Somatomedin B Domain Research Articles

Characterization of an Extensive Interface on Vitronectin for Binding to Plasminogen Activator Inhibitor-1: Adoption of Structure in an Intrinsically Disordered Region.

Small-angle neutron scattering (SANS) measurements were pursued to study human vitronectin, a protein found in tissues and the circulation that regulates cell adhesion/migration and proteolytic cascades that govern hemostasis and pericellular proteolysis. Many of these functions occur via interactions with its binding partner, plasminogen activator inhibitor-1 (PAI-1), the chief inhibitor of proteases that lyse and activate plasminogen. We focused on a region of vitronectin that remains uncharacterized from previous X-ray scattering, nuclear magnetic resonance, and computational modeling approaches and which we propose is involved in binding to PAI-1. This region, which bridges the N-terminal somatomedin B (SMB) domain with a large central β-propeller domain of vitronectin, appears unstructured and has characteristics of an intrinsically disordered domain (IDD). The effect of osmolytes was evaluated using circular dichroism and SANS to explore the potential of the IDD to undergo a disorder-to-order transition. The results suggest that the IDD favors a more ordered structure under osmotic pressure; SANS shows a smaller radius of gyration (Rg) and a more compact fold of the IDD upon addition of osmolytes. To test whether PAI-1 binding is also coupled to folding within the IDD structure, a set of SANS experiments with contrast variation were performed on the complex of PAI-1 with a vitronectin fragment corresponding to the N-terminal 130 amino acids (denoted the SMB-IDD because it contains the SMB domain and IDD in linear sequence). Analysis of the SANS data using the Ensemble Optimization Method confirms that the SMB-IDD adopts a more compact configuration when bound to PAI-1. Calculated structures for the PAI-1:SMB-IDD complex suggest that the IDD provides an interaction surface outside of the primary PAI-1-binding site located within the SMB domain; this binding is proposed to lead to the assembly of higher-order structures of vitronectin and PAI-1 commonly found in tissues.

Identification of a PAI-1-binding site within an intrinsically disordered region of vitronectin.

The serine protease inhibitor, plasminogen activator inhibitor Type-1 (PAI-1) is a metastable protein that undergoes an unusual transition to an inactive conformation with a short half-life of only 1-2 hr. Circulating PAI-1 is bound to a cofactor vitronectin, which stabilizes PAI-1 by slowing this latency conversion. A well-characterized PAI-1-binding site on vitronectin is located within the somatomedin B (SMB) domain, corresponding to the first 44 residues of the protein. Another PAI-1 recognition site has been identified with an engineered form of vitronectin lacking the SMB domain, yet retaining PAI-1 binding capacity (Schar, Blouse, Minor, Peterson. J Biol Chem. 2008;283:28487-28496). This additional binding site is hypothesized to lie within an intrinsically disordered domain (IDD) of vitronectin. To localize the putative binding site, we constructed a truncated form of vitronectin containing 71 amino acids from the N-terminus, including the SMB domain and an additional 24 amino acids from the IDD region. This portion of the IDD is rich in acidic amino acids, which are hypothesized to be complementary to several basic residues identified within an extensive vitronectin-binding site mapped on PAI-1 (Schar, Jensen, Christensen, Blouse, Andreasen, Peterson. J Biol Chem. 2008;283:10297-10309). Steady-state and stopped-flow fluorescence measurements demonstrate that the truncated form of vitronectin exhibits the same rapid biphasic association as full-length vitronectin and that the IDD hosts the elusive second PAI-1 binding site that lies external to the SMB domain of vitronectin.

Copper(II) Ions Increase Plasminogen Activator Inhibitor Type 1 Dynamics in Key Structural Regions That Govern Stability.

