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The crystal structure of the adenine nucleotide exchange factor GrpE in complex with the adenosine triphosphatase (ATPase) domain of Escherichia coli DnaK [heat shock protein 70 (Hsp70)] was determined at 2.8 angstrom resolution. A dimer of GrpE binds asymmetrically to a single molecule of DnaK. The structure of the nucleotide-free ATPase domain in complex with GrpE resembles closely that of the nucleotide-bound mammalian Hsp70 homolog, except for an outward rotation of one of the subdomains of the protein. This conformational change is not consistent with tight nucleotide binding. Two long a helices extend away from the GrpE dimer and suggest a role for GrpE in peptide release from DnaK.
Molecular chaperones play an essential role in protein folding by preventing the misfolding and aggregation of folding intermediates (1-3). Several classes of molecular chaperones have been conserved in evolution, including the members of the Hsp70, Hsp9O, and Hsp60 (chaperonin) families. Whereas the chaperonins form large oligomeric ring structures, members of the Hsp70 and Hsp90 families function as monomers or dimers.
DnaK, the Escherichia coli homolog of Hsp70, and the various eukaryotic Hsp70s act by binding and releasing extended peptide segments enriched in hydrophobic side chains. DnaK and its homologs are composed of an NH2-terminal 42-kD ATPase domain and a COOH-terminal 25-kD peptide binding domain, the structures of which are known (4, 5). The binding and release of peptides from DnaK is controlled by conformational changes induced by adenosine triphosphate (ATP) binding and hydrolysis in a mechanism that is not understood. In this reaction DnaK does not act alone but cooperates with two other factors, the chaperone DnaJ and the nucleotide exchange factor GrpE (6), in a manner that is analogous to the regulation of many guanosine triphosphate binding proteins.
The following model of the DnaK reaction cycle in protein folding is now emerging (7): ATP-bound DnaK is characterized by rapid peptide binding and release (8). DnaJ stimulates the hydrolysis of ATP by DnaK, resulting in the adenosine diphosphate (ADP)-bound state of DnaK, which binds peptide tightly (6, 7). Peptide release then requires the dissociation of ADP, which is catalyzed by GrpE, and ATP rebinding to DnaK then occurs (8, 9). Interestingly, whereas the function of DnaK and of mitochondrial Hsp70 critically depends on GrpE, the Hsp70...