Abstract: An essential cellular machinery that has been identified and studied only relatively recently is a collective of specialized proteins, molecular chaperones, that bind nonnative states of other proteins and assist them to reach a functional conformation, in most cases through the expenditure of ATP. Originally identified by their increased abundance following heat shock, chaperone proteins in general recognize exposed hydrophobic surfaces of nonnative species, surfaces that will ultimately be buried in the native state, and form noncovalent interactions with them, stabilizing them against irreversible multimeric aggregation. Release of polypeptide then follows, in many cases driven by an ATP-directed conformational change of the chaperone, permitting subsequent steps of polypeptide folding or biogenesis to occur. When such steps fail to proceed productively, recognition and rebinding by the same or another chaperone can occur, allowing another opportunity for a productive conformation to be reached. Different classes of molecular chaperones appear to be directed to binding specific nonnative states, the nature of which are beginning to be understood. For example, the two best-studied families, examined in detail below, the ubiquitous Hsp70 and Hsp60 (chaperonin) chaperones, recognize hydrophobic surface in the context of, respectively, extended and collapsed (globular) conformations, which are bound correspondingly either by local enclosure of the chain or by global enclosure of the polypeptide in a central cavity. Because there is not yet the detailed level of structural and mechanistic understanding for other recognized families of chaperones, they are not considered here, but important observations concerning binding, nucleotide use, and cellular actions are summarized in Table 1.Table 1Topology of Polypeptide Binding and Action of Chaperone Families Hsp70 chaperones, with their co-chaperones, comprise a set of abundant cellular machines that assist a large variety of protein folding processes in almost all cellular compartments. Historically, they were identified by induction under conditions of stress, during which they are now known to provide an essential action of preventing aggregation and assisting refolding of misfolded proteins. But they also play an essential role under normal conditions, including (1) assisting folding of some newly translated proteins; (2) guiding translocating proteins across organellar membranes through action at both the cis and trans sides; (3) disassembling oligomeric protein structures; (4) facilitating proteolytic degradation of unstable proteins; and in selected cases, (5) controlling the biological activity of folded regulatory proteins, including transcription factors (for a discussion of these actions, see69Morimoto R.I. Tissieres A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar, 40Hartl F.U. Molecular chaperones in cellular protein folding.Nature. 1996; 381: 571-580Crossref PubMed Scopus (2944) Google Scholar). All of these activities rely on the ATP-regulated association of Hsp70 with short hydrophobic segments in substrate polypeptides (23Flynn G.C. Pohl J. Flocco M.T. Rothman J.E. Peptide-binding specificity of the molecular chaperone BiP.Nature. 1991; 353: 726-730Crossref PubMed Scopus (610) Google Scholar; 83Rüdiger S. Buchberger A. Bukau B. Interaction of Hsp70 chaperones with substrates.Nature Struct. Biol. 1997; 4 (a): 342-349Crossref PubMed Scopus (264) Google Scholar, 84Rüdiger S. Germeroth L. Schneider-Mergener J. Bukau B. Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries.EMBO J. 1997; 16 (b): 1501-1507Crossref PubMed Scopus (598) Google Scholar), which prevents further folding or aggregation by shielding these segments. In Hsp70-assisted folding reactions, substrates undergo repeated cycles of binding/release (92Szabo A. Langer T. Schröder H. Flanagan J. Bukau B. Hartl F.U. The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system-DnaK, DnaJ and GrpE.Proc. Natl. Acad. Sci. USA. 1994; 91: 10345-10349Crossref PubMed Scopus (425) Google Scholar, 9Buchberger A. Schröder H. Hesterkamp T. Schönfeld H.