The structure of vitronectin, an adhesive protein that circulates in high concentrations in human plasma, was predicted through a combination of computational methods and experimental approaches. Fold recognition and sequence-structure alignment were performed using the threading program PROSPECT for each of three structural domains, i.e., the N-terminal somatomedin B domain (residues 1-53), the central region that folds into a four-bladed beta-propeller domain (residues 131-342), and the C-terminal heparin-binding domain (residues 347-459). The atomic structure of each domain was generated using MODELLER, based on the alignment obtained from threading. Docking experiments between the central and C-terminal domains were conducted using the program GRAMM, with limits on the degrees of freedom from a known inter-domain disulfide bridge. The docked structure has a large inter-domain contact surface and defines a putative heparin-binding groove at the inter-domain interface. We also docked heparin together with the combined structure of the central and C-terminal domains, using GRAMM. The predictions from the threading and docking experiments are consistent with experimental data on purified plasma vitronectin pertaining to protease sensitivity, ligand-binding sites, and buried cysteines.

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue-type plasminogen activator and urokinase, is known to convert readily to a latent form by insertion of the reactive center loop into a central ␤-sheet. Interaction with vitronectin stabilizes PAI-1 and decreases the rate of conversion to the latent form, but conformational effects of vitronectin on the reactive center loop of PAI-1 have not been documented. Mutant forms of PAI-1 were designed with a cysteine substitution at either position P1 or P9 of the reactive center loop. Labeling of the unique cysteine with a sulfhydrylreactive fluorophore provides a probe that is sensitive to vitronectin binding. Results indicate that the scissile P1-P1 bond of PAI-1 is more solvent exposed upon interaction with vitronectin, whereas the N-terminal portion of the reactive loop does not experience a significant change in its environment. These results were complemented by labeling vitronectin with an argininespecific coumarin probe which compromises heparin binding but does not interfere with PAI-1 binding to the protein. Dissociation constants of approximately 100 nM are calculated for the vitronectin/PAI-1 interaction from titrations using both fluorescent probes. Furthermore, experiments in which PAI-1 failed to compete with heparin for binding to vitronectin argue for separate binding sites for the two ligands on vitronectin.The adhesive glycoprotein, vitronectin, circulates in human plasma at concentrations of 200 -400 g⅐ml Ϫ1 and serves as a regulatory protein in humoral defense mechanisms by interacting with macromolecules in the reaction cascades of coagulation and fibrinolysis (reviewed in Refs. 1-3). The circulating form of vitronectin is a monomer of 72 kDa, and vitronectin is also found in a multimeric form in platelet releasates and in the extracellular matrix (4 -6). The anti-fibrinolytic protein, plasminogen activator inhibitor-1 (PAI-1), 1 is the major inhibitor of tissue-type plasminogen activator and urokinase-type plasminogen activator (7-11, reviewed in Refs. 12, 13). Like other serpins, PAI-1 has a reactive center loop that mimics the substrate of its target proteases (14, 15). The active conformation of PAI-1 is relatively unstable, so that the protein undergoes rapid conversion to a latent conformation which is characterized by the insertion of the reactive center loop into a central ␤-sheet within the molecule (16). Interactions between strands of the ␤-sheet and the reactive loop stabilize this conformation relative to the active conformation, in which the loop is thought to protrude from the surface of the molecule (7, 16).Binding to vitronectin results in a 2-3-fold increase in the half-life of active PAI-1 (17-19). In addition to stabilizing the active conformation of PAI-1, vitronectin also alters the protease specificity of the serpin so that the vitronectin⅐PAI-1 complex is endowed with the additional ability to inhibit thrombin (20,21). A vitronectin-binding site has been localized on the surface of PAI-1 using site-di...

A recombinant polypeptide corresponding to the C-terminal 129 amino acids of vitronectin exhibits heparin-binding affinity that is comparable to that of full-length vitronectin and is equally effective at neutralizing heparin anticoagulant activity. Results from this broad experimental approach argue that the behavior of the primary site is sufficient to account for the heparin binding activity of vitronectin and support an exposed orientation for the site in the structure of the native protein.

Suicide substrate beta, gamma-bidentate Rh(III)ATP (RhATP) was used to map the metal ion-binding site in yeast phosphoglycerate kinase (PGK). Cleavage of the RhATP-inactivated enzyme with pepsin and subsequent separation of peptides by reverse-phase high-performance liquid chromatography gave two Rh-nucleotide bound peptides. One of the peptides corresponded to the C-terminal residues of PGK, and the other to a part of helix V. Of the four glutamates present in the C-terminal peptide, Glu 398 may be a likely metal coordination site. Therefore, importance of the C-terminal residues in PGK catalysis may be attributed, in part to the coordination of metal ion of the metal-ATP substrate. Metal coordination may then align the C-terminal peptide to extend toward the N-terminal domain and form the "closed" active site. Results presented in this paper suggest that one or more side chains of the enzyme may be coordinated to the metal ion in the PGK.3-phospho-D-glycerate-RhATP complex, and that exchange-inert metal-ATP analogs could be used to determine metal coordination sites on kinases and other metal-ATP-utilizing enzymes.