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 tetr atricopeptide repeat (TPR) proteins is critical to its function as a molecu lar chaperone. We have used site-directed mutagenesis to clarify the struct ural basis for the binding of hsp90 to the TPR domain of phosphoprotein pho sphatase 5 (PP5). This TPR domain was chosen for study because its three-di mensional structure is known. We examined co-immunoprecipitation of hsp90 w ith wild type and mutant TPR constructs from transfected cells. Only mutati ons located on one face of the TPR domain affected hsp90 binding. This allo wed the identification of a binding groove. Three basic residues that are h ighly conserved in hsp90-binding TPR proteins extend prominently into this groove. Lys-97 and Arg-101 were absolutely required for hsp90 binding, whil e mutation of Arg-74 diminished, but did not abrogate, hsp90 binding. Mutat ion 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 acid ic residues in the hsp90 fragment. These data suggest conservation, among h sp90-binding TPR proteins, of a binding groove containing basic residues th at interact with acidic residues near the C terminus of hsp90.