Functional dissection of cdc37: characterization of domain structure and amino acid residues critical for protein kinase binding.

Hsp90 and its co-chaperone Cdc37 facilitate the folding and activation of numerous protein kinases. In this report, we examine the structure-function relationships that regulate the interaction of Cdc37 with Hsp90 and with an Hsp90-dependent kinase, the heme-regulated eIF2alpha kinase (HRI). Limited proteolysis of native and recombinant Cdc37, in conjunction with MALDI-TOF mass spectrometry analysis of peptide fragments and peptide microsequencing, indicates that Cdc37 is comprised of three discrete domains. The N-terminal domain (residues 1-126) interacts with client HRI molecules. Cdc37's middle domain (residues 128-282) interacts with Hsp90, but does not bind to HRI. The C-terminal domain of Cdc37 (residues 283-378) does not bind Hsp90 or kinase, and no functions were ascribable to this domain. Functional assays did, however, suggest that residues S127-G163 of Cdc37 serve as an interdomain switch that modulates the ability of Cdc37 to sense Hsp90's conformation and thereby mediate Hsp90's regulation of Cdc37's kinase-binding activity. Additionally, scanning alanine mutagenesis identified four amino acid residues at the N-terminus of Cdc37 that are critical for high-affinity binding of Cdc37 to client HRI molecules. One mutation, Cdc37/W7A, also implicated this region as an interpreter of Hsp90's conformation. Results illuminate the specific Cdc37 motifs underlying the allosteric interactions that regulate binding of Hsp90-Cdc37 to immature kinase molecules.