reduced peptide-binding affinity contributes to the lack of ATPase stimulation by peptide substrates in these Hsp70s.

Methyl-TROSY NMR Reveals Key Features of the Eukaryotic Hsp70 Allosteric Landscapes That Are Distinct from That of DnaK. NMR proved to be a powerful method to gain functionally important Fig. 4. Methyl-TROSY NMR reveals characteristics of the HspA1 and Hsc70 insights into the chaperone allosteric mechanism (8, 13, 24–26). 13C allosteric landscapes. Blowup of diagnostic regions of the Ile methyl-TROSY spectra of apo, ADP/NR-, ADP-, ATP/NR-, and ATP-bound states of HspA1 methyl transverse relaxation optimized spectroscopy (methyl- A B TROSY) NMR is particularly informative (13, 27–29), so we (residues 1 to 641) ( ) and Hsc70 (residues 1 to 646) ( ). Spectra of full-length proteins (in red) are overlaid on the spectra of the corresponding nucleotide- deployed this approach to explore the allosteric landscapes of bound states of the individual NBD(residues 1 to 390) for each Hsp70 (in eukaryotic Hsp70s and to compare their features to those of DnaK. δ blue). The docked state resonance (D) and undocked state resonance (U) are The Ile -methyl chemical shifts of the isolated NBD(residues labeled. Peptide NR is used as model substrate. The spectra of full Ile region 1 to 390) constructs in a given nucleotide-bound state provide a are shown in SI Appendix, Fig. S6. Additional panels with enlarged and la- reference point for the allosteric state in which the NBD and beled peaks are shown in SI Appendix, Fig. S9 (HspA1) and SI Appendix, Fig. SBD are independent of one another (i.e., undocked). We S10 (Hsc70).

Meng et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 27, 2021 HspA1 and Hsc70 populate two distinct slowly interconverting states, indicated by the presence of two resonances for many Ile methyls: One set of peaks is coincident with the domain- undocked conformation (Fig. 4 and SI Appendix, Figs. S6, S9, and S10); and nearly all peaks in the other set (with I28 of Hsc70 the only exception, SI Appendix, Fig. S11) overlay well with peaks for the ATP-bound state and, thus, can be attributed to the domain-docked conformation. The allosteric behaviors of HspA1 and Hsc70 do differ: The ADP-bound HspA1 construct is 80% docked based on the peak volumes of well-separated peaks, whereas ADP-bound Hsc70 populates the docked state significantly less (46%). The spectral quality of the ADP-bound HspA1 and Hsc70 is similar to their ATP-bound state, but not as good as their apo state or the isolated domains. Line shape broadening is observed for the ADP-bound and ATP-bound spectra. This broadening may be caused by a combination of slower tumbling of the molecules due to domain docking and the dynamic and heterogeneous features of the docked state. Addition of NR Fig. 5. Evolutionary residue changes on key interdomain interfaces mod- shifted the allosteric balance of both eukaryotic Hsp70s toward ulate Hsp70 allostery. (A) Sites probed by mutagenesis to study the origins of the undocked allosteric state: For Hsc70, the ADP/peptide- allosteric differences between HspA1 and DnaK on the NBD–SBD interface; bound chaperone is predominantly undocked, whereas a minor mutations are introduced in the background of the HspA1 A1(residues 1 to population (23%) of the docked form of HspA1 remains, even 555)y construct. Residues are colored as in Fig. 1 C and D.(B) Comparisons of when ADP and peptide are bound (Fig. 4 and SI Appendix, Figs. docked populations of NBD–SBD interface mutants and A1(residues 1 to 555)y S6, S9, and S10). These observed behaviors of HspA1 and Hsc70 in the ATP-bound state. The populations are calculated from NMR peak are distinct from observations of DnaK and other Hsp70s that volumes of I253, I331, and I343 in the undocked and docked states. The have been studied, all of which are completely undocked in the averages and SDs are calculated. The docked and undocked state resonances ADP-bound state (SI Appendix, Fig. S12) (13, 28, 30, 31). of