Proteins exist in two conformers. The trans conformation is favored by the most of the amino acids. The proline residue due to its unique geometry has a high probability of being in the cis conformation. Thus the cis/trans isomerisation of the peptide bond preceding the proline residue becomes a rate limiting step in the folding and unfolding of the proteins. The enzymes which catalyze this rate limiting step were discovered by Fischer in porcine kidney and called as peptidyl prolyl cis trans isomerases (PPIases). There are four families of the PPIases. They are the parvulins, cyclophilins, FKBPs and trigger factors. All the four families catalyze a common reaction and the give rise to a stable trans product. We therefore wanted to analyse if cross complementation exists across the PPIase families. Our analysis has shown that the prokaryotic and the PPIase domain of the eukaryotic parvulins show a high structural similarity. The catalytic residues were found to be conserved across the genera. Our study has shown that a single domain 92 amino acid long prokaryotic parvulin PpiC from E.coli could complement for the function of Ess1 in Saacharomyces cerevisiae. We have also shown that under conditions of over expression the carboxy terminus of NifM from Azotobacter vinelandii could functionally replace Ess1 in S. cerevisiae. However the complete nifM was unable to do so. We have shown that the amino terminus of NifM acts as a regulatory unit not only for the PPIase activity of its carboxy terminus domain but also for the PPIase activity of PpiC and human Pin1. Using random mutagenesis we have identified the potential docking sites on amino terminus of NifM. These sites are defined as the residues which are responsible for the regulatory activity of NifM. Further we have found that FKBPs which show a high similarity with human Pin1 was unable to isomerise substrate specific to the parvulins. Our analysis has shown, the substrate binding pocket in FKBP is large due to its aromatic nature. Hence it is unable FKBPs to complement for the function of the parvulins.

The peptidyl prolyl cis/trans isomerase, PPIase, has been the focus of numerous studies in the field of cell cycle regulation since proline-directed phosphorylation is an essential signaling mechanism that might arrest cancer proliferation. Pin1 is the first phosphorylation-dependent PPIase enzyme to be discovered. The Pin1 regulatory mechanism, acting on other mitotic proteins in vivo and in vitro, remains largely unknown. For the study of Pin1 function, two types of assays were used to identity ligands for Pin1: (1) The Enzyme-Linked Enzyme Binding Assay (ELEBA) for the identification of WW domain ligands, (2) a catalytic assay to identified inhibitors of Pin1 catalytic activity. The ELEBA offers a selective approach for detecting ligands that bind to the Pin1 WW domain from chemical libraries. By using the ELEBA, a pSer-Pro peptidomimetic library of 315 ligands was screened, identifying three promising ligands cis-D2, O2, and M18. Competitive Kd values for cis-D2, O2, and M18 were determined to be 263 ± 6.4, 206 ± 3.4, and 130 ± 3.0μM, respectively. Furthermore, we screened the pSer-Pro peptidomimetic library using a Pin1 discontinuous-catalytic assay to identify inhibitors of Pin1. Ligands D20 and K7 were identified to decrease more than 90% of the Pin1 catalytic activity. To investigate the nature of the Pin1 interaction with c-Myc, we synthesized and characterized four peptides corresponding to the c-Myc sequence. These peptides were used in NMR isomerization studies of Pin1 by our collaborator Dr. Jeffry Peng (University of Notre Dame). Preliminary work shows that Pin1 binds and isomerizes the Ac–LLPpTPPLSPS–NH₂ peptide at the cMyc pThr58 position. Finally, we measured a secondary kinetic isotope effect (2º KIE) to study the Pin1 catalytic mechanism of proline isomerization. The ratio of kH/kD for unlabeled and [d₃]Ser-labeled substrate gave a SKIE value of 1.34 ± 0.01. The normal 2º KIE value indicates that carbonyl-serine hybridization is not changing from sp² to sp³. This result supports substrate analogue inhibitor studies, and previous solvent and SKIE results on Pin1, that suggest a twisted amide mechanism assisted by a transient hydrogen bond in the transition state. / Ph. D.

Pin1 is an enzyme central to cell signaling pathways because it catalyzes the cis–trans isomerization of the peptide ω-bond in phosphorylated serine/threonine-proline motifs in many proteins. This regulatory function makes Pin1 a drug target in the treatment of various diseases. The effects of phosphorylation on Pin1 substrates and the basis for Pin1 recognition are not well understood. The conformational consequences of phosphorylation on Pin1 substrate analogues and the mechanism of recognition by the catalytic domain of Pin1 were determined using molecular dynamics simulations. Phosphorylation perturbs the backbone conformational space of Pin1 substrate analogues. It is also shown that Pin1 recognizes specific conformations of its substrate by conformational selection. Dynamical correlated motions in the free Pin1 enzyme are present in the enzyme of the enzyme–substrate complex when the substrate is in the transition state configuration. This suggests that these motions play a significant role during catalysis. These results provide a detailed mechanistic understanding of Pin1 substrate recognition that can be exploited for drug design purposes and further our understanding of the subtleties of post-translational phosphorylation and cis–trans isomerization. Results from accelerated molecular dynamics simulations indicate that catalysis occurs along a restricted path of the backbone configuration of the substrate, selecting specific subpopulations of the conformational space of the substrate in the active site of Pin1. The simulations show that the enzyme–substrate interactions are coupled to the state of the prolyl peptide bond during catalysis. The transition-state configuration of the substrate binds better than the cis and trans states to the catalytic domain of Pin1. This suggests that Pin1 catalyzes its substrate by noncovalently stabilizing the transition state. These results suggest an atomistic detail understanding of the catalytic mechanism of Pin1 that is necessary for the design of novel inhibitors and the treatment of several diseases. Additionally, a set of constant force biased molecular dynamics simulations are presented to explore the kinetic properties of a Pin1 substrate and its unphosphorylated analogue. The simulations indicate that the phosphorylated Pin1 substrate isomerizes slower than the unphosphorylated analogue. This is due to the lower diffusion constant for the phosphorylated Pin1 substrate.