Structure-based mutational analysis of S. Pombep13suc1
p13suc1 from schizosaccharomyces pombe is a member of a family of non-enzymatic cell cycle regulatory proteins called CKS for Cyclin-Dependent kinases Subunit. Other members of this family include CKS1 (S. cerevisiaeCyclin-Dependent Kinases are a class of Ser/Thr kinases that regulate the cell cycle. The suc1+ gene was initially identified as a seppresor of certain CDKl temperature sensitive mutations. Despite the wealth of crystallographic models available plus ample - albeit, sometimes conflicting - evidence from genetics and biochemical studies, an account of the exact physiological role of the CKS proteins remains an elusive goal. In a quest to identify the residues and hence the particular surface region involved in mediating protein-protein interactions with CDK2,1 employed the X-ray structure of Suc1 at 2.7A resolution as guide for a site-specific mutagenesis study. Comparative biochemical and biophysical characterisation of Suc1 and the mutants led to the conclusion that isoleucine-94 and Leucine-96 (located in the hydrophobic patch) are involved in mediating protein-protein interactions with GST-CDK2. This conclusion has since been confirmed by the publication of the X-ray structure of monomeric CksHs1l in a complex with CDK2 by Bourne et al., 1996 (Cell 84: 863-874). An extension of the mutational study to test the hypothesis that Suc1 may utilise conserved residues at the anion-binding site to mediate protein-protein interactions with the Anaphase Promoting Complex (APC) is described. X-ray data has been collected on wild type Suc1 crystals at 100K to 2.3Å resolution. The structure has been resolved and refined to a crystallographic R-factor of 22.6%. S. pombe Suc1 exists as a zinc-stabilised, non strand-exchanged dimer in both the 2.1Å and 2.3Å models. Structural analyses of two Suc1 folding mutants are also presented. The cyclins (A - H) are positive regulatory subunits of CDKs. They share a high degree of homology over a region of about 100 amino acid residues called the "cyclin box". The determination of the crystal structure of cyclin A3 (an N-terminal truncated version of bovine cyclin A) and a CDK2-cyclin A3 complex by other workers has revealed the mechanism of activation of CDKs by cyclins. In S. pombe, the CDKl-cyclin B heterodimeric complex constitutes the mitotic kinase. In order to understand the specificity underlying the CDK-cyclin interaction, I embarked on a structural study of S. pombe cyclin B and CDK1. Both full- length proteins have proven intractable to attempts to overproduce them in a soluble form in E. coli for crystallisation studies. A truncated version of cyclin B (similar to cyclin A3) was designed, cloned and overproduced in E. coli. The cyclin B3 protein was directed into inclusion bodies as insoluble aggregates. Extensive attempts - both in vivo and in vitro - to produce a soluble cyclin B3 proved unsuccessful. Finally, an E. coli co-expression system designed to overproduce CDK1-cyclin A3, CDK1-cyclin B3, CDK1-cyclin B and CDK1-Suc1 complexes is described.