Date of Award

8-2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Cynthia B. Peterson

Committee Members

Engin Serpersu, Liz Howell, Dan Roberts, Jeffrey M. Becker

Abstract

Active human Plasminogen Activator Inhibitor 1 (PAI-1) is most often found in complex with Vitronectin (VN), an ~62kDa glycoprotein. Research has shown PAI-1 and VN form higher order complexes in tissues, and our work indicates a 2:1 (PAI-1:VN) stoichiometry for these complexes. A logical model for PAI-1:VN interaction proposes that two PAI-1 molecules bind VN at separate sites. However, our small-angle neutron scattering (SANS) data suggest that there is a PAI-1: PAI-1:VN interaction, in which PAI-1 forms a dimer when in complex with VN. We tested this novel arrangement of PAI-1 within the complex by using a variety of biophysical methods. Through the use of VN binding deficient PAI-1 variants we were able to detect binding deficient PAI-1 in PAI-1:VN complexes, thus supporting the existence of a PAI-1:PAI-1:VN interaction. In addition to studying the PAI-1:VN complex assembly and macromolecular arrangement, we probed the disordered domain of VN in order to identify the effect of PAI-1 binding on the disordered nature of the domain. Additionally, we sought to examine the postulated binding site for PAI-1 in this domain. It is known that a class of proteins containing intrinsically disordered domains (IDDs) frequently undergoes a conformational change upon ligand binding. We present evidence that the disordered domain of VN can be classified as an IDD based on sequence composition and SANS data that demonstrate the IDD undergoes a disorder to order transition upon PAI-1 binding. Additionally, our SANS data support a model in which the IDD of VN interacts with the secondary binding site for VN on PAI-1. Overall, this work has greatly advanced the field, and has opened new paths of study for future research efforts in the Peterson lab.

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