Plasminogen activator inhibitor type 1 (PAI-1) regulates the fibrinolysis pathway by inhibiting the protease activity of plasminogen activators. PAI-1 works in concert with vitronectin (VN), an extracellular protein that aids in localization of active PAI-1 to tissues. The Peterson laboratory demonstrated that Cu(II) and other transition metals modulate the stability of PAI-1, exhibiting effects that are dependent on the presence or absence of the somatomedin B (SMB) domain of VN. The study presented here dissects the changes in molecular dynamics underlying the destabilizing effects of Cu(II) on PAI-1. We utilize backbone amide hydrogen/deuterium exchange monitored by mass spectrometry to assess PAI-1 dynamics in the presence and absence of Cu(II) ions with and without the SMB domain of VN. We show that Cu(II) produces an increase in dynamics in regions important for the function and overall stability of PAI-1, while the SMB domain elicits virtually the opposite effect. A mutant form of PAI-1 lacking two N-terminal histidine residues at positions 2 and 3 exhibits similar increases in dynamics upon Cu(II) binding compared to that of active wild-type PAI-1, indicating that the observed structural effects are not a result of coordination of Cu(II) to these histidine residues. Finally, addition of Cu(II) results in an acceleration of the local unfolding kinetics of PAI-1 presumed to be on pathway to the latency conversion. The effect of ligands on the dynamics of PAI-1 adds another intriguing dimension to the mechanisms for regulation of PAI-1 stability and function.

Interactions of Plasminogen Activator Inhibitor-1 with Vitronectin Involve an Extensive Binding Surface and Induce Mutual Conformational Rearrangements

In order to explore early events during the association of plasminogen activator inhibitor-1 (PAI-1) with its cofactor vitronectin, we have applied a robust strategy that combines protein engineering, fluorescence spectroscopy, and rapid reaction kinetics. Fluorescence stopped-flow experiments designed to monitor the rapid association of PAI-1 with vitronectin indicate a fast, concentration-dependent, biphasic binding of PAI-1 to native vitronectin but only a monophasic association with the somatomedin B (SMB) domain, suggesting that multiple phases of the binding interaction occur only when full-length vitronectin is present. Nonetheless, in all cases, the initial fast interaction is followed by slower fluorescence changes attributed to a conformational change in PAI-1. Complementary experiments using an engineered, fluorescently silent PAI-1 with non-natural amino acids showed that concomitant structural changes occur as well in native vitronectin. Furthermore, we have measured the effect of vitronectin on the rate of insertion of the reactive center loop into beta-sheet A of PAI-1 during reaction with target proteases. With a variety of PAI-1 variants, we observe that both full-length vitronectin and the SMB domain have protease-specific effects on the rate of loop insertion but that the two exhibit clearly different effects. These results support a model for PAI-1 binding to vitronectin in which the interaction surface extends beyond the region of PAI-1 occupied by the SMB domain. In support of this model are recent results that define a PAI-1-binding site on vitronectin that lies outside the somatomedin B domain (Schar, C. R., Blouse, G. E., Minor, K. H., and Peterson, C. B. (2008) J. Biol. Chem. 283, 10297-10309) and the complementary site on PAI-1 (Schar, C. R., Jensen, J. K., Christensen, A., Blouse, G. E., Andreasen, P. A., and Peterson, C. B. (2008) J. Biol. Chem. 283, 28487-28496).

Characterization of a Site on PAI-1 That Binds to Vitronectin Outside of the Somatomedin B Domain

Vitronectin and plasminogen activator inhibitor-1 (PAI-1) are proteins that interact in the circulatory system and pericellular region to regulate fibrinolysis, cell adhesion, and migration. The interactions between the two proteins have been attributed primarily to binding of the somatomedin B (SMB) domain, which comprises the N-terminal 44 residues of vitronectin, to the flexible joint region of PAI-1, including residues Arg-103, Met-112, and Gln-125 of PAI-1. A strategy for deletion mutagenesis that removes the SMB domain demonstrates that this mutant form of vitronectin retains PAI-1 binding (Schar, C. R., Blouse, G. E., Minor, K. M., and Peterson, C. B. (2008) J. Biol. Chem. 283, 10297-10309). In the current study, the complementary binding site on PAI-1 was mapped by testing for the ability of a battery of PAI-1 mutants to bind to the engineered vitronectin lacking the SMB domain. This approach identified a second, separate site for interaction between vitronectin and PAI-1. The binding of PAI-1 to this site was defined by a set of mutations in PAI-1 distinct from the mutations that disrupt binding to the SMB domain. Using the mutations in PAI-1 to map the second site suggested interactions between alpha-helices D and E in PAI-1 and a site in vitronectin outside of the SMB domain. The affinity of this second interaction exhibited a K(D) value approximately 100-fold higher than that of the PAI-1-somatomedin B interaction. In contrast to the PAI-1-somatomedin B binding, the second interaction had almost the same affinity for active and latent PAI-1. We hypothesize that, together, the two sites form an extended binding area that may promote assembly of higher order vitronectin-PAI-1 complexes.

