Identification of conserved residues required for the binding of a tetratricopeptide repeat domain to heat shock protein 90.
The sequential binding of heat shock protein 90 (hsp90) to a series of tetratricopeptide repeat (TPR) proteins is critical to its function as a molecular chaperone. We have used site-directed mutagenesis to clarify the structural basis for the binding of hsp90 to the TPR domain of phosphoprotein phosphatase 5 (PP5). This TPR domain was chosen for study because its three-dimensional structure is known. We examined co-immunoprecipitation of hsp90 with wild type and mutant TPR constructs from transfected cells. Only mutations located on one face of the TPR domain affected hsp90 binding. This allowed the identification of a binding groove. Three basic residues that are highly conserved in hsp90-binding TPR proteins extend prominently into this groove. Lys-97 and Arg-101 were absolutely required for hsp90 binding, while mutation of Arg-74 diminished, but did not abrogate, hsp90 binding. Mutation of Lys-32, another conserved basic residue in the binding groove, also blocked hsp90 binding. The TPR domain of PP5 bound specifically to a 12-kDa C-terminal fragment of hsp90. This binding was reduced by mutation of acidic residues in the hsp90 fragment. These data suggest conservation, among hsp90-binding TPR proteins, of a binding groove containing basic residues that interact with acidic residues near the C terminus of hsp90.
Department of Pharmacology, University of South Alabama, Mobile, Alabama 36688, USA.
L C
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Russell
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Whitt
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Chen
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Chinkers
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D017124:Conserved Sequence; D003593:Cytoplasm / Q000201:enzymology; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding
D018841:HSP90 Heat-Shock Proteins; D010455:Peptides; D010749:Phosphoprotein Phosphatases