-J. Bukau B. Substrate shuttling between the DnaK and GroEL systems indicates a chaperone network promoting protein folding.J. Mol. Biol. 1996; 261: 328-333Crossref PubMed Scopus (131) Google Scholar), frequently at a stoichiometry of a single Hsp70 monomer per substrate molecule. Hsp70 binding does not appear to induce global conformational changes in the substrate but, rather, appears to act locally. Substrates released from the chaperone undergo kinetic partitioning between folding to native state, aggregation, rebinding to Hsp70, and binding to other chaperones or proteases as part of a multidirectional folding network. Hsp70 proteins all consist of the same working parts: a highly conserved NH2-terminal ATPase domain of 44 kDa and a COOH-terminal region of 25 kDa, divided into a conserved substrate binding domain of 15 kDa and a less-conserved immediate COOH-terminal domain of 10 kDa (Figure 1). The molecular basis for Hsp70 binding to many nonnative proteins has been elucidated for the bacterial cytoplasmic homolog, DnaK, through biochemical and crystallographic studies. Studies with peptides have indicated that DnaK binds with greatest affinity to short hydrophobic segments in extended conformation (87Schmid D. Baici A. Gehring H. Christen P. Kinetics of molecular chaperone action.Science. 1994; 263: 971-973Crossref PubMed Scopus (404) Google Scholar, 111Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M. Hendrickson W.A. Structural analysis of substrate binding by the molecular chaperone DnaK.Science. 1996; 272: 1606-1614Crossref PubMed Scopus (982) Google Scholar). To qualify as a substrate, it thus seems a minimal requirement that a protein expose a single recognizable segment, either through local unfolding or as an intrinsically unfolded structural element, such as a loop. The crystal structure of the COOH-terminal substrate binding domain of DnaK complexed with a heptapeptide substrate, NRLLLTG (111Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M. Hendrickson W.A. Structural analysis of substrate binding by the molecular chaperone DnaK.Science. 1996; 272: 1606-1614Crossref PubMed Scopus (982) Google Scholar), reveals a β sandwich composed of two sheets of four strands each, followed in the primary structure by two α helices, A and B, which span back over the sandwich (Figure 2A). The top sheet emanates four loops, two of which (L1,2–L3,4) form the substrate binding pocket, a channel with a cross section of ∼5 × 7 Å. Along with loops flanking them at either side, they are stabilized by critical contacts with the overlying helix B, which may function as a lid in permitting entry and release of substrate (without directly contacting it) (Figure 2A and Figure 2B). The peptide in the substrate binding pocket pierces the narrow dimension of the domain and is contacted by DnaK only through its central five residues (Figure 2C and Figure 2D). The most extensive contacts are hydrophobic side-chain contacts between the loops and the three central leucines in the peptide (L3–5, Figure 2D). The central-most leucine side chain resides in a hydrophobic pocket in the floor of the channel and is surmounted by a hydrophobic “arch” that also interacts with the hydrophobic side chains of the neighboring leucine residues in the peptide. Seven hydrogen bonds are also observed between the peptide and DnaK, formed between the main chain of the peptide and, in most cases, the main chain of DnaK. These contacts, coupled with the hydrophobic specificity-determining pockets, dictate the requirement for an extended conformation of the bound peptide. The interactive surface of DnaK at the ends of the hydrophobic channel is negatively charged and favors the presence of basic residues at the end positions of the peptide (e.g., arginine at position 2). More globally, binding/enclosure of the extended peptide appears to require that the interacting polypeptide segment be separated from the remainder of the substrate protein by 10 Å or more, implying that the bound region of the polypeptide must be substantially unfolded. . A consensus motif recognized by DnaK in substrate polypeptides has been identified by screening a library of peptides derived from known protein sequences (84Rüdiger S. Germeroth L. Schneider-Mergener J. Bukau B. Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries.EMBO J. 1997; 16 (b): 1501-1507Crossref PubMed Scopus (598) Google Scholar) and comprises a hydrophobic core of 4–5 residues flanked by basic residues. The hydrophobic cores of individual peptides recognized by DnaK contain typically 2–4 hydrophobic residues, with Leu the most common, present in ∼90% of recognized peptides. Acidic residues are excluded from the cores and disfavored in the flanking regions. Such a motif occurs frequently within protein sequences, every 36 residues on average, and localizes preferentially to buried β strands of the corresponding folded proteins. The motif identified by this experimental approach corresponds remarkably well with the observed features of interaction of the NRLLLTG peptide from the structural study. ATP, bound by the NH2-terminal domain of Hsp70, is used to drive conformational changes in the COOH-terminal peptide binding domain that alter its affinity for substrates (Figure 3). The binding of ATP increases the dissociation constant for Hsp70–substrate complexes between 5- and 85-fold as a result of increases in koff of 2–3 orders of magnitude, coupled with increases in kon of ∼50-fold (73Palleros D.R. Reid K.L. Shi L. Welch W.J. Fink A.L. ATP-induced protein-Hsp70 complex dissociation requires K+ but not ATP hydrolysis.Nature. 