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 for normal hemostasis. After translocation into the endoplasmic reticulum, proVWF subunits dimerize through disulfide bonds between their C-terminal cystine 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 cysteines 1 and 4. Dimerization often is mediated by additional cysteines that differ among CK domain subfamilies. When expressed in a baculovirus system, recombinant VWF CK domains (residues 1957-2050) were secreted as dimers that were converted to monomers by selective reduction and alkylation of three unconserved cysteine residues: Cys(2008), Cys(2010), and Cys(2048). By partial reduction and alkylation, chemical and proteolytic digestion, mass spectrometry, and amino acid sequencing, the remaining intrachain disulfide bonds were characterized: Cys(1961)-Cys(2011) (), Cys(1987)-Cys(2041) (), Cys(1991)-Cys(2043) (), and Cys(1976)-Cys(2025). The mutation C2008A or C2010A prevented dimerization, whereas the mutation C2048A did not. Symmetry considerations and molecular modeling based on the structure of transforming growth factor-beta suggest that one or three of residues Cys(2008), Cys(2010), and Cys(2048) in each subunit mediate the covalent dimerization of proVWF.
Katsumi
A
A
Howard Hughes Medical Institute, Department of Medicine and Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Tuley
E A
EA
Bodó
I
I
Sadler
J E
JE
D000409:Alanine / Q000737:chemistry; D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002851:Chromatography, High Pressure Liquid; D003198:Computer Simulation; D003488:Cyanogen Bromide / Q000494:pharmacology; D003553:Cystine / Q000737:chemistry*; D019281:Dimerization; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010720:Phosphines / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011725:Pyridines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013820:Thermolysin / Q000494:pharmacology; D013997:Time Factors; D014841:von Willebrand Factor / Q000737:chemistry* / Q000235:genetics
D000409:Alanine; D003488:Cyanogen Bromide; D003553:Cystine; D004220:Disulfides; D004229:Dithiothreitol; D005033:Ethylmaleimide; D007202:Indicators and Reagents; D010720:Phosphines; D011725:Pyridines; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D013820:Thermolysin; D014841:von Willebrand Factor; C080938:tris(2-carboxyethyl)phosphine; C029351:4-vinylpyridine