Date of Award
Doctor of Philosophy
Biochemistry and Cellular and Molecular Biology
Elizabeth Howell, Engin Serpersu, Rebecca Prosser, Michael Best
Plasminogen activator inhibitor type-1 (PAI-1) specifically inhibits the proteases tissue type plasminogen activator and urokinase plasminogen activator to control the activation of fibrinolysis. Vitronectin interacts with PAI-1 primarily through the somatomedin B (SMB) domain to stabilize and localize PAI-1 to sites of injury. Our laboratory observed that transition metals such as copper2+ have VN dependent, reciprocal effects on how long PAI-1 remains active. We aim to determine the molecular basis for effects of copper2+ on PAI-1 activity. We employed a computational algorithm (MUG) to predict metal binding clusters, and introduced mutations hypothesized to create metal binding deficiency. We compared mutants to wild-type by: measurement of stability kinetics, thermodynamic parameters using isothermal titration calorimetry, and protein dynamics using hydrogen deuterium exchange. Active PAI-1 binds copper2+ in the low nanomolar range, while latent binds an order of magnitude weaker. In a mutant lacking the N-terminal histidines of PAI-1, we observed reduced copper2+ binding, but this does not abolish accelerated transition to the latent form. PAI-1 mutants lacking the carboxylate containing resides in the gate region, as well as a histidine of s4B proximal to the flexible joint region require more copper2+ than wild-type to promote accelerated latency formation, making these residues candidates for further metal binding characterization. SMB-PAI-1 complex binds copper2+ with comparable affinity and stoichiometry as PAI-1 alone. Finally, the SMB domain stabilizes PAI-1 by localized effects on dynamics in the same regions that are affected by copper2+. Thus, binding of SMB does not sterically interfere with copper binding to PAI-1, but rather negates copper2+ effects directly through changes in dynamics.
Bucci, Joel Cullen, "Pinpointing the Molecular Basis for Metal Ion Effects on Plasminogen Activator Inhibitor-1 (PAI-1). " PhD diss., University of Tennessee, 2016.