title: von Willebrand factor is dimerized by protein disulfide isomerase.
abstract: Multimeric von Willebrand factor (VWF) is essential for primary hemostasis. The biosynthesis of VWF high-molecular-weight multimers requires spatial separation of each step because of varying pH value requirements. VWF is dimerized in the endoplasmic reticulum by formation of disulfide bonds between the C-terminal cysteine knot (CK) domains of 2 monomers. Here, we investigated the basic question of which protein catalyzes the dimerization. We examined the putative interaction of VWF and the protein disulfide isomerase PDIA1, which has previously been used to visualize endoplasmic reticulum localization of VWF. Excitingly, we were able to visualize the PDI-VWF dimer complex by high-resolution stochastic optical reconstruction microscopy and atomic force microscopy. We proved and quantified direct binding of PDIA1 to VWF, using microscale thermophoresis and fluorescence correlation spectroscopy (dissociation constants KD = 236 ± 66 nM and KD = 282 ± 123 nM by microscale thermophoresis and fluorescence correlation spectroscopy, respectively). The similar KD (258 ± 104 nM) measured for PDI interaction with the isolated CK domain and the atomic force microscopy images strongly indicate that PDIA1 binds exclusively to the CK domain, suggesting a key role of PDIA1 in VWF dimerization. On the basis of protein-protein docking and molecular dynamics simulations, combined with fluorescence microscopy studies of VWF CK-domain mutants, we suggest the following mechanism of VWF dimerization: PDI initiates VWF dimerization by forming the first 2 disulfide bonds Cys2771-2773' and Cys2771'-2773. Subsequently, the third bond, Cys2811-2811', is formed, presumably to protect the first 2 bonds from reduction, thereby rendering dimerization irreversible. This study deepens our understanding of the mechanism of VWF dimerization and the pathophysiological consequences of its inhibition.
authors.affiliation: Faculty of Physics and Center for NanoScience, Ludwig Maximilian University, Munich, Germany;
authors.forename: Svenja
authors.initials: S
authors.lastname: Lippok
authors.affiliation: Molecular Biomechanics Group, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany;
authors.forename: Katra
authors.initials: K
authors.lastname: Kolšek
authors.affiliation: Faculty of Physics and Center for NanoScience, Ludwig Maximilian University, Munich, Germany;
authors.forename: Achim
authors.initials: A
authors.lastname: Löf
authors.affiliation: Max Planck Institute for the Structure and Dynamics of Matter, Hamburg, Germany; Microscopy and Image Analysis Technology Platform, Heinrich-Pette-Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany;
authors.forename: Dennis
authors.initials: D
authors.lastname: Eggert
authors.affiliation: Faculty of Physics and Center for NanoScience, Ludwig Maximilian University, Munich, Germany; Department of Chemistry, Division of Molecular Imaging and Photonics, Katholieke Universiteit Leuven-University of Leuven, Leuven, Belgium;
authors.forename: Willem
authors.initials: W
authors.lastname: Vanderlinden
authors.affiliation: Faculty of Physics and Center for NanoScience, Ludwig Maximilian University, Munich, Germany;
authors.forename: Jochen P
authors.initials: JP
authors.lastname: Müller
authors.affiliation: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany;
authors.forename: Gesa
authors.initials: G
authors.lastname: König
authors.affiliation: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany;
authors.forename: Tobias
authors.initials: T
authors.lastname: Obser
authors.affiliation: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany;
authors.forename: Karoline
authors.initials: K
authors.lastname: Röhrs
authors.affiliation: Medilys Laborgesellschaft mbH, Hemostaseology, Asklepios Klinik Altona, Hamburg, Germany; and.
authors.forename: Sonja
authors.initials: S
authors.lastname: Schneppenheim
authors.affiliation: Medilys Laborgesellschaft mbH, Hemostaseology, Asklepios Klinik Altona, Hamburg, Germany; and.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Budde
authors.affiliation: Fritz-Haber-Institut der Max-Planck-Gesellschaft, Berlin, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Baldauf
authors.affiliation: Molecular Biomechanics Group, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany;
authors.forename: Camilo
authors.initials: C
authors.lastname: Aponte-Santamaría
authors.affiliation: Molecular Biomechanics Group, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany;
authors.forename: Frauke
authors.initials: F
authors.lastname: Gräter
authors.affiliation: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany;
authors.forename: Reinhard
authors.initials: R
authors.lastname: Schneppenheim
authors.affiliation: Faculty of Physics and Center for NanoScience, Ludwig Maximilian University, Munich, Germany;
authors.forename: Joachim O
authors.initials: JO
authors.lastname: Rädler
authors.affiliation: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany;
authors.forename: Maria A
authors.initials: MA
authors.lastname: Brehm
mesh_terms: D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008853:Microscopy; D018625:Microscopy, Atomic Force; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D014841:von Willebrand Factor / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004220:Disulfides; D050505:Mutant Proteins; D014841:von Willebrand Factor; D019704:Protein Disulfide-Isomerases; D003545:Cysteine