The Hsp70 interdomain linker is a dynamic switch that enables allosteric communication between two structured domains
2017; Elsevier BV; Volume: 292; Issue: 36 Linguagem: Inglês
10.1074/jbc.m117.789313
ISSN1083-351X
AutoresCharles A. English, Woody Sherman, Wenli Meng, Lila M. Gierasch,
Tópico(s)Bacillus and Francisella bacterial research
ResumoHsp703 molecular chaperones play key roles in cellular protein homeostasis by binding to exposed hydrophobic regions of incompletely folded or aggregated proteins. This crucial Hsp70 function relies on allosteric communication between two well-structured domains: an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain (SBD), which are tethered by an interdomain linker. ATP or ADP binding to the NBD alters the substrate-binding affinity of the SBD, triggering functionally essential cycles of substrate binding and release. The interdomain linker is a well-structured participant in the interdomain interface in ATP-bound Hsp70s. By contrast, in the ADP-bound state, exemplified by the Escherichia coli Hsp70 DnaK, the interdomain linker is flexible. Hsp70 interdomain linker sequences are highly conserved; moreover, mutations in this region compromise interdomain allostery. To better understand the role of this region in Hsp70 allostery, we used molecular dynamics simulations to explore the conformational landscape of the interdomain linker in ADP-bound DnaK and supported our simulations by strategic experimental data. We found that while the interdomain linker samples many conformations, it behaves as three relatively ordered segments connected by hinges. As a consequence, the distances and orientations between the NBD and SBD are limited. Additionally, the C-terminal region of the linker forms previously unreported, transient interactions with the SBD, and the predominant linker-docking site is available in only one allosteric state, that with high affinity for substrate. This preferential binding implicates the interdomain linker as a dynamic allosteric switch. The linker-binding site on the SBD is a potential target for small molecule modulators of the Hsp70 allosteric cycle. Hsp703 molecular chaperones play key roles in cellular protein homeostasis by binding to exposed hydrophobic regions of incompletely folded or aggregated proteins. This crucial Hsp70 function relies on allosteric communication between two well-structured domains: an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain (SBD), which are tethered by an interdomain linker. ATP or ADP binding to the NBD alters the substrate-binding affinity of the SBD, triggering functionally essential cycles of substrate binding and release. The interdomain linker is a well-structured participant in the interdomain interface in ATP-bound Hsp70s. By contrast, in the ADP-bound state, exemplified by the Escherichia coli Hsp70 DnaK, the interdomain linker is flexible. Hsp70 interdomain linker sequences are highly conserved; moreover, mutations in this region compromise interdomain allostery. To better understand the role of this region in Hsp70 allostery, we used molecular dynamics simulations to explore the conformational landscape of the interdomain linker in ADP-bound DnaK and supported our simulations by strategic experimental data. We found that while the interdomain linker samples many conformations, it behaves as three relatively ordered segments connected by hinges. As a consequence, the distances and orientations between the NBD and SBD are limited. Additionally, the C-terminal region of the linker forms previously unreported, transient interactions with the SBD, and the predominant linker-docking site is available in only one allosteric state, that with high affinity for substrate. This preferential binding implicates the interdomain linker as a dynamic allosteric switch. The linker-binding site on the SBD is a potential target for small molecule modulators of the Hsp70 allosteric cycle. Hsp70 3The abbreviations used are: Hsp70, 70-kDa heat shock protein; NBD, nucleotide-binding domain; SBD, substrate-binding domain; MD, molecular dynamics; MSA, multiple-sequence alignment; DS, dominant state; PDB, Protein Data Bank. 3The abbreviations used are: Hsp70, 70-kDa heat shock protein; NBD, nucleotide-binding domain; SBD, substrate-binding domain; MD, molecular dynamics; MSA, multiple-sequence alignment; DS, dominant state; PDB, Protein Data Bank. molecular chaperones maintain cellular protein homeostasis by binding to exposed hydrophobic regions of incompletely folded and aggregated proteins and then allosterically shifting between states with high and low substrate affinities in response to the binding of adenine nucleotides. This mechanism enables Hsp70 chaperones to facilitate initial folding of newly synthesized proteins, protect substrates from aggregation, and facilitate assembly of complexes. Extensive work using the Escherichia coli Hsp70 DnaK as a model has shown that when Hsp70 chaperones are bound to ADP, their N-terminal nucleotide-binding domain (NBD) is disengaged (“undocked”) from the C-terminal substrate-binding domain (SBD), the helical lid of the SBD is closed over the substrate-binding cleft, and the substrate on/off binding rates to the SBD are slow with high binding affinity for substrates (1.Mayer M.P. Kityk R. Insights into the molecular mechanism of allostery in Hsp70s.Front. Mol. Biosci. 