I253, I331, and I343 of A1(residues 1 to 555)y and all other interface In the case of DnaK, binding of peptide substrate to the ATP- mutants are displayed in SI Appendix, Fig. S14. bound state favors SBD helical lid closure and partial NBD–SBD undocking to form the allosterically active state (12, 13). This ATP/peptide-bound state of DnaK is largely undocked, which is are not conserved between HspA1 and DnaK, mutation of the also confirmed by the limited proteolysis data presented above. residue in HspA1 to the one in DnaK should favor domain However, ATP/peptide-bound HspA1, based on methyl-TROSY undocking of ATP-bound HspA1. We evaluated the populations NMR data, even in the presence of a 50-fold molar excess of of docked conformations by integrating the peak volumes of I253, I331, and I343 resonances of docked and undocked species peptide substrate NR, adopts a predominantly docked confor- Materials and Methods mation (Fig. 4 and SI Appendix, Figs. S6 and S9). This behavior is for each variant in the ATP-bound state ( SI Appendix consistent with the observation that peptide substrate cannot and , Fig. S13) and compared them to that of A1 stimulate the ATPase of HspA1, since the activation of the (residues 1 to 555)y. β α ATPase by substrate requires undocking between NBD and SBD R509 is in the loop connecting the -SBD and the -lid. Se- while the linker must remain bound to NBD (13). For Hsc70, quence alignment shows that R509 is highly conserved between adding peptide substrate to the ATP-bound state favors domain eukaryotic cytosolic and endoplasmic reticulum (ER) Hsp70s undocking (Fig. 4 and SI Appendix, Figs. S6 and S10); the pop- (e.g., HspA1, Hsc70, and BiP) but is missing in prokaryotic Hsp70s ulation of undocked conformation is 64% (which includes fully (e.g., DnaK) and in mitochondrial Hsp70s. The ATP-bound and partially undocked species). This is consistent with the fact structure of BiP shows that this Arg forms two hydrogen bonds that peptide substrates can stimulate the ATPase rate of Hsc70 with D178 (D152 in HspA1) in the NBD (Fig. 5 and SI Appendix, only moderately. Fig. S13A) (32). As expected, the ATP-bound R509E mutant has a reduced population of the docked conformation (42% docked) Evolutionary Residue Changes on Key Interdomain Interfaces Account relative to ATP-bound A1(residues 1 to 555)y (84% docked) and for the Change of Allostery Between Eukaryotic Hsp70s and DnaK. favors the undocked conformation. Mutation I164L, which per- Sequence alignments demonstrate that there are numerous turbs the hydrophobic cluster formed between the NBD and the evolutionary residue changes among Hsp70s on the two key al- α-lid in the docked state of HspA1 and DnaK (Fig. 5 and SI losteric interdomain interfaces—that is, the NBD–SBD interface Appendix, Fig. S13B), significantly favors domain undocking in and the β-SBD–α-lid interface (Fig. 1 C and D and SI Appendix, both of these Hsp70s. Mutation of V163 in the same hydro- Figs. S1–S3). We hypothesized that residue changes on the key phobic cluster to the corresponding DnaK residue (Arg) also allosteric interfaces are particularly influential to the Hsp70 al- favors domain undocking, with 61% docked population in the losteric mechanisms. In particular, given the preference for the ATP-bound state. In DnaK, a soft mutation in the linker, L390V, domain-docked state in HspA1 relative to DnaK, we anticipated has been shown to stabilize the NBD–β-SBD interface in the that the residue changes responsible for their distinct allosteric docked state (13). A comparable mutation in HspA1, L392V, landscapes would either stabilize the NBD–SBD interface or exhibits the same effect to favor domain docking (Fig. 5 and SI destabilize the β-SBD–α-lid interface. To test this hypothesis, we Appendix, Fig. S13C). L390 (L392 in HspA1) interacts with T215 made HspA1 variants with a V163R, I164L, I216T, L392V, or in the NBD hydrophobic cleft in the ATP-bound DnaK struc- R509E mutation (Fig. 5 and SI Appendix, Fig. S13) on