A mutation in the human cyclin-dependent kinase interacting protein, CksHs2, interferes with cyclin-dependent kinase binding and biological function, but preserves protein structure and assembly.
A mutation directing an amino acid substitution in the conserved beta-hinge 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 hexameric assembly of CksHs2. However, such an effect was seen only at high, non-physiological pH. Three-dimensional structures of the E63Q hexameric mutant protein were determined to 2.6 A resolution in a P4(3)2(1)2 space group and 2.1 A in the C2 space group isostructural with wild-type, and both were shown to be virtually identical to the refined 1.7 A wild-type structure. Thus, the E63Q mutation did not alter the wild-type structure and assembly of CksHs2 but, surprisingly, disrupted the essential biological function of the protein and significantly reduced its ability to bind to cyclin-dependent kinases. The Kd of wild-type CksHs2 for CDK2 was 5.05 x 10(-8) M, whereas 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, suggest that glutamine 63 is likely to be directly involved in cyclin-dependent kinase binding in vitro and in vivo.
Watson
M H
MH
Department of Molecular Biology, Scripps Research Institute, La Jolla, CA 92037, USA.
Bourne
Y
Y
Arvai
A S
AS
Hickey
M J
MJ
Santiago
A
A
Bernstein
S L
SL
Tainer
J A
JA
Reed
S I
SI
D001483:Base Sequence; D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000378:metabolism; D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins; D017124:Conserved Sequence; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
C093090:CKS1B protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D011994:Recombinant Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C083689:CKS2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases