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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

Academic Article
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Overview

authors

  • Watson, M. H.
  • Bourne, Y.
  • Arvai, A. S.
  • Hickey, M. J.
  • Santiago, A.
  • Bernstein, S. L.
  • Tainer, John
  • Reed, Steven

publication date

  • September 1996

journal

  • Journal of Molecular Biology  Journal

abstract

  • 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.

subject areas

  • Base Sequence
  • Binding Sites
  • CDC2 Protein Kinase
  • CDC2-CDC28 Kinases
  • Carrier Proteins
  • Cell Cycle Proteins
  • Conserved Sequence
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinases
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation
  • Protein Conformation
  • Protein Kinases
  • Protein-Serine-Threonine Kinases
  • Recombinant Proteins
  • Saccharomyces cerevisiae
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Identity

International Standard Serial Number (ISSN)

  • 0022-2836

Digital Object Identifier (DOI)

  • 10.1006/jmbi.1996.0490

PubMed ID

  • 8800213
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Additional Document Info

start page

  • 646

end page

  • 657

volume

  • 261

issue

  • 5

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