William Holmes

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.

A strategy for predicting the ligand binding competence of recombinant orphan nuclear receptors using biophysical characterization

Publisher Summary Nuclear receptors have been historically associated with the steroid hormone receptors, for example, estrogen and glucocorticoid receptors, by virtue of DNA binding domain sequence homology comprising two zinc finger motifs. Many of these are orphan receptors, having no defined ligand. The nuclear receptors present tempting targets in the pursuit of a systems based research approach as so many at present have been cloned. However, when recombinant forms of a receptor are available before its cognate ligand has been identified, confusion arises on weather an orphan receptor is active for use in vitro assays. Researchers now have access to unparalleled amounts of DNA sequence and genetic data. Families of homologous gene products can be studied with the intent of connecting specific proteins to various disease conditions. Specifically, to apply this type of strategy in studies, the problem has been approached with two premises in the chapter. First, the recombinant constructs of orphan nuclear receptors are engineered to contain domains with hypothetical functional homology to receptors with known activities and ligands. In particular, a lot of knowledge has been gained concerning retinoid X receptor a (RXRα) and the domains necessary for DNA binding, retinoid binding, and self/hetero-association. For the purpose of this study, PPARα, PPARδ, PPARγ, RXRα, and LXRα constructs were created to contain the putative ligand binding domains (LBD). The amino acid residues within this conserved contiguous region have been shown to be both necessary and sufficient to demonstrate ligand binding competence for RXRα and other nuclear receptors. Structurally, the LBDs are composed primarily of multiple α-helicies. Second, each nuclear receptor is characterized using a variety of biophysical techniques. This chapter has presented data that suggests that the usefulness of a recombinant protein can be determined before an appropriate ligand is available. These results are an encouraging start in the attempt to predict the binding competency of recombinant orphan nuclear receptors.