文摘

The N-terminal cysteine-rich somatomedin B (SMB) domain (residues 1-44) of the humanglycoprotein 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 recombinantSMB (rSMB) are organized into four disulfide bonds in a linear uncrossed pattern (Cys⁵-Cys⁹, Cys¹⁹-Cys²¹, Cys²⁵-Cys³¹, and Cys³²-Cys³⁹). In the present study, we use an alternative method to show thatthis disulfide bond arrangement remains a major preferred one in solution, and we determine the solutionstructure of the domain using NMR analysis. The solution structure shows that the four disulfide bondsare tightly packed in the center of the domain, replacing the traditional hydrophobic core expected for aglobular 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 hydrophobicsurface consists mainly of side chains from the loop formed by the Cys²⁵-Cys³¹ disulfide bond, and issurrounded by conserved acidic and basic side chains, which are likely to contribute to the specificity ofthe intermolecular interactions of this domain. Interestingly, the overall fold of the molecule is compatiblewith several arrangements of the disulfide bonds. A number of different disulfide bond arrangementswere able to satisfy the NMR restraints, and an extensive series of conformational energy calculationsperformed in explicit solvent confirmed that several disulfide bond arrangements have comparablestabilization energies. An experimental demonstration of the presence of alternative disulfide conformationsin active rSMB is provided by the behavior of a mutant in which Asn¹⁴ is replaced by Met. This mutanthas the same PAI-1 binding activity as rVN1-51, but its fragmentation pattern following cyanogen bromidetreatment is incompatible with the linear uncrossed disulfide arrangement. These results suggest that activeforms of the SMB domain may have a number of allowed disulfide bond arrangements as long as theCys²⁵-Cys³¹ disulfide bond is preserved.