Warren J. Rocque

Crystal Structure and Mutational Analysis of the Human CDK2 Kinase Complex with Cell Cycle–Regulatory Protein CksHs1

The 2.6 Angstrom crystal structure for human cyclin-dependent kinase 2(CDK2) in complex with CksHs1, a human homolog of essential yeast cell cycle-regulatory proteins suc1 and Cks1, reveals that CksHs1 binds via all four beta strands to the kinase C-terminal lobe. This interface is biologically critical, based upon mutational analysis, but far from the CDK2 N-terminal lobe, cyclin, and regulatory phosphorylation sites. CDK2 binds the Cks single domain conformation and interacts with conserved hydrophobic residues plus His-60 and Glu-63 in their closed beta-hinge motif conformation. The beta hinge opening to form the Cks beta-interchanged dimer sterically precludes CDK2 binding, providing a possible mechanism regulating CDK2-Cks interactions. One face of the complex exposes the sequence-conserved phosphate-binding region on Cks and the ATP-binding site on CDK2, suggesting that CKs may target CDK2 to other phosphoproteins during the cell cycle.

Crystallization of protein-ligand complexes.

Methods presented for growing protein–ligand complexes fall into the categories of co-expression of the protein with the ligands of interest, use of the ligands during protein purification, cocrystallization and soaking the ligands into existing crystals.

The formation of a covalent complex between a dipeptide ligand and the src SH2 domain.

The X-ray crystal structure of the src SH2 domain revealed the presence of a thiol residue (Cys 188) located proximal to the phosphotyrosine portion of a dipeptide ligand. An aldehyde bearing ligand (1) was designed to position an electrophilic carbonyl group in the vicinity of the thiol. X-ray crystallographic and NMR examination of the complex formed between (1) and the src SH2 domain revealed a hemithioacetal formed by addition of the thiol to the aldehyde group with an additional stabilizing hydrogen bond between the acetal hydroxyl and a backbone carbonyl.