2015; 2: 58Crossref PubMed Google Scholar, 2.Gierasch L.M. Hsp70 molecular machines: versatile modular nanomachines that mediate multiple biological functions.in: Gierasch L.M. Horwich A.L. Slingsby C. Wickner S. Agard D. Structure and Action of Molecular Chaperones. World Scientific, Singapore2016: 1-48Crossref Scopus (2) Google Scholar3.Swain J.F. Dinler G. Sivendran R. Montgomery D.L. Stotz M. Gierasch L.M. Hsp70 chaperone ligands control domain association via an allosteric mechanism mediated by the interdomain linker.Mol. Cell. 2007; 26: 27-39Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). By contrast, when the chaperone is bound to ATP, the NBD docks onto the SBD, and interfaces are formed between the β-subdomain of the SBD and the NBD, and between the helical lid and the NBD (Fig. 1A) (4.Kityk R. Kopp J. Sinning I. Mayer M.P. Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones.Mol. Cell. 2012; 48: 863-874Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 5.Qi R. Sarbeng E.B. Liu Q. Le K.Q. Xu X. Xu H. Yang J. Wong J.L. Vorvis C. Hendrickson W.A. Zhou L. Liu Q. Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP.Nat. Struct. Mol. Biol. 2013; 20: 900-907Crossref PubMed Scopus (184) Google Scholar). The NBD and SBD of Hsp70s are connected via an interdomain linker. The interdomain linker participates in the allosteric transitions between the docked and undocked states in several ways. First, in the docked, ATP-bound state, a portion of the linker containing a highly conserved, hydrophobic sequence (VLLL in DnaK) binds to a pocket beneath the crossing helices of the NBD and adopts a β-strand conformation, adding to a small β-sheet in subdomain IIA of the actin-like fold of the NBD (4.Kityk R. Kopp J. Sinning I. Mayer M.P. Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones.Mol. Cell. 2012; 48: 863-874Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 5.Qi R. Sarbeng E.B. Liu Q. Le K.Q. Xu X. Xu H. Yang J. Wong J.L. Vorvis C. Hendrickson W.A. Zhou L. Liu Q. Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP.Nat. Struct. Mol. Biol. 2013; 20: 900-907Crossref PubMed Scopus (184) Google Scholar) (Fig. 1B). Substrate binding to the SBD results in an increase in the ATPase activity of the NBD, thus enabling rapid transitions between the high- and low-affinity states. Strikingly, binding of the linker to the NBD is sufficient to shift the NBD conformation and stimulate the rate of hydrolysis of ATP to ADP even in the absence of the SBD (3.Swain J.F. Dinler G. Sivendran R. Montgomery D.L. Stotz M. Gierasch L.M. Hsp70 chaperone ligands control domain association via an allosteric mechanism mediated by the interdomain linker.Mol. Cell. 2007; 26: 27-39Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 6.Vogel M. Mayer M.P. Bukau B. Allosteric regulation of Hsp70 chaperones involves a conserved interdomain linker.J. Biol. Chem. 2006; 281: 38705-38711Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), showing that the binding of the linker to the NBD is a key event in allosteric communication between the NBD and the SBD. Second, the linker contributes to allosteric functioning in the undocked state by acting as a flexible tether and enabling the domains to behave as independent units. The tethered domains adopt conformational states that are essentially the same as the structures they assume when isolated from the rest of the system (3.Swain J.F. Dinler G. Sivendran R. Montgomery D.L. Stotz M. Gierasch L.M. Hsp70 chaperone ligands control domain association via an allosteric mechanism mediated by the interdomain linker.Mol. Cell. 2007; 26: 27-39Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 7.Bertelsen E.B. Chang L. Gestwicki J.E. Zuiderweg E.R. Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 8471-8476Crossref PubMed Scopus (356) Google Scholar). When acting as a tether, the interdomain linker samples an ensemble of structures, apparently behaving as an intrinsically disordered segment: It is proteolytically labile (8.Buchberger A. Theyssen H. Schröder H. McCarty J.S. Virgallita G. Milkereit P. Reinstein J. Bukau B. Nucleotide-induced conformational changes in the ATPase and substrate binding domains of the DnaK chaperone provide evidence for interdomain communication.J. Biol. Chem. 1995; 270: 16903-16910Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 9.Kamath-Loeb A.S. Lu C.Z. Suh W.C. Lonetto M.A. Gross C.A. Analysis of three DnaK mutant proteins suggests that progression through the ATPase cycle requires conformational changes.J. Biol. Chem. 1995; 270: 30051-30059Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and gives rise to sharp resonances with little dispersion in NMR spectra (3.Swain J.F. Dinler G. Sivendran R. Montgomery D.L. Stotz M. Gierasch L.M. Hsp70 chaperone ligands control domain association via an allosteric mechanism mediated by the interdomain linker.Mol. Cell. 2007; 26: 27-39Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 10.Zhuravleva A. Clerico E.M. Gierasch L.M. An interdomain energetic tug-of-war creates the allosterically active state in Hsp70 molecular chaperones.Cell. 2012; 151: 1296-1307Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). However, the linker imposes some restrictions on the relative orientations of the NBD and SBD, as shown by NMR analysis of the ADP-bound DnaK in which interdomain paramagnetic relaxation data were consistent with restriction of NBD and SBD relative orientations to a 35° cone (7.Bertelsen E.B. Chang L. Gestwicki J.E. Zuiderweg E.R. Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 8471-8476Crossref PubMed Scopus (356) Google Scholar) (Fig. 1C). Teleologically, one could argue that these restrictions limit the amount of conformational space searched to find the transition pathway to the docked state (i.e. reducing the entropy loss upon interconverting between states, thereby facilitating the transitions). Hence, the conformational sampling of the interdomain linker must be finely tuned throughout the Hsp70 allosteric cycle. A detailed picture of the conformational landscape of the Hsp70 interdomain linker is lacking, particularly in the ADP-bound state, where the linker populates an ensemble of conformations, and the adjacent domains are structured but undocked. It is crucial that the linker behavior be examined in the presence of its neighboring domains, because transient interactions such as those proposed based on mutagenesis of the linker (6.Vogel M. Mayer M.P. Bukau B. Allosteric regulation of Hsp70 chaperones involves a conserved interdomain linker.J. Biol. Chem. 2006; 281: 38705-38711Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) may play an important role in shaping the linker conformational ensemble. Thus, the full Hsp70 molecule (70 kDa) must be analyzed to derive an accurate structural ensemble of the interdomain linker in the undocked state. To retain the key atomic level details necessary to precisely characterize interfaces between the linker and the domains, we used all-atom molecular dynamics (MD) of the intact DnaK system. Other groups have deployed powerful computational approaches to pose questions about the DnaK system, and their findings have been interpreted in terms of its allosteric function, although the linker conformational landscape was not the primary focus of these studies. For example, Morra and co-workers (11.Chiappori F. Merelli I. Colombo G. Milanesi L. Morra G. Molecular mechanism of allosteric communication in Hsp70 revealed by molecular dynamics simulations.PLoS Comput. Biol. 2012; 8: e1002844Crossref PubMed Scopus (46) Google Scholar, 12.Chiappori F. Merelli I. Milanesi L. Colombo G. Morra G. An atomistic view of Hsp70 allosteric crosstalk: from the nucleotide to the substrate binding domain and back.Sci. Rep. 2016; 6: 23474Crossref PubMed Scopus (30) Google Scholar) simulated the initial steps in the allosteric transition of DnaK from its ADP-bound to its ATP-bound state. For ADP and substrate-bound DnaK (12.Chiappori F. Merelli I. Milanesi L. Colombo G. Morra G. An atomistic view of Hsp70 allosteric crosstalk: from the nucleotide to the substrate binding domain and back.Sci. Rep. 2016; 6: 23474Crossref PubMed Scopus (30) Google Scholar), these authors observed a state with the two domains collapsed on one another and several contacts between the linker and the SBD. Stetz and Verkhiver (13.Stetz G. Verkhivker G.M. Dancing through life: molecular dynamics simulations and network-centric modeling of allosteric mechanisms in Hsp70 and Hsp110 chaperone proteins.PLoS One. 2015; 10: e0143752Crossref PubMed Scopus (39) Google Scholar, 14.Stetz G. Verkhivker G.M. Computational analysis of residue interaction network and coevolutionary relationships in the Hsp70 chaperones: a community-hopping model of allosteric regulation communication.PLoS Comput. Biol. 2017; 13: e1005299Crossref PubMed Scopus (65) Google Scholar) applied a novel network model that incorporates evolutionary sequence information and MD-derived trajectories to elucidate potential allosteric communication mechanisms. Their approach identified several candidate residue interaction patterns that may be important in allostery. Finally, Penkler et al. (15.Penkler D. Sensoy Ö. Atilgan C. Tastan Bishop Ö. Perturbation-response scanning reveals key residues for allosteric control in Hsp70.J. Chem. Inf. Model. 