Our major goal is to provide a stereochemical foundation for the biological roles played by a novel mitotic regulator known as Pin1. The human peptidyl-prolyl cis-trans isomerase (PPIase) Pin1 is a conserved mitotic regulator essential for the G2 to M transition of the eukaryotic cell cycle. We have determined the high resolution x-ray crystal structure of Pin1 complexed with a proline containing substrate. In addition, we have begun a functional analysis of Pin1 in vitro and in vivo in order to correlate the observed structural features of this novel PPIase with its role in regulating entry and exit from mitosis. The crystallographic structure together with pH titration studies and mutagenesis of an active site cysteine suggest a catalytic mechanism that includes general acid-base and covalent catalysis during peptide bond isomerization. Pin1 displays a strong preference for an acidic residue N-terminal to the isomerized proline bond due to interaction of this acidic side chain with a conserved basic cluster at the entrance to the PPIase domain's active site. Structural work in progress, confirms that optimal interaction with this basic cluster is achieved with phosphothreonine or phosphoserine containing substrates. The known specificity of cyclin-dependent protein kinases (CDKs) is for threonine or serine residues amino terminal to proline. Pin1, with an active site structure ideally suited for binding phosphorylated -SP- or -TP- containing substrates, likely regulates CDK targets during progression from the inter- to the mitotic phase of the cell cycle. In particular, PinI directly regulates the activity of the mitotic phosphatase CDC25C by binding to its hyperphosphorylated N-terminal region. This region rich in CDK sites, is phosphorylated, and this phosphorylation is essential for CDC25C activity. With regard to this proposal, we will employ a two tiered approach involving a combination of macromolecular x-ray crystallography for high resolution structural analysis of Pin1 and a number of Pin1 mutants in complex with substrates including CDC25C, and in vitro functional analysis of Pin1's substrate specificity and mechanism of action. In addition to the structural work, we will determine the kinetic and thermodynamic parameters governing Pin1-target protein interactions. In addition, we will complete the structural elucidation of the tetramerization domain of a Pin1 interacting kinase known as NIMA and begin crystallization and structural elucidation of Pin1 complexed to this domain of the NIMA kinase. Finally, we propose to design, synthesize and test several structurally based Pin1 inhibitors as new anti-mitotic agents.
| Verdecia, Mark A; Joazeiro, Claudio A P; Wells, Nicholas J et al. (2003) Conformational flexibility underlies ubiquitin ligation mediated by the WWP1 HECT domain E3 ligase. Mol Cell 11:249-59 |
