Proline Isomerase Dynamics and the Modulation of Tau Function by Pin1.

Peptidyl-prolyl isomerases (PPIases) constitute a superfamily of enzymes that catalyze the cis/trans isomerization of prolyl bonds. A number of natural substrates for PPIases have recently been identified, pointing to a biological role for the isomerase activity of these enzymes beyond their originally envisioned role as folding catalysts. The first goal of this dissertation was to elucidate the molecular mechanism by which the prolyl isomerase Pin1 can regulate the function of one of its biological substrates, the microtubule-associated protein Tau. Tau proteins are essential factors for the regulation of microtubule dynamics in neuronal cells. Misregulation of the Tau: microtubule interaction in vivo results in the collapse of the neuronal cytoskeleton and is a hallmark of a number of neurodegenerative ailments including Alzheimer's disease. The microtubule stabilizing function of Tau is impaired by hyper-phosphorylation. Pin1 has been previously found to restore the function of phosphorylated Tau in vitro. Chapter 2 of this thesis details a series of experiments determining that Pin1 selectively isomerizes phosphorylated Tau at a functionally relevant site resulting in a partial rescue of Tau function. Our spectroscopic study of phosphorylated Tau also suggests that Pin1 catalysis serves to disturb the acquired local structure of Tau upon phosphorylation. The second goal of this dissertation was to characterize the internal dynamics of the Pin1 isomerase domain during turnover. Chapter 3 describes a series of NMR relaxation measurements of the backbone amides of the Pin1 catalytic domain revealing protein motions in the millisecond timescale related to both the substrate binding and to the isomerization step. These results, together with a model of the enzyme: substrate complex derived from our NMR structural data, suggest a reaction trajectory for isomerization by Pin1. Interestingly the internal dynamics of the Pin1 catalytic domain during turnover are similar to the dynamics of the free enzyme leading to our hypothesis that the protein motions relevant for enzyme catalysis are "encoded" in the enzyme structure and exist in the free state. The question of pre-existing functional motions in enzymes is also addressed in chapter 4, which describes a similar study undertaken on a prolyl isomerase from a different sub-family: cyclophilin A. The results from the relaxation measurements on cyclophilin A parallel the results from the Pin1 isomerase experiments, indicating that intrinsic dynamics necessary for catalysis may be a common feature among prolyl isomerases.