Preliminary crystallographic analysis of the Cks protein p13(suc1P90AP92A) from Schizosacharromyces pombe.
The p13(suc1) is the fission yeast member of the Cks (Cdc28-dependant kinase subunit) family of proteins. The Cks proteins bind to and are required for the function of cyclin-dependant kinase (Cdk) proteins during cell cycle progression in eukaryotic cells. Two conformations of Cks have been detected crystallographically; a compact monomer with the C-terminal fourth beta-strand inserted into the core of the molecule between strands 2 and 3, and a strand-exchanged dimer where the fourth beta-strand is inserted into the core of the dimer partner in an equivalent position. There is a highly conserved "hinge" region consisting of the motif PEP, N-terminal to the fourth beta-strand. In the monomer this motif constitutes a beta-turn, while in the dimeric structure it is extended, allowing strand exchange. The mutant protein p13(suc1P90AP92A), in which alanine residues replace both prolines of the turn, provides an opportunity to examine the role of the prolines in this hinge region and how they may allow for the formation of strand-exchanged dimers by Cks proteins. We have expressed and purified this mutant protein. Two millimolar p13(suc1P90AP92A) crystallised in 50 mM tris(hydroxymethyl)aminomethane pH 7.5, 30% poly(ethylene glycol) 1500. Diffraction data were collected at room temperature on an MAR345 image plate using Cu Kalpha radiation from a Rigaku RU200 rotating-anode generator source to 2.70A. The crystal has unit cell parameters a=b=75.1 A, c=34.9 A, alpha=beta=90 degrees , gamma=120 degrees. Diffraction data were indexed to the space group P6 and systematic absences 00l indicate a screw axis consistent with P6(3).
Kelly
Joyanne A
JA
Pharmacology Unit, School of Pharmacology and Medicine, The University of Western Australia, 35 Stirling Highway, Crawley, Perth, WA 6009, Australia.
Williams
Elizabeth A
EA
Wilce
Matthew C J
MC
D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000737:chemistry*; D018360:Crystallography, X-Ray; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012568:Schizosaccharomyces / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014961:X-Ray Diffraction
D018797:Cell Cycle Proteins; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; C051655:Suc1 protein, S pombe; D011392:Proline; D000409:Alanine