Defining the Native Disulfide Topology in the Somatomedin B Domain of Human Vitronectin

The N-terminal 44 amino acid residues of the human plasma glycoprotein vitronectin, known as the somatomedin B (SMB) domain, mediates the interaction between vitronectin and plasminogen activator inhibitor 1 (PAI-1) in a variety of important biological processes. Despite the functional importance of the Cys-rich SMB domain, how its four disulfide bridges are arranged in the molecule remains highly controversial, as evidenced by three different disulfide connectivities reported by several laboratories. Using native chemical ligation and orthogonal protection of selected Cys residues, we chemically synthesized all three topological analogs of SMB with predefined disulfide connectivities corresponding to those previously published. In addition, we oxidatively folded a fully reduced SMB in aqueous solution, and prepared, by CNBr cleavage, the N-terminal segment of 51 amino acid residues of intact vitronectin purified from human blood. Proteolysis coupled with mass spectrometric analysis and functional characterization using a surface plasmon resonance based vitronectin-PAI-1-SMB competition assay allowed us to conclude that 1) only the Cys(5)-Cys(21), Cys(9)-Cys(39), Cys(19)-Cys(32), and Cys(25)-Cys(31) connectivity is present in native vitronectin; 2) only the native disulfide connectivity is functional; and 3) the native disulfide pairings can be readily formed during spontaneous (oxidative) folding of the SMB domain in vitro. Our results unequivocally define the native disulfide topology in the SMB domain of human vitronectin, providing biochemical as well as functional support to the structural findings on a recombinant SMB domain by Read and colleagues (Zhou, A., Huntington, J. A., Pannu, N. S., Carrell, R. W., and Read, R. J. (2003) Nat. Struct. Biol. 10, 541-544).

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.

A Model for the Three-Dimensional Structure of Human Plasma Vitronectin from Small-Angle Scattering Measurements

Small-angle X-ray scattering (SAXS) measurements were used to characterize vitronectin, a circulatory protein found in human plasma that functions in regulating cell adhesion and migration, as well as proteolytic cascades that affect blood coagulation, fibrinolysis, and pericellular proteolysis. SAXS measurements were taken over a 3-fold range of protein concentrations, yielding data that characterize a monodisperse system of particles with an average radius of gyration of 30.3 +/- 0.6 A and a maximum linear dimension of 110 A. Shape restoration was applied to the data to produce two models of the solution structure of the ligand-free protein. A low-resolution model of the protein was generated that indicates the protein to be roughly peanut-shaped. A better understanding of the domain structure of vitronectin resulted from low-resolution models developed from available high-resolution structures of the domains. These domains include the N-terminal domain that was determined experimentally by NMR [Mayasundari, A., Whittemore, N. A., Serpersu, E. H., and Peterson, C. B. (2004) J. Biol. Chem. 279, 29359-29366] and the docked structure of the central and C-terminal domains that were determined by computational threading [Xu, D., Baburaj, K., Peterson, C. B., and Xu, Y. (2001) Proteins: Struct., Funct., Genet. 44, 312-320]. This model provides an indication of the disposition of the central domain and C-terminal heparin-binding domains of vitronectin with respect to the N-terminal somatomedin B (SMB) domain. This model constructed from the available domain structures, which agrees with the low-resolution model produced from the SAXS data, shows the SMB domain well separated from the central and heparin-binding domains by a disordered linker (residues 54-130). Also, binding sites within the SMB domain are predicted to be well exposed to the surrounding solvent for ease of access to its various ligands.