1993; 365: 664-666Crossref PubMed Scopus (340) Google Scholar, 87Schmid D. Baici A. Gehring H. Christen P. Kinetics of molecular chaperone action.Science. 1994; 263: 971-973Crossref PubMed Scopus (404) Google Scholar, 65McCarty J.S. Buchberger A. Reinstein J. Bukau B. The role of ATP in the functional cycle of the DnaK chaperone system.J. Mol. Biol. 1995; 249: 126-137Crossref PubMed Scopus (333) Google Scholar, 96Theyssen H. Schuster H.-P. Bukau B. Reinstein J. The second step of ATP binding to DnaK induces peptide release.J. Mol. Biol. 1996; 263: 657-670Crossref PubMed Scopus (191) Google Scholar, 75Pierpaoli E.V. Sandmeier E. Baici A. Schönfeld H.-J. Gisler S. Christen P. The power stroke of the DnaK/DnaJ/GrpE molecular chaperone system.J. Mol. Biol. 1997; 269: 757-768Crossref PubMed Scopus (107) Google Scholar). The ATPase cycle of Hsp70 can thus be viewed, in its simplest form, as an alternation between two states: the ATP-bound state, with low affinity and fast exchange rates for substrates (substrate binding pocket open), and the ADP-bound state, with high affinity and slow exchange rates for substrates (substrate binding pocket closed) . From the kinetic parameters of these two states, it is clear that the rapid association of Hsp70 with substrates can only occur in the ATP state, because substrate binding to the ADP state is too slow on the time scale of folding reactions. Dissection of ATP binding reveals that it occurs in two steps: first, the rapid formation of a weak complex, followed by a slower structural rearrangement (37Ha J.-H. McKay D.B. Kinetics of nucleotide-induced changes in the tryptophane fluorescence of the molecular chaperone Hsc70 and its subfragments suggest the ATP-induced conformational change follows initial ATP binding.Biochemistry. 1995; 34: 11635-11644Crossref PubMed Scopus (73) Google Scholar, 96Theyssen H. Schuster H.-P. Bukau B. Reinstein J. The second step of ATP binding to DnaK induces peptide release.J. Mol. Biol. 1996; 263: 657-670Crossref PubMed Scopus (191) Google Scholar), leading to an overall Kd for ATP in the submicromolar range (29Gao B. Greene L. Eisenberg E. Characterization of nucleotide-free uncoating ATPase and its binding to ATP, ADP and ATP analogues.Biochemistry. 1994; 33: 2048-2054Crossref PubMed Scopus (45) Google Scholar, 36Ha J.-H. McKay D.B. ATPase kinetics of recombinant bovine 70 kDa heat shock cognate protein and its amino-terminal ATPase domain.Biochemistry. 1994; 33: 14625-14635Crossref PubMed Scopus (67) Google Scholar, 96Theyssen H. Schuster H.-P. Bukau B. Reinstein J. The second step of ATP binding to DnaK induces peptide release.J. Mol. Biol. 1996; 263: 657-670Crossref PubMed Scopus (191) Google Scholar). The second step probably reflects the precise locking-in of the nucleotide in the binding pocket, essential for hydrolysis (see below), and is kinetically coupled to the release or exchange of a previously bound polypeptide. The subsequent conversion of the ATP–peptide–Hsp70 ternary complex to the ADP state then stabilizes the chaperone–peptide interaction. The importance of this step has been demonstrated by the finding that mutant Hsp70 proteins, arrested in the ATP-bound state due to defects in hydrolysis, are completely deficient in chaperone activities (38Ha, J.-H., Johnson, E.R., McKay, D.B., Sousa, M.C., Takeda, S., and Wilbanks, S.M. (1997). Structure and properties of the 70-kilodalton heat-shock proteins. In Molecular Chaperones in the Life Cycle of Proteins, A.L. Fink and Y. Goto, eds. (New York: Marcel Dekker Inc.), pp. 35–122.Google Scholar). Hydrolysis of ATP is the rate-limiting step in the ATPase cycle of Hsp70 proteins in isolation (28Gao B. Yumiko E. Greene L. Eisenberg E. Nucleotide binding properties of bovine brain uncoating ATPase.J. Biol. Chem. 1993; 268: 8507-8513Abstract Full Text PDF PubMed Google Scholar, 65McCarty J.S. Buchberger A. Reinstein J. Bukau B. The role of ATP in the functional cycle of the DnaK chaperone system.J. Mol. Biol. 1995; 249: 126-137Crossref PubMed Scopus (333) Google Scholar, 48Karzai A.W. McMacken R. A bipartite signaling mechanism involved in DnaJ-mediated activation of the Escherichia coli DnaK protein.J. Biol. Chem. 1996; 271: 11236-11246Crossref PubMed Scopus (189) Google Scholar, 96Theyssen H. Schuster H.