The broad, long-term objective of the proposed research is to develop a method to enable crystal structure studies of adhesion proteins in their force activated, extended-state conformation. Atomic level knowledge of the physiological (or pathological) state of adhesion complexes should increase our understanding of the regulation of force activated protein-ligand complexes and enable more effective drugs to treat and prevent disease. Specifically, the applicant will study the interaction between the A1-domain of Von Willebrand Factor (VWF-A1) and platelet glycoprotein GPIb a-domain (GPIba). This interaction is activated by shear forces in hemodynamic flow and is important for hemostasis. We hypothesize the existence of a force-activated, extended-state complex between VWF-A1 and GPIba. Without a priori knowledge of the extended-state structure, obtaining crystal structures of these complexes is currently impossible since there is no physiologically meaningful way to apply force to a protein crystal. Therefore, we propose to develop a method to identify mutations that stabilize the extended-state complex with the purpose of obtaining these elusive crystal structures in the absence of force.
The specific aims of the research are to (1) Find mutants with enhanced affinity for both VWF-A1 and GPIba. Since it is not obvious what mutations need to be made to stabilize the extended-state, the applicant will use a combination of random and focused mutagenesis coupled with yeast surface display to screen for high affinity binders (i.e., activated). The applicant will then (2) Combine mutations in VWF-A1 and GPIba to uncover potential synergistic effects. Next, the applicant will (3) Characterize the binding affinity and kinetics of the putative extended-state mutant pairs using flow cytometry and surface plasmon resonance. (4) The mutations'effects on adhesion dynamics and mechanical stability will be studied in parallel plate flow chamber studies. Last, the applicant will (5) Crystallize the extended-state complex for structural elucidation and comparison to wild-type and Von Willebrand Disease mutants.
Von Willebrand Factor plays a crucial role in the body's ability to cease bleeding from wounds. In order to better understand this process, and diseases where wounds cannot cease bleeding, we seek molecular level knowledge of how Von Willebrand Factor performs its hemostatic role. This understanding may lead to more effective treatments for bleeding disorders.
|Blenner, Mark A; Dong, Xianchi; Springer, Timothy A (2014) Structural basis of regulation of von Willebrand factor binding to glycoprotein Ib. J Biol Chem 289:5565-79|