Assignment of the Four Disulfides in the N-terminal Somatomedin B Domain of Native Vitronectin Isolated from Human Plasma

The primary sequence of the N-terminal somatomedin B (SMB) domain of native vitronectin contains 44 amino acids, including a framework of four disulfide bonds formed by 8 closely spaced cysteines in sequence patterns similar to those found in the cystine knot family of proteins. The SMB domain of vitronectin was isolated by digesting the protein with endoproteinase Glu-C and purifying the N-terminal 1-55 peptide by reverse-phase high performance liquid chromatography. Through a combination of techniques, including stepwise reduction and alkylation at acidic pH, peptide mapping with matrix-assisted laser desorption ionization mass spectrometry and NMR, the disulfide bonds contained in the SMB domain have been determined to be Cys(5):Cys(9), Cys(19):Cys(31), Cys(21):Cys(32), and Cys(25):Cys(39). This pattern of disulfides differs from two other connectivities that have been reported previously for recombinant forms of the SMB domain expressed in Escherichia coli. This arrangement of disulfide bonds in the SMB domain from native vitronectin forms a rigid core around the Cys(19): Cys(31) and Cys(21):Cys(32) disulfides. A small positively charged loop is created at the N terminus by the Cys(5): Cys(9) cystine. The most prominent feature of this disulfide-bonding pattern is a loop between Cys(25) and Cys(39) similar to cystine-stabilized alpha-helical structures commonly observed in cystine knots. This alpha-helix has been confirmed in the solution structure determined for this domain using NMR (Mayasundari, A., Whittemore, N. A., Serpersu, E. H., and Peterson, C. B. (2004) J. Biol. Chem. 279, 29359-29366). It confers function on the SMB domain, comprising the site for binding to plasminogen activator inhibitor type-1 and the urokinase receptor.

The Solution Structure of the N-terminal Domain of Human Vitronectin: PROXIMAL SITES THAT REGULATE FIBRINOLYSIS AND CELL MIGRATION

The three-dimensional structure of an N-terminal fragment comprising the first 51 amino acids from human plasma vitronectin, the somatomedin B (SMB) domain, has been determined by two-dimensional NMR approaches. An average structure was calculated, representing the overall fold from a set of 20 minimized structures. The core residues (18-41) overlay with a root mean square deviation of 2.29 +/- 0.62 A. The N- and C-terminal segments exhibit higher root mean square deviations, reflecting more flexibility in solution and/or fewer long-range NOEs for these regions. Residues 26-30 form a unique single-turn alpha-helix, the locus where plasminogen activator inhibitor type-1 (PAI-1) is bound. This structure of this helix is highly homologous with that of a recombinant SMB domain solved in a co-crystal with PAI-1 (Zhou, A., Huntington, J. A., Pannu, N. S., Carrell, R. W., and Read, R. J. (2003) Nat. Struct. Biol. 10, 541-544), although the remainder of the structure differs. Significantly, the pattern of disulfide cross-links observed in this material isolated from human plasma is altogether different from the disulfides proposed for recombinant forms. The NMR structure reveals the relative orientation of binding sites for cell surface receptors, including an integrin-binding site at residues 45-47, which was disordered and did not diffract in the co-crystal, and a site for the urokinase receptor, which overlaps with the PAI-1-binding site.

Disulfide bonding arrangements in active forms of the somatomedin B domain of human vitronectin.

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.

Kinetic Analysis of the Interaction between Vitronectin and the Urokinase Receptor

Although the urokinase receptor (uPAR) binds to vitronectin (VN) and promotes the adhesion of cells to this matrix protein, the biochemical details of this interaction remain unclear. VN variants were employed in BIAcore experiments to examine the uPAR-VN interaction in detail and to compare it to the interaction of VN with other ligands. Heparin and plasminogen bound to VN fragments containing the heparin-binding domain, indicating that this domain was functionally active in the recombinant peptides. However, no significant binding was detected when uPAR was incubated with this domain, and neither heparin nor plasminogen competed with it for binding to VN. In fact, uPAR only bound to fragments containing the somatomedin B (SMB) domain, and monoclonal antibodies (mAbs) that bind to this domain competed with uPAR for binding to VN. Monoclonal antibody 8E6 also inhibited uPAR binding to VN, and this mAb was shown to recognize sulfated tyrosine residues 56 and 59 in the region adjacent to the SMB domain. Destruction of this site by acid treatment eliminated mAb 8E6 binding but had no effect on uPAR binding. Thus, there appears to be a single binding site for uPAR in VN, and it is located in the SMB domain and is distinct from the epitope recognized by mAb 8E6. Inhibition of uPAR binding to VN by mAb 8E6 probably results from steric hindrance.

