Structural Analysis Of Pin1 And Its Homologues

The reversible phosphorylation of proteins on serine or threonine residues preceding proline(Ser/Thr-Pro) is a major cellular signaling mechanism.Recent identification of the novel prolyl isomerase Pinl that specifically isomerizes only the phosphorylated Ser/Thr-Pro bonds in certain proteins led us to propose a new signaling mechanism,whereby prolyl isomerization catalytically induces conformational changes in proteins following phosphorylation to regulate protein function.Emerging data indicates that such conformational changes have profound effects on catalytic activity,dephosphorylation,protein-protein interactions, subcellular location and/or turnover.Furthermore,this postphosphorylation mechanism might play an important role in cell growth control and diseases such as cancer and Alzheimer's disease.Very little is known about its catalytic mechanism and its interaction with its biological substrates.Therefore,in the current studies,we have focused on the structural and functional studies on Pinl mutants and its homologues.We constructed four Pinl mutants:one in the WW domain(W34A) and three in the PPIase domain(K63A,C113A,M130A). Pinl wild type and mutants' protein were express and purification.Crystals of these Pinl proteins are obtained by the hanging drop vapor diffusion method.The results indicate mutant Pinl-W34 destabilize the interactions between the substrate(PEG 400) and Pinl and thus abolished the binding of substrate.We also find PEG 400 molecule has a very similar orientation with the Pinl-WT in the catalytic PPIase domain mutants structure such as C113A and M130A.From our mutants K63A,C113A and M130A,suggesting the second sulfate ion is most likely the representative of the second phosphate binding site.In a summary,these studies also provided new information on potential residues in substrate binding and a deeper understanding of how Pinl interacts with its biological target proteins,which could perhaps help effective rational drug design.