-P. Bukau B. Reinstein J. The second step of ATP binding to DnaK induces peptide release.J. Mol. Biol. 1996; 263: 657-670Crossref PubMed Scopus (191) Google Scholar) and likely results in dramatic conformational changes in Hsp70 that convert it to the high affinity, slow exchange state, which sequesters substrate protein. The final step in the ATPase cycle, the release of ADP and Pi, does not induce detectable conformational changes but allows the subsequent rapid binding of ATP, which reestablishes the starting point. Although nucleotide exchange is 10–20-fold faster than the rate of hydrolysis in the unstimulated cycle (65McCarty J.S. Buchberger A. Reinstein J. Bukau B. The role of ATP in the functional cycle of the DnaK chaperone system.J. Mol. Biol. 1995; 249: 126-137Crossref PubMed Scopus (333) Google Scholar, 96Theyssen H. Schuster H.-P. Bukau B. Reinstein J. The second step of ATP binding to DnaK induces peptide release.J. Mol. Biol. 1996; 263: 657-670Crossref PubMed Scopus (191) Google Scholar), it can become rate-limiting when the hydrolysis step is stimulated by co-chaperones (see below). The nearly identical structures of the ATPase domains of DnaK and Hsc70 consist of two large, globular subdomains (I and II), separated by a deep central cleft and connected by two crossed α helices (Figure 3B and Figure 3C). Both subdomains and the connecting helices contribute to forming the binding pocket for nucleotide and the required Mg2+ and K+ ions at the bottom of the cleft (20Flaherty K.M. Deluca-Flaherty C. McKay D.B. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein.Nature. 1990; 346: 623-628Crossref PubMed Scopus (782) Google Scholar). The nucleotide is positioned in the active site by interactions with two phosphate-binding loops and a hydrophobic adenosine binding pocket (20Flaherty K.M. Deluca-Flaherty C. McKay D.B. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein.Nature. 1990; 346: 623-628Crossref PubMed Scopus (782) Google Scholar), together with contacts with the Mg2+ ion, which is coordinated by several side chains of Hsc70. Based on structural studies of wild-type and mutant Hsc70 proteins in the presence of a variety of nucleotides and metals, McKay and coworkers have proposed a mechanism for ATP hydrolysis (21Flaherty K.M. Wilbanks S.M. DeLuca-Flaherty C. McKay D.B. Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. II. Structure of the active site with ADP or ATP bound to wild type or mutant ATPase fragment.J. Biol. Chem. 1994; 269: 12899-12907Abstract Full Text PDF PubMed Google Scholar, 71O'Brien M.C. Flaherty K.M. McKay D.B. Lysine 71 of the chaperone protein Hsc70 is essential for ATP hydrolysis.J. Biol. Chem. 1996; 271: 15874-15878Crossref PubMed Scopus (109) Google Scholar): structural rearrangement of Hsp70 during ATP binding leads to adjustment of the position of the γ-phosphate so that a bidentate complex is formed between the β- and γ-phosphate oxygens and Mg2+, permitting an in-line attack by a water (or OH−) that is hydrogen-bonded to Lys-71 (Figure 3C). Precise geometry of the nucleotide and the surrounding residues requires the correct positioning of the Mg2+ ion, established in part by the binding of two K+ ions nearby. This accounts for the absolute requirement of K+ for ATP hydrolysis and chaperone activity of Hsp70 proteins (73Palleros D.R. Reid K.L. Shi L. Welch W.J. Fink A.L. ATP-induced protein-Hsp70 complex dissociation requires K+ but not ATP hydrolysis.Nature. 1993; 365: 664-666Crossref PubMed Scopus (340) Google Scholar). The molecular mechanism by which the chemical energy of ATP is used to perform mechanical work, that is, the opening and closing of the substrate binding pocket, is poorly understood. The available atomic structures of the Hsc70 ATPase domain do not indicate large-scale motions in response to either nucleotide binding or hydrolysis. Either there are subtle conformational changes in the ATPase domain that are amplified to produce dramatic changes in the rest of the chaperone or the crystallized ATPase domain fragments do not reflect conformational changes occurring in the full-length protein in response to the nucleotide. Supporting the latter possibility, biochemical demonstration of nucleotide-dependent conformational changes in Hsp70 proteins requires the physical connection of the NH2-terminal ATPase domain with the adjacent substrate binding domain (8Buchberger 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: 16903-16910Crossref PubMed Scopus (212) Google Scholar). In particular, several studies suggest that ATP binding triggers an association of the ATPase domain with the substrate binding domain and that this causes further conformational changes within the substrate binding domain itself (8Buchberger 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: 16903-16910Crossref PubMed Scopus (212) Google Scholar, 37Ha J.