A Method for Defining Binding Sites Involved in Protein–Protein Interactions: Analysis of the Binding of Plasminogen Activator Inhibitor 1 to the Somatomedin Domain of Vitronectin

Plasminogen activator inhibitor type-1 (PAI-1) is bound to vitronectin (VN) in plasma and in the extracellular matrix. We previously employed a domain-swapping approach to show that the high-affinity binding site for PAI-1 in VN is contained within residues 12–30 in the amino-terminal somatomedin B (SMB) domain. In this study, we attempt to further delineate the location of this site by employing a novel approach that is based on the use of monoclonal antibodies (Mabs) together with site-directed mutagenesis. Six separate Mabs were identified that bound to the SMB domain and competed with PAI-1 for binding to VN. The relative affinity of each of the Mabs, and of PAI-1 itself, for binding to individual variants of SMB (prepared by alanine scanning mutagenesis), was then determined and compared in competitive binding experiments. Three separate, partially overlapping Mab epitopes within SMB were defined by these studies, and the PAI-1 binding site was localized to the region between residues 24 and 37. When considered together with the domain swapping data, these studies suggest that the PAI-1 binding site is contained within a common seven-residue region (i.e., residues 24–30) in the SMB domain.

Plasminogen activator inhibitor-1 regulates cell adhesion by binding to the somatomedin B domain of vitronectin.

Plasminogen activator inhibitor-1 (PAI-1) binds to the somatomedin B (SMB) domain of vitronectin. It inhibits the adhesion of U937 cells to vitronectin by competing with the urokinase receptor (uPAR; CD87) on these cells for binding to the same domain. Although the inhibitor also blocks integrin-mediated cell adhesion, the molecular basis of this effect is unclear. In this study, the effect of the inhibitor on the adhesion of a variety of cells (e.g., U937, MCF7, HT-1080, and HeLa) to vitronectin was assessed, and the importance of the SMB domain in these interactions was determined. Although PAI-1 blocked the adhesion of all of these cells to vitronectin-coated wells, it did not block adhesion to a variant of vitronectin which lacked the SMB domain. Interestingly, HT-1080 and U937 cells attached avidly to microtiter wells coated with purified recombinant SMB (which does not contain the RGD sequence), and this adhesion was again blocked by the inhibitor. These results affirm that PAI-1 can inhibit both uPAR- and integrin-mediated cell adhesion, and demonstrate that the SMB domain of vitronectin is required for these effects. They also show that multiple cell types can employ uPAR as an adhesion receptor. The use of purified recombinant SMB should help to further define this novel adhesive pathway, and to delineate its relationship with integrin-mediated adhesive events.

Modulation of type-1 plasminogen activator inhibitor-induced vitronectin multimerization

Summary We recently reported that the interaction of active type-1 plasminogen activator inhibitor (PAI-1) with native vitronectin (Vn) present in unfractionated plasma induced the generation of conformationally altered, multimeric Vn. Here, the role of disulfide bridging in Vn and the role of the somatomedin B (SMB) domain of Vn in PAI-1-induced multimerization were characterized. Blocking of free SH-groups in Vn with the simultaneous addition of active PAI-1 prevented disulfide-linked Vn multimerization. In contrast, conformational changes and non-covalent stabilized multimerization were not affected. Moreover, pretreatment of Vn with dithionitrobenzoic acid (DTNB), followed by extensive dialysis to remove SH-reactive compounds, blocked disulfide-linked Vn multimerization. These observations point to the importance of free reactive cysteine(s) already present in native plasma Vn for the covalent stabilization of PAI-1-induced Vn multimers. Moreover, non-covalently and covalently-stabilized Vn multimerization and conformational changes were specifically prevented by recombinant Vn polypeptides encompassing the N-terminal SMB domain. The latter polypeptides may be of importance in elucidating the importance of PAI-1-induced Vn multimerization in complex biological systems.