2017; 57: 1359-1374Crossref PubMed Scopus (47) Google Scholar) used MD to study structural perturbations brought about by the binding and unbinding of nucleotides and substrates. They report the residues that undergo the largest, induced structural perturbations and infer that these are probably critical to allostery. Among these were residues Lys387–Val389, Leu391, and Asp393 in the linker, consistent with the present work. They also reported several linker interactions with the SBD, but no specific contacts were described. Here, we have focused on the conformational behavior that underlies a crucial role of the linker in allostery in the elusive conformationally dynamic undocked state of DnaK. Our work reveals that the DnaK interdomain linker conformational landscape comprises a limited set of conformations, even in the undocked, ADP-bound state. In addition to the well-defined structure seen in crystal structures of ATP-bound DnaK, in the undocked state, the linker behaves as a hinged tether, with locally ordered segments connected by hinge residues, thus enabling some relative motion of the two tethered domains but restricting both the interdomain distance and the relative orientations of the domains significantly. We further show that in the ADP-bound state of DnaK, the conserved hydrophobic segment of the linker forms specific but transient contacts with the SBD. The picture that emerges from this analysis presents the interdomain linker as a dynamic switch that performs a key role in interdomain allostery through alternative interactions with either the NBD or the SBD and restricted interdomain relative orientations. This improved understanding of the interdomain linker interactions broadens the potential to interfere in Hsp70 function by targeting specific sites with small molecule modulators, which could be used as chemical probes or as the basis for the development of novel therapeutics. In addition, our observations offer an explanation for several evolutionarily conserved features of the interdomain linker. Based on inspection of prokaryotic Hsp70 sequences and domain X-ray crystal structures, we defined the DnaK linker region as 384GDVKDVLLLDVT395 because Gly384 terminates a highly conserved α-helix in the NBD (PDB code 1DKG) (16.Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F. Kuriyan J. Crystal structure of the nucleotide exchange factor GrpE bound to the ATPase domain of the molecular chaperone, DnaK.Science. 1997; 276: 431-435Crossref PubMed Scopus (413) Google Scholar), and the residue following Thr395 (Pro396) is the first residue belonging to secondary structure associated with the SBD (PDB code 1DKX) (17.Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Structural analysis of substrate binding by the molecular chaperone DnaK.Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1059) Google Scholar). The electron density of the linker is not well-resolved in X-ray crystal structures of DnaK domains, arguing that it is a flexible interdomain tether. We compared 640 Hsp70 interdomain linker sequences using a multiple-sequence alignment (MSA) (18.Smock R.G. Rivoire O. Russ W.P. Swain J.F. Leibler S. Ranganathan R. Gierasch L.M. An interdomain sector mediating allostery in Hsp70 molecular chaperones.Mol. Syst. Biol. 2010; 6: 414Crossref PubMed Scopus (104) Google Scholar). We find that many eukaryotic interdomain linkers are longer than those from prokaryotic Hsp70s; the increased length arises from an insertion of three residues between positions 384 and 387 and one residue between residue 387 and 388 (numbered according to the E. coli DnaK sequence). A few fungal Hsp70 interdomain linker sequences are one residue longer than most eukaryotic, and a few are outliers (e.g. the sequence from white spruce is one residue longer than the sequence of prokaryotes). Fig. 2 shows a representative set of Hsp70 interdomain linker sequences and the sequence conservation logos (19.Crooks G.E. Hon G. Chandonia J.-M. Brenner S.E. WebLogo: a sequence logo generator.Genome Res. 2004; 14: 1188-1190Crossref PubMed Scopus (8235) Google Scholar) calculated for the full MSA. Many residues in the linker sequence are highly conserved. In particular, Gly384, Asp388, Leu391, Asp393, and Val394 are over 80% conserved in the MSA. Two regions can readily be identified in the interdomain linker: first, an N-terminal region that is predominantly hydrophilic, 384GDVKD388 in DnaK. This region is moderately conserved among Hsp70s, with variation in length but retention of hydrophilic character with the exception of Val386, which is highly conserved (68% hydrophobic). This region is followed by a strongly conserved (at least 90% conserved in residue type) hydrophobic region, 389VLLLDVT395 in DnaK. In the last three-residue region, 393DVT395 in E. coli DnaK, the DV sequence is nearly 100% conserved, and Thr395 is nearly always Thr or Ala, with occasional substitution by other small uncharged, non-aromatic residues. Secondary structure predictions for the linker sequence suggest that whereas the hydrophilic region is random coil, the 389VLLLD393 region is predicted to be an extended strand (20.Rost B. Sander C. Combining evolutionary information and neural networks to predict protein secondary structure.Proteins. 