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Structure of a Bag/Hsc70 Complex: Convergent Functional Evolution of Hsp70 Nucleotide Exchange Factors

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Science  23 Feb 2001:
Vol. 291, Issue 5508, pp. 1553-1557
DOI: 10.1126/science.1057268

Abstract

Bag (Bcl2-associated athanogene) domains occur in a class of cofactors of the eukaryotic chaperone 70-kilodalton heat shock protein (Hsp70) family. Binding of the Bag domain to the Hsp70 adenosine triphosphatase (ATPase) domain promotes adenosine 5′-triphosphate–dependent release of substrate from Hsp70 in vitro. In a 1.9 angstrom crystal structure of a complex with the ATPase of the 70-kilodalton heat shock cognate protein (Hsc70), the Bag domain forms a three-helix bundle, inducing a conformational switch in the ATPase that is incompatible with nucleotide binding. The same switch is observed in the bacterial Hsp70 homolog DnaK upon binding of the structurally unrelated nucleotide exchange factor GrpE. Thus, functional convergence has allowed proteins with different architectures to trigger a conserved conformational shift in Hsp70 that leads to nucleotide exchange.

The evolutionary conserved members of the Hsp70 family play essential roles in preventing misfolding and aggregation of newly synthesized or unfolded proteins (1–3). Coordinated binding and release of substrates by these molecular chaperones is strictly dependent on their ATPase activity. Nucleotide binding to the NH2-terminal ATPase domain of Hsp70 regulates the substrate binding properties of its COOH-terminal peptide-binding domain by an unknown mechanism (4,5). Hsp70 binds adenosine 5′-triphosphate (ATP) with high affinity and slowly hydrolyzes it to adenosine 5′-diphosphate (ADP). ATP-bound Hsp70 has low affinity for substrate, whereas the ADP-bound form has high affinity. Substrate binding to Hsp70/ATP stimulates ATP hydrolysis (6), resulting in a more stable complex of Hsp70/ADP with bound substrate. ATP hydrolysis is also stimulated by Hsp40 proteins, an evolutionary conserved family of Hsp70 co-chaperones, which promote substrate binding to Hsp70 (7, 8). Release of substrate from the complex is dependent on the exchange of bound ADP for ATP. In prokaryotes (and mitochondria), this reaction is promoted by GrpE, a nucleotide exchange factor for the bacterial Hsp70 homolog DnaK (9–12). Given the high level of conservation of Hsp70 and Hsp40 proteins, it seems likely that the cytosol of eukaryotes would contain a GrpE homolog. However, no nucleotide exchange factor for Hsp70 has been definitively identified, although such a function has been proposed for the Bcl2-associated athanogene 1 (Bag-1) protein (13).

Bag-1 was first identified in the mammalian cytosol by virtue of its interaction with the anti-apoptotic protein Bcl-2 and was shown to promote cell survival (14). The Bag family is characterized by the Bag domain that mediates direct interaction with the Hsp70 ATPase domain (15, 16) and directly interacts with a number of client proteins, including the protein kinase Raf-1 (17), growth factor receptors (18), and the retinoic acid receptor (19). All Bag-1 isoforms (S, M, and L) contain a ubiquitin homology domain, thought to contact the proteasome (20). Bag-3 lacks the ubiquitin homology domain but contains a WW domain (W, Trp) (21). These unique sequence elements likely target individual Bag family members to their unique partners and reflect their specific roles in different cellular processes such as protein folding and degradation, signal transduction, and apoptosis. Bag-1 was shown to stimulate the ATPase rate of Hsp70 in an Hsp40-dependent manner and to promote substrate release from the chaperone. These findings led to the proposal that Bag-1 is a nucleotide exchange factor for Hsp70 (13), a view that has been controversially discussed (22).

The minimal Bag domain of Bag-1 was identified by limited proteolysis [residues 151–264 of Bag-1M (Web fig. 1) (23)]. This construct has the same affinity for Hsc70 (the constitutively expressed cytosolic isoform of Hsp70) or its ATPase domain as the Bag isoform Bag-1M [dissociation constant K d 1 to 3 μM; measured by isothermal titration calorimetry (Web table 1) (23)]. Binding affinity is substantial reduced in the presence of ADP or ATP, matching the characteristics of the full-length proteins (24). Hsp40-dependent ATP hydrolysis by Hsc70 is stimulated 10-fold in the presence of the Bag domain or Bag-1M (Fig. 1A) (25), and both the Bag domain and Bag-1M trigger efficient release of radiolabeled Hsc70 from a model substrate [partially denatured glucocorticoid receptor ligand binding domain (LBD)] (26) (Fig. 1B). Bag-dependent dissociation of the Hsc70/LBD complex requires the presence of ATP; binding of Bag-1 to Hsc70 alone does not trigger substrate release (Fig. 1B). Taken together, the Bag domain used for crystallization is fully functional and accounts for the effects of Bag-1M on Hsp70 activity.