the NBD– ture. This Thr becomes an Ile (I216) in HspA1. Thus, the in- SBD interface in the homology model of HspA1, based on the creased hydrophobicity in this site is expected to contribute to known structures of DnaK. The mutations were based on A1 the elevated domain-docking preference in HspA1. This expec- (residues 1 to 555)y construct, and their docking/undocking be- tation is confirmed by that fact that the I216T variant of HspA1 haviors were compared with A1(residues 1 to 555)y in the ATP- mutant populates the docked conformation to a lesser extent bound state. These mutations were designed to perturb the (73%) in the ATP-bound state. docking/undocking equilibrium in a way that can be rationalized The HspA1(residues 1 to 641) I164L and R509E mutants by the nature of the mutation. Mutation of conserved residues display significantly higher basal ATPase rates than that of wild should perturb the populations of the docked and undocked type (SI Appendix, Fig. S15). This is consistent with these two states in a similar pattern for HspA1 and DnaK; for residues that mutations’ favoring domain undocking of HspA1 and increasing

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1811105115 Meng et al. Downloaded by guest on September 27, 2021 the population of the allosterically active (higher ATPase) state. In addition, we observed that the ATPase rates of both mutants can be stimulated modestly by peptide p5, with 1.5-fold stimulation for I164L and 1.7-fold for R509E (SI Appendix, Fig. S15). By comparison, no stimulation from peptide substrate is observed for the wild-type protein. In the SBD of DnaK, there are three pairs of salt bridges formed between the β-SBD and the α-lid. Examining the dis- tances between the participating atoms in these salt bridges in the HspA1 SBD demonstrates that two of them are weakened (longer) compared with those in DnaK (SI Appendix, Fig. S16A and Table S2). The weaker salt bridges in HspA1 at the β-SBD– α-lid interface are expected to favor NBD–SBD docking. We made the DnaK(residues 1 to 638) D540A,K548A mutant, which removes the salt bridges, and compared its methyl-TROSY spectrum with the spectrum of DnaK(residues 1 to 638) in the ATP/peptide-bound states (SI Appendix, Fig. S16B). Indeed, the data demonstrate that domain docking is favored in DnaK(residues 1 to 638) D540A,K548A (SI Appendix, SI Discussion). Thus, the two salt bridges stabilize the β-SBD–α-lid interface, and the decreased salt bridge strength in HspA1 compared with DnaK can favor domain docking. Discussion Fig. 6. Evolutionary tuning of Hsp70 energy landscapes. Schematic illustra- The evolutionary features of the allosteric landscapes of Hsp70s tion of allosteric landscapes of HspA1, Hsc70, and DnaK in different ligand- underlie their functional diversities. In this work, we investigated bound states. D represents the docked state; U represents the undocked state; the allosteric landscapes of the eukaryotic cytoplasmic Hsp70s and P represents the partially docked state observed in ATP/peptide-bound HspA1 and Hsc70 and compared them to that of the E. coli DnaK, with the NBD and SBD largely undocked and with the linker still bound Hsp70 DnaK. From our domain dissection, biochemical assays, to NBD (13). The energy landscapes are drawn based on NMR, ATPase, and BIOPHYSICS AND

and methyl-TROSY NMR results, we found marked differences limited proteolysis data; the barrier heights and well depths are only quali- COMPUTATIONAL BIOLOGY between the allosteric landscapes of eukaryotic Hsp70s and that tative. Binding of ATP favors NBD–SBD docking of all three Hsp70s. Peptide of DnaK. In general, eukaryotic Hsp70s favor NBD–SBD do- substrate stimulates the ATPase activity of DnaK significantly and favors NBD– main docking significantly more than DnaK does (schematically SBD undocking of ATP-bound DnaK to form a domain undocked/linker-bound, illustrated in Fig. 6). Strikingly, ADP-bound HspA1 adopts a allosterically active P state (13). In contrast, peptide