The Reduced, Denatured Somatomedin B Domain of Vitronectin Refolds into a Stable, Biologically Active Molecule ² Yuichi Kamikubo, ‡ Gerard Kroon, § Scott A. Curriden, ‡ H. Jane Dyson,* ,§ and David J. Loskutoff ‡ Department of Cell Biology, DiVision of Vascular Biology, and Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037 ReceiVed NoVember 7, 2005; ReVised Manuscript ReceiVed January 17, 2006 ABSTRACT: The high-affinity binding site in human vitronectin (VN) for plasminogen activator inhibitor-1 (PAI-1) has been localized to the NH 2 -terminal cysteine-rich somatomedin B (SMB) domain (residues 1-44). A number of published structural and biochemical studies show conflicting results for the disulfide bonding pattern and the overall fold of the SMB domain, possibly because this domain may undergo disulfide shuffling and/or conformational changes during handling. Here we show that bacterially expressed recombinant SMB (rSMB) can be refolded to a single form that shows maximal activity in binding to PAI-1 and to a conformation-dependent monoclonal antibody (mAb 153). The oxidative refolding pathway of rSMB can be followed in the presence of glutathione redox buffers. This approach allowed the isolation and analysis of a number of intermediate folding species and of the final stably folded species at equilibrium. Competitive surface plasmon resonance analysis demonstrated that the stably refolded rSMB regained biological activity since it bound efficiently to PAI-1 and to mAb 153. In contrast, none of the folding intermediates bound to PAI-1 or to mAb 153. We also show by NMR analysis that the stably refolded rSMB is identical to the material used for the solution structure determination [Kamikubo et al. (2004) Biochemistry 43, 6519] and that it binds specifically to mAb 153 via an interface that includes the three aromatic side chains previously implicated in binding to PAI-1. Vitronectin (VN) is a 75 kDa adhesive glycoprotein found in the circulation and extracellular matrix of many tissues (1). It plays a significant role in a number of physiological processes such as cell adhesion, cell migration, modulation of the immune system, and regulation of blood coagulation and fibrinolysis (1, 2). This range of functions seems to reflect the ability of VN to interact with numerous partner proteins (3). One important binding partner for VN is type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of both tissue- and urokinase-type plasminogen activators. Although PAI-1 is a member of the serine protease inhibitor (serpin) superfamily, it has several unique properties that distinguish it from other serpins (4). For example, PAI-1 is conformationally unstable (5), and it rapidly decays into an inactive “latent” form in solution by spontaneous insertion of its reactive center loop (RCL) into the main -sheet of the molecule (6-8). Moreover, PAI-1 is a trace protein in blood, and it circulates in complex with VN (9-12). PAI-1 binds to VN with high affinity, and this interaction stabilizes the inhibitor in its active conformation by restricting move- ment of the RCL of the inhibitor and preventing its insertion (13, 14). The binding of PAI-1 to VN also alters the adhesive properties of VN, and it is now clear that PAI-1 regulates cell adhesion and migration on VN (15-18). The interaction between PAI-1 and VN may also be clinically important since elevated levels of active PAI-1 are not only associated with several thrombotic diseases such as myocardial infarc- tion and deep vein thrombosis (19) but also indicate a poor prognosis for survival in several metastatic human cancers (20). These observations suggest that the diverse physiologi- cal functions of VN may be mediated, at least in part, through these effects on PAI-1 (4, 21). The high-affinity binding site for PAI-1 has been localized to the somatomedin B (SMB) domain at the N-terminus (amino acid residues 1-44) of VN (22-24). The SMB domain contains eight Cys residues arranged into four disulfide bonds, and correct disulfide linkages in this domain are required for PAI-1 binding (25-27). Several recent studies provide insights into the structure of this important domain. For example, we recently isolated an active form of recombinant SMB (rSMB) from transformed Escherichia coli and presented data to suggest that the four disulfide bonds were arranged consecutively in a linear uncrossed pattern (Cys 5 -Cys 9 , Cys 19 -Cys 21 , Cys 25 -Cys 31 , and Cys 32 - Cys 39 ) (see Figure 1A) (25). The solution structure of this active form of rSMB was determined by NMR spectroscopy (28) and suggested that the four disulfide bonds were tightly packed in the center of the domain, replacing the traditional hydrophobic core expected for a globular protein (Figure 1B). The few non-cysteine hydrophobic side chains (e.g., Leu 24 , Tyr 27 , and Tyr 28 ) form a cluster on the outside of the domain, providing a distinctive binding surface for PAI-1 (Figure 1B). This hydrophobic surface consists mainly of side chains from ² This work was supported by NIH Grants HL31950 to D.J.L. and CA27489 to H.J.D. * Correspondence should be addressed to this author: phone, 858 784 2223; fax, 858 784 9822; e-mail, dyson@scripps.edu. ‡ Department of Cell Biology, Division of Vascular Biology, The Scripps Research Institute. § Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute. 3297 Biochemistry 2006, 45, 3297-3306 10.1021/bi052278f CCC: $33.50 © 2006 American Chemical Society Published on Web 02/16/2006