-H. McKay D.B. Kinetics of nucleotide-induced changes in the tryptophane fluorescence of the molecular chaperone Hsc70 and its subfragments suggest the ATP-induced conformational change follows initial ATP binding.Biochemistry. 1995; 34: 11635-11644Crossref PubMed Scopus (73) Google Scholar, 105Wilbanks S.M. Chen L. Tsuruta H. Hodgson K.O. McKay D.B. Solution small-angle X-ray scattering study of the molecular chaperone hsc70 and its subfragments.Biochemistry. 1995; 34: 12095-12106Crossref PubMed Scopus (92) Google Scholar), although the precise changes that open the substrate binding pocket remain unknown. Whatever the coupling mechanism, differences in the structure of the substrate binding domain in two crystal forms of DnaK have led Hendrickson and coworkers to propose a structural basis for the ATP-induced opening of the binding pocket (111Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M. Hendrickson W.A. Structural analysis of substrate binding by the molecular chaperone DnaK.Science. 1996; 272: 1606-1614Crossref PubMed Scopus (982) Google Scholar). One structure has a “kink” at residues 536–538 of the lid-forming α helix B (indicated by orange segment in Figure 2A), and consequently, the subdomain COOH-terminal to the substrate binding domain has rotated upwards by 11°, causing a loss of stabilizing contacts between α helix B and the outer loops. It has been proposed that this represents the initial stages of release by a “latch” mechanism and that further movement of the “lid” opens up the substrate binding pocket and triggers substrate release. The steady-state turnover rate of the unstimulated Hsp70 ATPase is too slow (between 0.02 and 0.2 min−1) to drive the chaperone activities of Hsp70, even in the presence of substrates, which typically stimulate the ATPase activity 2–10-fold (22Flynn G.C. Chappell T.G. Rothman J.E. 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For the DnaK system, GrpE and DnaJ together stimulate the ATP turnover rate at least several hundred-fold at saturating conditions (65McCarty J.S. Buchberger A. Reinstein J. Bukau B. The role of ATP in the functional cycle of the DnaK chaperone system.J. Mol. Biol. 1995; 249: 126-137Crossref PubMed Scopus (333) Google Scholar), which may be more than is necessary to support chaperone function. The effects of DnaJ and GrpE have to be balanced to optimize the equilibrium between substrate binding and release; this is achieved in vivo by coregulation of expression of their genes. A structure of the stable complex between a dimer of NH2 terminally truncated GrpE and the ATPase domain of DnaK (Figure 4B) shows that GrpE triggers nucleotide exchange by a contact through one GrpE subunit that opens the nucleotide binding cleft of DnaK, as manifested by a 14° rotation (purple arrows in Figure 4B) of the IIB subdomain of the DnaK ATPase domain (relative to its position in the ADP-bound structure of the Hsc70 ATPase domain) (39Harrison C.J. Hayer-Hartl M. Di Liberto M. Hartl F.-U. 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 (394) Google Scholar). This motion displaces DnaK residues (Ser-274, Lys-270, and Glu-267) that provide crucial hydrogen bonds to the adenine and ribose rings of bound ADP. It is intriguing that GrpE homologs appear to be lacking in the cytosol, nucleus, and endoplasmic reticulum of eukaryotic cells. A nucleotide exchange factor thus seems dispensable for at least some chaperone activities of cytosolic Hsp70 homologs, such as Ssa1p of yeast (57Levy E.J. McCarty J. Bukau B. Chirico W.J. Conserved ATPase and luciferase refolding activities between bacteria and yeast Hsp70 chaperones and modulator.FEBS Lett. 1995; 368: 435-440Abstract Full Text PDF PubMed Scopus (67) Google Scholar) and Hsp70/Hsc70 of the mammalian cytosol (24Freeman B.C. Morimoto R.I. The human cytosolic molecular chaperones hsp90, hsp70 (hsc70) and hdj-1 have distinct roles in recognition of a non-native protein and protein refolding.EMBO J. 1996; 15: 2969-2979Crossref PubMed Google Scholar). For Ssa1p, it appears that its DnaJ co-chaperone, Ydj1p, stimulates not only ATP hydrolysis but also product release (112Ziegelhoffer T. Lopez-Buesa P. Craig E. The dissociation of ATP from hsp70 of Saccharomyces cerevisiae is stimulated by both Ydj1p and peptide substrates.J. Biol. 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