Abstract
Cyclin-dependent kinase subunit (CKS) proteins bind to cyclin-dependent kinases and target various proteins to phosphorylation and proteolysis during cell division. Crystal structures showed that CKS can exist both in a closed monomeric conformation when bound to the kinase and in an inactive C-terminal beta-strand-exchanged conformation. With the exception of the hinge loop, however, both crystal structures are identical, and no new protein interface is formed in the dimer. Protein engineering studies have pinpointed the crucial role of the proline 90 residue of the p13(suc1) CKS protein from Schizosaccharomyces pombe in the monomer-dimer equilibrium and have led to the concept of a loaded molecular spring of the beta-hinge motif. Mutation of this hinge proline into an alanine stabilizes the protein and prevents the occurrence of swapping. However, other mutations further away from the hinge as well as ligand binding can equally shift the equilibrium between monomer and dimer. To address the question of differential affinity through relief of the strain, here we compare the ligand binding of the monomeric form of wild-type S. pombe p13(suc1) and its hinge mutant P90A in solution by NMR spectroscopy. We indeed observed a 5-fold difference in affinity with the wild-type protein being the most strongly binding. Our structural study further indicates that both wild-type and the P90A mutant proteins adopt in solution the closed conformation but display different dynamic properties in the C-terminal beta-sheet involved in domain swapping and protein interactions.
Highlights
Progression through the cell cycle is tightly regulated at all levels with a central role for the CDK1 complex
A small subunit called cyclin-dependent kinase subunit (CKS) is essential for cell cycle progression [3], and the cell is unable to enter or exit mitosis depending on the time at which the CKS is removed [4]
The conserved CKS fold further contains a four-stranded -sheet flanked by two ␣-helices where the four-stranded -sheet has on one side a patch of conserved hydrophobic residues that bind tightly to the C-terminal domain of the kinase as revealed by the crystal structure of the human CKSHs11⁄7CDK2 complex [15]
Summary
Progression through the cell cycle is tightly regulated at all levels with a central role for the CDK1 complex. A small subunit called cyclin-dependent kinase subunit (CKS) is essential for cell cycle progression [3], and the cell is unable to enter or exit mitosis depending on the time at which the CKS is removed [4] This small protein does not modulate directly the catalytic function of the CDK complex, it is involved in the interaction with regulators of the kinase complex [5]. Solution NMR Study of the Monomeric Form of p13suc Protein the CDK complexes [19, 20] As this hinge region switches from a turn (in the closed globular form) to an extended conformation (in the open exchanged form), the protein in its dimeric form can no longer interact with the kinase [15]. Very recently it was shown that single point mutations of residues as far as 20 Å from the hinge motif or, alternatively, ligand binding can shift the equilibrium between both forms toward the monomeric form [21]
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