1994; 19: 55-72Crossref PubMed Scopus (1337) Google Scholar). In the ATP-bound structures of DnaK (PDB codes 4B9Q (4.Kityk R. Kopp J. Sinning I. Mayer M.P. Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones.Mol. Cell. 2012; 48: 863-874Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar) and 4JN4 (5.Qi R. Sarbeng E.B. Liu Q. Le K.Q. Xu X. Xu H. Yang J. Wong J.L. Vorvis C. Hendrickson W.A. Zhou L. Liu Q. Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP.Nat. Struct. Mol. Biol. 2013; 20: 900-907Crossref PubMed Scopus (184) Google Scholar)) and the human Hsp70 that resides in the endoplasmic reticulum (BiP) (PDB code 5E84 (21.Yang J. Nune M. Zong Y. Zhou L. Liu Q. Close and allosteric opening of the polypeptide-binding site in a human Hsp70 chaperone BiP.Structure. 2015; 23: 2191-2203Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar)), the interdomain linker adopts a well-defined structure in which the conserved C-terminal hydrophobic sequence, VLLL (Fig. 2), is incorporated into subdomain IIA of the NBD as an antiparallel fourth β-strand on the edge of the small sheet (Fig. 1B). The crystal structure of the ATP-bound form of BiP also provides a picture of the conformation adopted by a mammalian interdomain linker with its additional five residues in the hydrophilic-dominated region (21.Yang J. Nune M. Zong Y. Zhou L. Liu Q. Close and allosteric opening of the polypeptide-binding site in a human Hsp70 chaperone BiP.Structure. 2015; 23: 2191-2203Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The hydrophilic-dominated region of the linker for both Hsp70s forms a solvent-exposed loop (Fig. 1B) with a fold that has been termed a “niche-4l motif” (22.Torrance G.M. Leader D.P. Gilbert D.R. Milner-White E.J. A novel main chain motif in proteins bridged by cationic groups: the niche.J. Mol. Biol. 2009; 385: 1076-1086Crossref PubMed Scopus (21) Google Scholar). This motif generally allows for metal ion binding, but in this case, it enables a large amount of the surface area of the loop to be solvent-exposed. In contrast, the hydrophobic-dominated region adopts a β-sheet configuration. This arrangement shields the hydrophobic region from the solvent. It is noteworthy that all of these structures represent a low-energy state adopted when the given Hsp70 is ATP-bound and under the conditions of crystallization (i.e. in the crystal lattice and for the particular construct crystallized). In one structure (PDB code 4JN4 (5.Qi R. Sarbeng E.B. Liu Q. Le K.Q. Xu X. Xu H. Yang J. Wong J.L. Vorvis C. Hendrickson W.A. Zhou L. Liu Q. Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP.Nat. Struct. Mol. Biol. 2013; 20: 900-907Crossref PubMed Scopus (184) Google Scholar)), several mutations were incorporated into loops of DnaK to facilitate crystallization of the ATP-bound form (5.Qi R. Sarbeng E.B. Liu Q. Le K.Q. Xu X. Xu H. Yang J. Wong J.L. Vorvis C. Hendrickson W.A. Zhou L. Liu Q. Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP.Nat. Struct. Mol. Biol. 2013; 20: 900-907Crossref PubMed Scopus (184) Google Scholar), and in the other structure (PDB code 4B9Q (4.Kityk R. Kopp J. Sinning I. Mayer M.P. Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones.Mol. Cell. 2012; 48: 863-874Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar)), a disulfide was introduced between the α-helical lid and the NBD to achieve crystallization. To verify that the linker would adopt the conformation seen in these crystal structures when in the context of wild-type DnaK and relieved of the constraints of the crystal lattice, we ran 15-ns MD simulations to relax the docked ATP-bound crystal structure of full-length, wild-type DnaK with implicit solvent (see “Experimental procedures” for details). We found that the linker retains the backbone conformation seen in the crystal structures, and most side chains do not change their conformations significantly, with the exception of the Lys387 side chain, which fluctuated among several solvent-exposed conformations. To explore the conformational ensemble of the interdomain linker in the context of the NBD and SBD, we ran all-atom MD simulations on the undocked ADP-bound state of DnaK (for details, see “Experimental procedures”). A major consideration in calculating the conformational landscapes for molecules as large as DnaK is the completeness of the conformational exploration. To assess whether our simulations fully explored conformational space without becoming trapped in a local minimum, we ran three independent trajectories from different starting structures. The first trajectory, lasting 600 ns, started from the reported NMR-derived ADP-bound DnaK structure (PDB code 2KHO) (7.Bertelsen E.B. Chang L. Gestwicki J.E. Zuiderweg E.R. Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 8471-8476Crossref PubMed Scopus (356) Google Scholar). In the second, a 200-ns simulation, the initial NBD and SBD structures were unchanged, but the linker was computationally altered to an α-helical conformation. A third, short (200-ns) simulation was run with the linker initially in a β-sheet conformation, again retaining the NBD and SBD structures from PDB entry 2KHO (7.Bertelsen E.B. Chang L. Gestwicki J.E. Zuiderweg E.R. Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 8471-8476Crossref PubMed Scopus (356) Google Scholar) while altering their relative orientations. In all three trajectories, the linker rapidly (within 25 ns of the start of the simulation) began to populate the same states (supplemental Fig. S1). Furthermore, no new states were populated in any of the additional simulations, suggesting that the relevant regions of the conformational landscape were fully explored. The overall ADP–DnaK structure behaved very similarly in our MD trajectories as it did in the model that emerged from NMR studies carried out by Bertelsen et al. (7.Bertelsen E.B. Chang L. Gestwicki J.E. Zuiderweg E.R. Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 8471-8476Crossref PubMed Scopus (356) Google Scholar) (supplemental Table S1). In particular, the NBD and SBD were observed to reorient with respect to each another as though tethered by a flexible linker; however, in agreement with the conclusions of the NMR studies, our careful analysis of the conformational explorations of the linker revealed that the relative motion of the two domains was restricted. Moreover, the linker end-to-end distance varied from 15 to 30 Å with 95% of the population between 15 and 19 Å in a near Gaussian distribution (see supplemental Fig. S2). Thus, the conformational states populated by the interdomain linker play a dual function: They restrict both the relative orientations sampled by the NBD and SBD and the interdomain distance fluctuations. The predominant secondary structures adopted by linker residues are shown in Fig. 3. For comparison purposes, we also simulated the linker in the absence of the NBD and SBD. We found that the interdomain linker in the absence of any influence from neighboring domains has a high propensity to adopt a significant amount of helical secondary structure (supplemental Fig. S3), indicating that it possesses its own inherent structural properties and that its behavior in the full chaperone is strongly influenced by adjacent NBD and SBD. Although it is somewhat structurally restricted, the interdomain linker in ADP-bound DnaK resembles an intrinsically disordered region in that many conformational states are visited over the course of the trajectory. The conformational ensemble of the linker is depicted in an overlay of 60 structures from every 10 ns of the 600-ns MD trajectory (Fig. 4A, top). Strikingly, when different regions of the linker were superimposed separately, we found that the apparently disordered linker is in fact a composite of three relatively ordered regions with persistent conformational preferences (see Fig. 4A): residues 384–386 (part of the N-terminal hydrophilic region); residues 388–390 (the hydrophobic central region); and residues 392–395 (encompassing the conserved C-terminal sequence LDVT). Between the ordered regions are hinge points (at residues Lys387 and Leu391), which allow the linker to adopt multiple conformations that comprise an ensemble, but a restricted ensemble. We overlaid linker conformations to determine how many distinct states are allowed under the restrictions imposed by the hinge residues. From the overlays in Fig. 4A, we see that the hydrophobic central region is the most ordered of the three regions and adopts an α-helical conformation. The N-terminal region possesses some limited flexibility, mostly concentrated in the initial two residues, Gly384 and Asp385, and samples (ϕ, ψ) angles near (80°, 20°) and (−75°, −25°), respectively. By contrast, the C-terminal region clearly comprises alternative conformational states, although its overlay shows some clustering. By projecting the sampled conformational space on coordinates that sensitively report on excursions of this region, we found one major energy minimum and three minor minima (Fig. 4B). The dominant state (DS) represents 80% of the population and may therefore be the most relevant state for allosteric communication. The overlays for each of the linker energy minima are shown in Fig. 4C alo
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