Molecular and Cellular Biology of Plasminogen Activation

i004i
Interactions between PAI-1 and Vitronectin: Two Proteins,
Two Sites, and Two Phases
Schar CR 1 , Jensen JK 2 , Blouse GE 2 , Minor KH 1 , Andreasen PA 2 , Peterson CB* 1
1Department of Biochemistry, Cellular, and Molecular Biology, University of Tennessee, Knoxville,
Tennessee, USA;
2Laboratory of Cellular Protein Science, Department of Molecular and Structural Biology, University of
Aarhus, Aarhus, Denmark
Presenting author e-mail: cbpeters@utk.edu
We have generated a mutant form of vitronectin that lacks the well-characterized N-terminal
somatomedin B domain, known to house the primary high-affinity site for binding of PAI-1 to
vitronectin. Residual binding of PAI-1 to this deletion mutant of vitronectin is observed. Also, we
have used a large battery of mutant forms of PAI-1 to evaluate the specific interactions required
for binding. With these reagents, the second binding site for vitronectin on PAI-1 was mapped
to a region around helix D rich in charged amino acids. We have used kinetic and equilibrium
measurements with surface plasmon resonance to study PAI-1 to full-length and the truncated
mutant of vitronectin that is missing the somatomedin B domain. Clearly, the interaction of
vitronectin and PAI-1 at the second site is weaker than the primary interaction between the
somatomedin B domain and the flexible joint region that lies between helices D, E and F on
PAI-1. Most notably, the off rate for binding is much faster, comparable to that for latent PAI-1
dissociation from vitronectin. Interestingly, latent PAI-1 binds nearly as well at the second site
as does active PAI-1, consistent with less dramatic structural changes in the helix D region upon
conversion to the latent structure compared to those that occur with expansion of the central beta
sheet that affect the primary binding site interactions with the somatomedin B domain. These
characteristic features of binding at the two sites are consistent with FRET experiments and
stopped-flow fluorescence measurements that reveal biphasic binding between vitronectin and
PAI-1.
16 X I t h I n t e r n a t i o n a l W o r k s h o p o n