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.

Among the peptidyl prolyl isomerases, the Cyclophilin family of proteins has been linked to various cellular activities such as regulation of homeostasis, mitochondrial permeability, and cell death. Their functionality spans throughout the cell and throughout all cell types as different isoforms. Previous studies done on Cyclophilin A revealed an interesting contact ensemble when bound to a substrate. Because of the similarity of CypA to its homologues, it is believed that they too will exhibit the same contact dynamics. We have defined the dynamics of cyclophilin isoforms through Molecular Dynamics simulations and determined their contact dynamics, characterizing their contact ensembles, and their relative dynamical conservation to each other.

Mycobacterium tuberculosis, the causative pathogen of tuberculosis, currently infects one-third of the world’s population, resulting in two million deaths annually. This clearly shows that tuberculosis is one of the most serious diseases of our times. The often unpleasant side effects from the current drugs, combined with the difficulty of ensuring patient compliance, and the emergence of drug-resistant and multidrug-resistant strains, makes the need for new and better drugs urgent. In this thesis, all the steps, from cloning, purification, crystallization, to activity determination, and structure determination are presented for two different M. tuberculosis enzymes. The structures, which were modeled from X-ray crystallographic data, provide the framework for structure-based drug design. Here, new potential inhibitors can be tailor-made based on the specific interactions in the enzyme’s active site. The bacteria have two different peptidyl-prolyl cis-trans isomerases that catalyze the isomerization of peptide bonds preceding proline residues, a process of high importance for correct folding. Here we present the structure of peptidyl-prolyl cis-trans isomerase A, an enzyme present inside the bacteria, and distinguish it from the B form of the enzyme, which is membrane bound, placing its active site outside the bacteria. The enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step within the non-mevalonate pathway, which leads to the production of isopentenyl diphosphate. This compound is the precursor of various isoprenoids, vital to all living organisms. In humans, isopentenyl diphosphate is produced via a different pathway, indicating that all the enzymes within the non-mevalonate pathway may be suitable drug targets in M. tuberculosis. Several structures of both wild type and mutant 1-deoxy-D-xylulose-5-phosphate reductoisomerase in complex with different substrates, and also with the known inhibitor fosmidomycin, provide valuable information not only to the field of drug design, but also, in this case, into the catalysis.

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