title: Disulfide bonding arrangements in active forms of the somatomedin B domain of human vitronectin.
abstract: The N-terminal cysteine-rich somatomedin B (SMB) domain (residues 1-44) of the human glycoprotein vitronectin contains the high-affinity binding sites for plasminogen activator inhibitor-1 (PAI-1) and the urokinase receptor (uPAR). We previously showed that the eight cysteine residues of recombinant SMB (rSMB) are organized into four disulfide bonds in a linear uncrossed pattern (Cys(5)-Cys(9), Cys(19)-Cys(21), Cys(25)-Cys(31), and Cys(32)-Cys(39)). In the present study, we use an alternative method to show that this disulfide bond arrangement remains a major preferred one in solution, and we determine the solution structure of the domain using NMR analysis. The solution structure shows that the four disulfide bonds are tightly packed in the center of the domain, replacing the traditional hydrophobic core expected for a globular protein. The few noncysteine hydrophobic side chains form a cluster on the outside of the domain, providing a distinctive binding surface for the physiological partners PAI-1 and uPAR. The hydrophobic surface consists mainly of side chains from the loop formed by the Cys(25)-Cys(31) disulfide bond, and is surrounded by conserved acidic and basic side chains, which are likely to contribute to the specificity of the intermolecular interactions of this domain. Interestingly, the overall fold of the molecule is compatible with several arrangements of the disulfide bonds. A number of different disulfide bond arrangements were able to satisfy the NMR restraints, and an extensive series of conformational energy calculations performed in explicit solvent confirmed that several disulfide bond arrangements have comparable stabilization energies. An experimental demonstration of the presence of alternative disulfide conformations in active rSMB is provided by the behavior of a mutant in which Asn(14) is replaced by Met. This mutant has the same PAI-1 binding activity as rVN1-51, but its fragmentation pattern following cyanogen bromide treatment is incompatible with the linear uncrossed disulfide arrangement. These results suggest that active forms of the SMB domain may have a number of allowed disulfide bond arrangements as long as the Cys(25)-Cys(31) disulfide bond is preserved.
authors.affiliation: Department of Cell Biology, Division of Vascular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Kamikubo
authors.forename: Roberto
authors.initials: R
authors.lastname: De Guzman
authors.forename: Gerard
authors.initials: G
authors.lastname: Kroon
authors.forename: Scott
authors.initials: S
authors.lastname: Curriden
authors.forename: Jaap G
authors.initials: JG
authors.lastname: Neels
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Churchill
authors.forename: Philip
authors.initials: P
authors.lastname: Dawson
authors.forename: Stanisław
authors.initials: S
authors.lastname: Ołdziej
authors.forename: Anna
authors.initials: A
authors.lastname: Jagielska
authors.forename: Harold A
authors.initials: HA
authors.lastname: Scheraga
authors.forename: David J
authors.initials: DJ
authors.lastname: Loskutoff
authors.forename: H Jane
authors.initials: HJ
authors.lastname: Dyson
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012996:Solutions; D013002:Somatomedins / Q000235:genetics / Q000378:metabolism*; D019096:Vitronectin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D004220:Disulfides; C524898:PLAUR protein, human; D017395:Plasminogen Activator Inhibitor 1; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D012996:Solutions; D013002:Somatomedins; D019096:Vitronectin; C016756:somatomedin B; D003545:Cysteine