Figure 1

Functional characterization of Bag-1M and the Bag domain. (A) Bag-1M and the Bag domain stimulate the ATPase activity of Hsc70 in a Hsp40-dependent manner. Hsc70 (3 μM) was incubated at 30°C with Hsp40 (3 μM) and Bag-1M or isolated Bag domain (3 μM) as indicated. The amount of ATP hydrolyzed was quantitated (9, 25). (B) Bag-1M and the Bag domain stimulate Hsc70 release from substrate polypeptide in a nucleotide-dependent manner. Release of35S-methionine–labeled Hsc70 from partially denatured immobilized LBD of the glucocorticoid receptor was measured upon incubation with ovalbumin (Ova), Bag-1M, or Bag domain (5 μM each) for 10 min at 25°C in the presence or absence of ATP/Mg2+ (2 mM) (26, 33). S, supernatant fractions containing released Hsc70; P, pellet fractions containing LBD-bound Hsc70. Supernatants and pellets were analyzed by SDS–polyacrylamide gel electrophoresis, followed by phosphoimaging. The bar diagram shows the amounts of Hsc70 released from LBD expressed in percentage of total bound Hsc70. Error bars in (A) and (B) indicate SD of three independent experiments.

We determined the crystal structure of the Bag domain in complex with the ATPase domain of Hsc70 at 1.9 Å resolution (Table 1) (27). The Bag domain forms a three-helix bundle (Fig. 2A). Helices 2 and 3 contact subdomains IB and IIB of the ATPase (Fig. 2, A and B). Binding of the monomeric Bag domain to the ATPase domain is mediated by electrostatic interactions, mainly exploiting residues Glu212, Asp222, Arg237, and Gln245 in Bag-1 (Fig. 2C). These residues are highly conserved in all known Bag proteins, and their individual replacement with alanine results in Bag variants with substantially decreased activity in ATPase and substrate release assays, consistent with reduced affinities for the Hsc70 ATPase domain (28). A structure-based sequence search reveals Bag proteins in the cytosol of all eukaryotic species (Fig. 2D). The main residues of Hsc70 involved in the interaction are Arg261and Glu283. All Hsc70 residues involved in the interaction with Bag (Fig. 2C) are absolutely conserved in all cytosolic forms of eukaryotic Hsp70 proteins, correlating with the occurrence of Bag proteins in the cytosol, but are highly divergent in DnaK and BiP (the Hsp70 paralog in the endoplasmic reticulum), proteins that either rely on GrpE as a nucleotide exchange factor (DnaK) or are thought to function independently of an exchange factor (BiP).

Figure 2

The Bag domain/Hsc70 complex. (A) The backbones of the Bag domain (red) and the ATPase domain of Hsc70 (green) shown in a ribbons representation generated with the program Bobscript (38). (B) The electrostatic potential of the Bag domain/Hsc70 (residues 5–381) complex modeled onto the accessible molecular surface as calculated and visualized with GRASP (39). Red and blue indicate negative and positive charges, respectively. Orientation as in (A). (C) Schematic diagram of the interactions between Hsc70 and the Bag domain. The diagram was produced by LIGPLOT (40). Red, residues of Bag-1M; green, residues of Hsc70. (D) Structure-based sequence alignment of Bag do- main proteins from different species. Conserved residues forming the interaction surface with the Hsc70 ATPase domain are highlighted in red, and residues important for packing interactions are shown in blue (41). GenBank accession numbers are as follows: human Bag-1M human (hum), Q99933; human Bag-3/CAIR/BIS, AAD16122; human Bag-4, AAD16123; human Bag-5, AAD16124; Caenorhabditis elegans (C.e) Bag-1, AAD16125;Schizosaccharomyces pombe (S.p) Bag-1B, AAD16127;Saccharomyces cerevisiae (S.c) Snl1p, NP_012248;Arabidopsis thaliana (A.t) putative protein, CAB87278; andDrosophila melanogaster (D.m) gene product, AAF49807.

Table 1

Data collection, phasing, and refinement statistics. Data collection values are as defined in the program SCALEPACK (34), the MAD phasing values are as defined in the program SOLVE (35), and the model refinement values are as defined in the program CNS (37). In data from the DESY x-ray source, Bijvoiet pairs have been separated.

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Although the Bag domain and GrpE of Escherichia coli are structurally unrelated (Fig. 3, A and B), they interact with the same subdomains, IB and IIB, of their respective Hsp70 ATPase domain. However, in contrast to the Bag domain, GrpE forms a dimer that mainly employs a β strand subdomain, in addition to contacts from its α helical region, to bind to the ATPase domain of DnaK (Fig. 3B) (12). Hydrophobic contacts from the β strand region of GrpE reach deep into the nucleotide-binding cleft and contribute substantially to the tight interaction of the two proteins. Different residues are contacted in the Hsp70 ATPase domains of the two complexes. GrpE also interacts with a loop in DnaK that is not conserved in eukaryotic cytosolic forms of Hsp70 (Fig. 4B).

