Date of Award


Degree Type


Degree Name

Doctor of Philosophy




Dr. David Litchfield


The enzyme Pinl is a peptidyl-prolyl isomerase (PPIase) that structurally consists of an N-terminal WW interaction domain and a C-terminal catalytic or PPIase domain. The understanding of how these two domains work together and perform the many functions discovered in cells is incomplete. Therefore, we hypothesize that Pinl acts as an extra regulatory step in signalling pathways by first binding to targets with its WW domain and then catalyzing the isomerization of those same proteins using its PPIase domain.

To gain insights into how Pinl performs its molecular function, we mutated specific residues of Pinl and examined both their ability to support viability in yeast and isomerase activity. We determined that in the phospho-specific binding loop of the PPIase domain K63 was the most important basic residue for Pinl function and activity while only one contact from R68 or R69 was needed. Furthermore, by mutating the Pinl catalytic residue, Cl 13, to both D and S, we showed that a negative charge at this position is more important than a nucleophile. Finally, extensive mutagenesis of two conserved active site histidines, H59 and HI57, determined that these two residues are not as important as Cl 13 for Pinl function or activity. Instead, protein stability experiments suggested a structural role for both histidines. Collectively, these results led us to propose a new non-covalent mechanism for Pinl catalysis.

We also examined the binding of Pinl mutants to full-length targets and surprisingly found that a binding-deficient mutation in the PPIase domain, R68/69A, had a lower affinity for most Pinl targets. We also discovered two classes of Pinl binding proteins: Class I proteins bind both the WW and PPIase domains of Pinl while Class II

proteins mainly bind the WW. We proposed, based upon structures of the WW and PPIase domains bound to peptides, that the differences between Class I and Class II binding may relate to the presence of a proline at the +1 position. These results have provided novel insights into the binding of the two domains of Pin 1 to full-length targets. Taken together, these studies have expanded the knowledge of the molecular mechanisms of Pinl action in cells.



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