Mutual A domain interactions in the force sensing protein von Willebrand factor.
The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets.
Department of Applied Experimental Biophysics, Institute of Biophysics, Johannes Kepler University, Linz, Austria.
Sandra
S
Posch
Molecular Biomechanics Group, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany.
Camilo
C
Aponte-Santamaría
Department of Physics, FU Berlin, Berlin, Germany.
Richard
R
Schwarzl
Center for Advanced Bioanalysis GmbH (CBL), Linz, Austria.
Andreas
A
Karner
Department of Physics, FU Berlin, Berlin, Germany.
Matthias
M
Radtke
Molecular Biomechanics Group, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany.
Frauke
F
Gräter
Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Tobias
T
Obser
Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Gesa
G
König
Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Maria A
MA
Brehm
Department of Applied Experimental Biophysics, Institute of Biophysics, Johannes Kepler University, Linz, Austria.
Hermann J
HJ
Gruber
Department of Physics, FU Berlin, Berlin, Germany.
Roland R
RR
Netz
Theory Department, Fritz-Haber-Institut der Max-Planck-Gesellschaft, Berlin, Germany.
Carsten
C
Baldauf
Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Reinhard
R
Schneppenheim
Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Frankfurt/Main, Germany.
Robert
R
Tampé
Department of Applied Experimental Biophysics, Institute of Biophysics, Johannes Kepler University, Linz, Austria; Center for Advanced Bioanalysis GmbH (CBL), Linz, Austria. Electronic address: peter.hinterdorfer@jku.at.
Peter
P
Hinterdorfer
D001665:Binding Sites; D001792:Blood Platelets / Q000737:chemistry*; D006801:Humans; D055595:Mechanical Phenomena; D018625:Microscopy, Atomic Force; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D011487:Protein Conformation; D000072417:Protein Domains; D000072760:Single Molecule Imaging; D014841:von Willebrand Factor / Q000737:chemistry*
D014841:von Willebrand Factor
Atomic force microscopy; Brownian dynamics simulation; Molecular dynamics simulation; Primary hemostasis; Single molecule force spectroscopy; von Willebrand factor