The Solution Structure of the N-terminal 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 Vitronectin is a multidomain plasma glycoprotein involved in a variety of biological processes such as cell adhesion, cell migration, modulation of the immune system, and regulation of blood coagulation and fibrinolysis (1, 2). Many regulatory functions of vitronectin result from its ability to interact with plasminogen activator inhibitor 1 (PAI-1), 2 a member of the serine protease inhibitor superfamily that inhibits both tissue-and urinary-type plasminogen activators (3-6). PAI-1 plays important roles in thrombosis and fibrinolysis and has been implicated in hemostasis, angiogenesis, and tumor metastasis (7-11). Low abundant PAI-1 circulates in blood complexed with vitronectin (12); unliganded PAI-1 undergoes rapid "selfinactivation" into an inactive "latent" form in which the inhibitory reactive-site loop inserts as a new strand into the main ␤-sheet of the molecule (13-15). Ample evidence suggests that vitronectin regulates the activity of PAI-1 and PAI-1-mediated cellular events by stabilizing the active form of the inhibitor and delaying its conformational transition to the latent state (16 -18). Conversely, PAI-1 mediates, via binding, vitronectin-dependent cell adhesion and migration (19 -23). Thus, molecular recognition between vitronectin and PAI-1 is of great importance in biology.Loskutoff and colleagues (24 -27) first pinpointed a high affinity PAI-1-binding site in vitronectin to the N-terminal somatomedin B (SMB) domain of the adhesive glycoprotein. The SMB domain consists of 44 amino acid residues (28 -31), including eight conserved cysteines that form four functionally indispensable intramolecular disulfide bridges (24). Kamikubo et al. (32) reported that an N-terminal fragment of VN of 97 amino acid residues, expressed in the cytoplasm of Escherichia coli and purified by immuno-affinity chromatography, showed activity in PAI-1 binding and antibody recognition simi...

Section: Discussionmentioning

confidence: 99%

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

Zou

Yuan

et al. 2007

“…Steric effects may account for the small degree of inhibition observed with the SMB-PAI-1 complex binding to r⌬sBVN because of the observed proximity of the SMB domain-binding site and the second binding site. The main PAI-1 binding determinants in the SMB domain are near a single-turn ␣-helix housing residues 26 -30 and stabilized by an extensively disulfide-bonded domain structure (15,26). Although the core of the SMB domain, including residues 18 -41, is well structured, NMR measurements (26) and x-ray crystallography (15) indicate that the flanking regions extending beyond residue 42 are more disordered.…”

Section: R Smentioning

confidence: 99%

“…Two versions of the SMB domain were used. One form (residues 1-51) was purified from a cyanogen bromide digest of human vitronectin as described (26). Another form (residues 1-47) was produced as a recombinant protein in Pichia pastoris and was a kind gift from Michael Ploug (Finsen Laboratory, Copenhagen, Denmark).…”

Section: Methodsmentioning

confidence: 99%

“…Although the core of the SMB domain, including residues 18 -41, is well structured, NMR measurements (26) and x-ray crystallography (15) indicate that the flanking regions extending beyond residue 42 are more disordered. It is therefore also possible that the slight SMB domain inhibition is due to the overlap of the SMB domain (residues 1-51 for the cyanogen bromide fragment (26) or residues 1-47 for the recombinant SMB (29)) and r⌬sBVN (residues and that this flexible region (amino acids [42][43][44][45][46][47] in the isolated SMB domain partially interferes with the binding of the PAI-1-SMB complex to fulllength vitronectin and r⌬sBVN.…”

Section: R Smentioning

confidence: 99%

See 1 more Smart Citation

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

Schar

Jensen

Christensen

et al. 2008

Self Cite

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 ␣-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 ϳ100-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.Vitronectin is a circulatory protein that interacts with several other plasma components that regulate coagulation and fibrinolysis, cell lysis via the complement cascade, and cell binding and migration in an integrin-dependent and urokinase-type plasminogen activator receptor-dependent fashion (1, 2). An interaction with vitronectin that has been widely studied occurs with the serine protease inhibitor (or serpin), PAI-1, 2 the primary inhibitor of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Most circulating PAI-1 is found in a complex with vitronectin (3). The formation of the complex between vitronectin and PAI-1 is presumed to be physiologically significant in several respects. Binding of vitronectin to PAI-1 stabilizes the active conformation of the serpin, increasing the half-life for its conversion to the inactive, so-called latent state, and maintains its protease inhibitory properties for a prolonged period (4, 5). PAI-1 binds to the N-terminal ϳ50-amino acid somatomedin B (SMB) domain of vitronectin. The binding of PAI-1 competes with the binding of the urokinase-type plasminogen activator receptor to this domain and with the binding of integrins to the adjacent RGD sequence (for a review, see Ref. 6). Moreover, PAI-1 has been reported to induce oligomerization of vitronectin (7-10).Th...

“…The partially reduced isoforms were subjected to peptide mapping using mass spectrometry, and the polypeptide was characterized by 1 H NMR to identify the individual disulfide pairings. Our results indicate that the domain scaffold with its intricate network of disulfides is important to orient the single ␣-helix within this domain (18) properly for binding of ligands including PAI-1 and uPAR. The SMB domain thus represents a new member of the cystine-stabilized helix (CSH) family (34).…”

mentioning

confidence: 97%

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

Horn

Hurst

Mayasundari

et al. 2004

Self Cite

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 Vitronectin is a large glycoprotein with wide ranging distribution and function. The hallmark feature of the vitronectin structure is a series of distinct functional domains that allow it to interact both with itself and with a number of other ligands in a variety of environments including the circulation, extracellular matrix, and platelets (1-4). Of particular interest is the N-terminal somatomedin B (SMB) 1 domain of vitronectin. This domain contains the high affinity binding site for the serpin PAI-1. The interaction between PAI-1 and vitronectin is important to the function of both proteins in thrombolysis, cell adhesion, and pericellular proteolysis (1, 5-9). 2 Equally important for the adhesive properties of vitronectin are binding sites for cell-surface receptors, including integrins and uPAR, that are housed within this small N-terminal domain (2, 10 -15).Because it is known that the SMB domain provides a high affinity binding site for PAI-1 and that this interaction stabilizes PAI-1 in its physiologically active form, the structure of the SMB domain of vitronectin has been hotly pursued. Computational predictions for the structure of this domain using a threading algorithm were challenging compared with other domains from vitronectin (16), as there were no reported structures at the time for homologues of this small domain containing four disulfides. Nevertheless, there are over 100 homologues in the sequence data base, suggesting that this folding motif has been conserved in evolution. Only recently have three-dimensional structures describing the SMB domain become available from two different approaches. First, an x-ray structure has been reported on a co-crystal of PAI-1 and a recombinant form of the SMB domain expressed in Escherichia coli (17). Subsequently, we completed the determination of a solution structure for the SMB domain purified from circulating vitronectin that was isolated from human plasma (18). Although the two structures differ in overall fold, they share a common feature, a single ␣-helix that contains key amino acids for PAI-1 binding. Not surprisingly, a recent report using NMR on a recombinant SMB arrived at a similar structure to that observed in the co-crystal with PAI-1 (19).Key to understanding the structure of the SMB domain is defining the correct dis...