substrate addition shows predominantly docked conformation (80%), and ADP-bound only minor effects on the ATP-bound state of HspA1 and cannot stimulate its Hsc70 is 46% docked. These behaviors are distinct from other ATPase activity. Thus, we show a very shallow energy well for the P state of Hsp70s that have been studied, all of which are completely or HspA1. Peptide substrate favors partial domain undocking of ATP-bound Hsc70 and stimulates its ATPase moderately. Therefore, we conclude that largely undocked in the ADP-bound state: DnaK (6, 7), BiP the P state is partially populated in ATP/peptide-bound Hsc70. We show the (ER-resident Hsp70) (28, 30), and Ssc1 (mitochondrial Hsp70) depths of the P states of the eukaryotic Hsp70s in dashed lines, because we (31). The ATP-bound state of HspA1 and Hsc70 both adopt the lack direct data to distinguish undocked from partially undocked and we rely docked conformation, which is the same as DnaK. However, on peptide ATPase activation. ADP-bound DnaK is completely undocked, model peptide substrates have different effects on the ATP- whereas ADP-bound HspA1 is predominantly docked and ADP-bound Hsc70 bound state of eukaryotic Hsp70s and DnaK. In the case of falls in between. The depths of the wells for undocked and docked states in DnaK, the ATP/peptide-bound state adopts a partially docked our landscapes reflect this striking distinction among the Hsp70s. A fuller conformation (“P” state), with the NBD and SBD largely discussion of the rationale for depicting the energy landscapes is described in undocked and the linker bound to NBD, which is correlated to SI Appendix, SI Materials and Methods. ATPase stimulation (13). In contrast, the ATP/peptide-bound HspA1 remains docked upon addition of high concentration of peptide substrate, and ATPase data show that addition of model Our previous studies demonstrated that the allosteric properties substrate peptides does not stimulate its ATPase. Thus, we of DnaK result from an energetic tug-of-war between two inter- – β –α speculate the P state is not populated in the ATP/peptide-bound domain interfaces: the NBD SBD interface and the -SBD -lid state of HspA1 (Fig. 6). For Hsc70, NMR data show that the interface. Mutations in DnaK foreshadowed that the allosteric model substrate peptide favors domain undocking (36% remains landscape of Hsp70s would be highly tunable by modulating these docked in the ATP/peptide-bound Hsc70), and peptide sub- two interfaces. By mutational studies, we demonstrate that the strates stimulate its ATPase moderately (approximately two- evolutionary residue changes occurring on these key interfaces can fold). Therefore, based on these results, we speculate that the P account for the observed differences in the allosteric behavior of state is populated in the ATP/peptide-bound state of Hsc70, but HspA1 and DnaK. Mutation of conserved residues causes similar to a lesser extent than in the case of DnaK (Fig. 6). While shifts between the populations of the docked and undocked states peptide substrate has a very minor effect on ATP-bound HspA1, for HspA1 and DnaK. In addition, it is also possible to turn DnaK it can shift ADP-bound HspA1 from 80% docked to 77% into HspA1 by tuning the NBD–SBD interface and the β-SBD– undocked. We attribute these different responses to peptide of α-lid interface. For example, a comparison of the SBD struc- ATP-bound HspA1 and ADP-bound HspA1 to be due to the tures of DnaK and HspA1 suggests that two of the three pairs of effect of nucleotide. The affinity of peptide for the ADP-bound salt bridges on the β-SBD–α-lid interface are weakened in HspA1 state of HspA1 is higher than the affinity for the ATP-bound compared with DnaK (SI Appendix, SI Discussion and Fig. S15). state; therefore, its influence on the allosteric equilibrium is The DnaK D540A,K548A mutant exhibits favored domain greater. Also, we conclude that ADP and ATP exert different docking compared with wild type, providing a good example of effects on the energetics of the docked state of HspA1. Although turning DnaK into HspA1. The hydrophobic cluster formed be- both nucleotides favor domain docking of HspA1, the ATP- tween NBD and α-lid in the docked state could also be a good bound HspA1 docked conformation is more energetically fa- target; weakening this cluster would make HspA1 more DnaK- vorable than the ADP-bound HspA1 docked conformation. like. For example, the V163R mutant in HspA1 favors domain

