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The cdc2 kinase subunit (cks) proteins play an essential function in the control of mitosis through their molecular complexes with the cdc2 kinase. In this work, we characterize the conformational state(s) in solution of the cks proteins p13suc1 from Schizosaccharomyces pombe and p9cksphy from Physarum polycephalum. Monomers of p13suc1 and p9cksphy were found to be markedly nonglobular, presumably with a long, nonfolded C-terminal moiety. This was in contrast to the previously published structure of p13suc1, derived from crystallographic studies on a zinc-promoted p13suc1 dimer, in which the individual p13suc1 subunits had a globular conformation. This disparity was resolved when we found that the globular p13suc1 fold undergoes a conformational transition into nonglobular monomers upon dissociation of the dimers following chelation of the zinc ions by ethylenediaminetetraacetic acid (EDTA). We also found that p13suc1, but not p9cksphy, forms stable dimers in the absence of metal ions. The topology of these EDTA-insensitive dimers likely resembles that of the human p9ckshs2 protein, characterized by beta 4 strand exchange from each nonglobular monomer.

The mammalian p34cdc2 protein kinase, a universal cell cycle regulator, complements cdc2/CDC28 temperature-sensitive mutations in yeasts. We report the biochemical characterisation of two substitutions of human cdc2 at lysine 33, a residue involved in nucleotide binding, that differently alter the fission yeast cell cycle. K33A-hscdc2 and K33R-hscdc2 mutants are both catalytically inactive, but overexpression of K33R-cdc2 is lethal while K33A-cdc2 is not. We show that human K33R-cdc2 acts as a dominant negative allele that associates yeast cdc13/cyclinB and therefore renders endogeneous Schizosaccharomyces pombe cdc2 unactivatable. These results are discussed on the light of the molecular modeling of the mutants in the cdc2 model structure.

The cks proteins (for cdc2 kinase subunit) are essential cell cycle regulators. They interact strongly with the mitotic cdc2 kinase, but the mechanism and the biological function of this association still await understanding. The oligomerization state in solution of two members of this ubiquitous protein family, the suc1 gene product from the fission yeast and the newly cloned cksphy gene product from the myxomycete Physarum, was investigated by small-angle X-ray scattering (SAXS) and biochemical methods. We found that the major molecular species are monodispersed monomeric proteins. Minor amounts of dimeric suc1 proteins were also found, but no equilibrium between the two forms was observed and surprisingly, the hexameric assemblies observed in the crystal structure of the human ckshs2 homolog were not detected. These apparent discrepancies between proteins that display cross-complementation address the question of the control of the cks oligomerization process and its link to the biological function.