Figure 3

Structural comparison of (A) Bag domain/Hsc70 and (B) DnaK/GrpE complexes. The ATPase domains of Hsc70 and DnaK are colored green, the Bag domain is red, and GrpE is yellow.

Figure 4

Nucleotide exchange mechanism of Bag and GrpE proteins. (A and B) Superposition of Hsc70 (residues 5–381) in complex (A) with the Bag domain with the nucleotide-bound domain of Hsc70 (PDB code 3Hsc) and (B) with the ATPase domain of DnaK in complex with its nucleotide exchange factor GrpE (PDB code 1DKG). Color code represents the rmsd of the superpositions calculated and visualized by the SwissPDB Viewer (blue, low degree of deviation; red, high degree of deviation). A loop region in domain IIB of DnaK not present in Hsc70 is seen in red (maximal rmsd). (C) Superposition of the side chains in Hsc70 involved in ATP binding and hydrolysis. The nucleotide-bound state is drawn in gray; the conformation of the respective residues in the Bag domain/Hsc70 (residues 5–381) complex is drawn in orange (41). (D) A 2F oF c simulated annealing omit map of the Hsc70 region involved in ATP binding and hydrolysis calculated in the absence of the indicated residues (contour level at 1σ) (41).

The consequence of Bag-1 binding to the Hsp70 ATPase domain is a 14° rotation of subdomain IIB about a hinge in the region of Leu228 and Leu309 relative to the structure of Hsc70 with bound ATP [Fig. 4A and Web fig. 2A (23)] (29). In this region, the highest root mean square deviation (rmsd) between the two structures is observed [Web fig. 3 (23)]. Despite striking structural differences between the Bag domain and GrpE, the conformation of the Hsc70 ATPase in complex with Bag is remarkably similar to the conformation of the ATPase domain of DnaK in complex with GrpE [Fig. 4B and Web fig. 2B (23)] (12). In both cases, this conformation is incompatible with high-affinity nucleotide binding. Thus, the eukaryotic Bag domain has evolved convergently to induce a nucleotide release mechanism that has been conserved in Hsp70 proteins.

The high-resolution crystal structure allows the analysis of the conformational changes in positions that are critical for nucleotide binding and hydrolysis by superimposing relevant residues from Hsc70 bound to ATP (29) and Hsc70 bound to Bag [Fig. 4C and Web fig. 4 (23)]. The main changes in the ATPase domain induced by binding of the Bag domain occur in residues of subdomains IA and IIB that orient the adenosine moiety of the nucleotide. The positions of other residues in the binding cleft and the residues involved in catalysis of ATP hydrolysis in subdomains IB and IIA are not significantly altered. The structural transition results in a movement of Glu268, Lys271, and Ser275 in domain IIB and Thr13, Thr14, and Tyr15 in subdomain IA away from their position in the nucleotide-bound structure. This leads to an opening of the nucleotide-binding cleft. Additionally, the side chain of Arg272 assumes an alternative conformation when compared to the nucleotide-bound state. In the latter state, Arg272and Arg342 form a clamp that fixes the adenine ring through hydrophobic interactions and the π-electron system of their guanidinium groups.

The Bag domain promotes nucleotide release from Hsp70 by opening the nucleotide-binding cleft upon binding to the ADP-bound state of Hsp70. Because of the excess of ATP over ADP and Bag proteins in the eukaryotic cytosol, ATP will enter the nucleotide-binding pocket and displace bound Bag protein, resulting in an acceleration of nucleotide exchange. In the presence of Hsp40, which stimulates ATP hydrolysis, nucleotide exchange becomes rate limiting; in the absence of Hsp40, ATP hydrolysis is rate limiting. Thus, an acceleration of the ATPase is only observed in the presence of both Hsp40 and Bag-1 (Fig. 1, B and C). The nucleotide binding and release kinetics of the ATPase cycle are almost identical for Hsp70 and DnaK (10, 30, 31). Given the strict dependence of DnaK on an efficient nucleotide exchange mechanism, it seems likely that a nucleotide exchange factor is necessary for at least some Hsp70 functions in the eukaryotic cytosol, as supported by the occurrence of Bag proteins in all eukaryotes.

On the basis of our structural analysis, it is clear that Bag proteins function as nucleotide exchange factors for Hsp70 by stabilizing the nucleotide-free state of the ATPase. A similar mechanism has been described for the protein Sos, the nucleotide exchange factor of the guanosine triphosphatase Ras (32). In both cases, efficient release and rebinding of nucleotides are achieved by a conformational change that is induced in the enzyme by insertion of two α helices from the exchange factor into the nucleotide-binding cleft.

Bag and GrpE represent two nucleotide exchange factors for Hsp70 proteins with different structures that have been optimized through functional conversion. By binding to different regions of their partner proteins, both GrpE and the Bag domain trigger a conserved switch in the Hsp70 ATPase domain. It remains to be established whether this conserved switch is exploited by yet unidentified additional exchange factors.

  • * To whom correspondence should be addressed. E-mail: ismail{at}moarefi.com

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