The reduced, denatured somatomedin B domain of vitronectin refolds into a stable, biologically active molecule.
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.
Kamikubo
Yuichi
Y
Department of Cell Biology, Division of Vascular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Kroon
Gerard
G
Curriden
Scott A
SA
Dyson
H Jane
HJ
Loskutoff
David J
DJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation / Q000502:physiology*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013002:Somatomedins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D019096:Vitronectin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D013002:Somatomedins; D019096:Vitronectin; C016756:somatomedin B; D005978:Glutathione