Date of Award

8-2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Cynthia Peterson

Committee Members

Elizabeth Howell, Engin Serpersu, Rebecca Prosser, Michael Best

Abstract

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.

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