A MUTATION IN THE HUMAN CYCLIN-DEPENDENT KINASE INTERACTING PROTEIN, CKSHS2, INTERFERES WITH CYCLIN-DEPENDENT KINASE BINDING AND BIOLOGICALFUNCTION, BUT PRESERVES PROTEIN-STRUCTURE AND ASSEMBLY

A mutation directing an amino acid substitution in the conserved P-hin ge region of one of the human Cks isoforms, CksHs2, was constructed by site-directed mutagenesis. Replacement of glutamine for glutamate 63 (E63Q) was predicted to stabilize the beta-interchanged dimeric and he xameric assembly of CksHs2. However, such an effect was seen only at h igh, non-physiological pH. Three-dimensional structures of the E63Q he xameric mutant protein were determined to 2.6 Angstrom resolution in a P4(3)2(1)2 space group and 2.1 Angstrom in the C-2 space group isostr uctural with wild-type, and both were shown to be virtually identical to the refined 1.7 Angstrom wild-type structure. Thus, the E63Q mutati on did not alter the wild-type structure and assembly of CksHs2 but, s urprisingly, disrupted the essential biological function of the protei n and significantly reduced its ability to bind to cyclin-dependent ki nases. The K-d Of wild-type CksHs2 for CDK2 was 5.05 x 10(-8) M, where as the affinity of the mutant protein for CDK2 was too low to allow a determination. These data, coupled with the observation that monomeric but not hexameric CksHs2 interacts with cyclin-dependent kinases, sug gest that glutamine 63 is likely to be directly involved in cyclin-dep endent kinase binding in vitro and in vivo. (C) 1996 Academic Press Li mited