title: Solution NMR study of the monomeric form of p13suc1 protein sheds light on the hinge region determining the affinity for a phosphorylated substrate.
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.
authors.affiliation: CNRS-Université de Lille 2 UMR 8525, Institut Pasteur de Lille-Institut de Biologie de Lille, 59019 Lille Cedex, France.
authors.forename: Benoît
authors.initials: B
authors.lastname: Odaert
authors.forename: Isabelle
authors.initials: I
authors.lastname: Landrieu
authors.forename: Klaas
authors.initials: K
authors.lastname: Dijkstra
authors.forename: Gea
authors.initials: G
authors.lastname: Schuurman-Wolters
authors.forename: Peter
authors.initials: P
authors.lastname: Casteels
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Wieruszeski
authors.forename: Dirk
authors.initials: D
authors.lastname: Inze
authors.forename: Ruud
authors.initials: R
authors.lastname: Scheek
authors.forename: Guy
authors.initials: G
authors.lastname: Lippens
mesh_terms: D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D018797:Cell Cycle Proteins; D019281:Dimerization; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D006859:Hydrogen / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins; D013696:Temperature
chemical_list: D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D008024:Ligands; D029702:Schizosaccharomyces pombe Proteins; C051655:Suc1 protein, S pombe; D006859:Hydrogen; D000409:Alanine