Localization of disulfide bonds in the cystine knot domain of human von Willebrand factor

von Willebrand factor (VWF) is a multimeric glycoprotein that is required f or normal hemostasis. After translocation into the endoplasmic reticulum, p roVWF subunits dimerize through disulfide bonds between their C-terminal cy stine knot-like (CK) domains. CK domains are characterized by six conserved cysteines, Disulfide bonds between cysteines 2 and 5 and between cysteines 3 and 6 define a ring that is penetrated by a disulfide bond between cyste ines 1 and 4. Dimerization often is mediated by additional cysteines that d iffer among CK domain subfamilies. When expressed in a baculovirus system, recombinant VWF CK domains (residues 1957-2050) were secreted as dimers tha t were converted to monomers by selective reduction and alkylation of three unconserved cysteine residues: Cys(2008), CyS2010 and Cys(2048). BY partia l reduction and alkylation, chemical and proteolytic digestion, mass spectr ometry, and amino acid sequencing, the remaining intrachain disulfide bonds were characterized: Cys(1961)-Cys(2011) (1-4), Cys(1987)-Cys(2041) (2-5), Cys(1991)-Cys(2045) (3-6), and Cys(1976)- Cys(2025). The mutation C2008A or C2010A prevented dimerization, whereas the mutation C2048A did not. Symmet ry considerations and molecular modeling based on the structure of transfor ming growth factor-beta suggest that one or three of residues Cys(2008), Cy s(2010), and Cys(2048) in each subunit mediate the covalent dimerization of proVWF.