Meng et al. PNAS Latest Articles | 5of6 Downloaded by guest on September 27, 2021 undocking of HspA1. The corresponding R159V mutant in DnaK the two eukaryotic cytosolic Hsp70s, HspA1 and Hsc70, exhibit should favor domain docking of DnaK (more HspA1-like). Our residue variation on this interface; V163 in HspA1 becomes findings clearly show that evolutionary residue changes on inter- T163 in Hsc70. Thus, it may be possible to design small mole- faces key for allosteric communication in Hsp70s can tune their cules that recognize one of the two eukaryotic cytosolic Hsp70s on allosteric energy landscapes (Fig. 6). What are the functional this interface and perturb its allostery while leaving the other one consequences of this tuning? Distinct allosteric landscapes of unaffected. eukaryotic Hsp70s can be crucial for the diversity and specificity of The physiological implications of the different allosteric Hsp70 functions. While prokaryotic cells have highly streamlined landscapes of the prokaryotic and eukaryotic Hsp70s are not yet protein homeostasis networks with multifunctional chaperones clear; however, studies have shown that the SBD of Hsp70s can deployed for a range of functions, eukaryotes have evolved distinct adopt opened conformations to accommodate and bind folded chaperone networks to perform specialized functions (33, 34). structures or large substrates (30, 36, 37). The favored domain Eukaryotic Hsp70s interact with a broad spectrum of substrates; docking and the consequent SBD opening of eukaryotic Hsp70s poising them more delicately between docked and undocked observed in this study might be important for them to accom- states leads them to be further tunable by the affinity for a given modate an array of large substrates, given that eukaryotic pro- substrate. Moreover, their tunability enables them to engage in teomes exhibit increased complexity with a higher proportion of specific cochaperone partnerships with different Hsp40s and large proteins and more complex protein folds as compared with nucleotide-exchange factors (NEFs) for particular functions; this prokaryotes (38, 39). specificity of chaperone/cochaperone teams is consistent with the much more expanded repertoires of Hsp40s and NEFs present in Materials and Methods eukaryotes compared to prokaryotes (35). In addition to the implications of evolutionary tuning of allosteric landscapes, the Construct design, protein expression and purification, NMR spectroscopy, deeper understanding afforded by our findings may invite novel limited proteolysis experiment, ATPase assay, substrate binding assay, sequence alignment, homology model, molecular graphics, and determining approaches to the design of small-molecule modulators of Hsp70 the energy landscapes of HspA1, Hsc70, and DnaK are described in detail in SI functions. Targeting the regions shown to tune the allosteric be- Appendix, SI Materials and Methods. havior provides new target sites for designed inhibitors and acti- vators. For example, our mutagenesis data demonstrates that the –α ACKNOWLEDGMENTS. We thank Alexandra Pozhidaeva for critical reading allostery of HspA1 is very sensitive to the NBD -lid interface. of the manuscript and Anne Gershenson, Charles English, and Abhay Thakur Two mutations on this interface, V163R and I164L, can perturb for helpful discussions. This work was supported by National Institutes of thepopulationofdockedconformationsignificantly.Inparticular, Health Grants GM027616 and GM118161 (to L.M.G.).

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