Reversible membrane binding of gamma-carboxyglutamic acid (Gla)-containing coagulation factors requires Ca(2+)-binding to 10-12 Gla residues. Here we describe the solution structure of the Ca(2+)-free Gla-EGF domain pair of factor x which reveals a striking difference between the Ca(2+)-free and Ca(2+)-loaded forms. In the Ca(2+)-free form Gla residues are exposed to solvent and Phe 4, Leu 5 and Val 8 form a hydrophobic cluster in the interior of the domain. In the Ca(2+)-loaded form Gla residues ligate Ca2+ in the core of the domain pushing the side-chains of the three hydrophobic residues into the solvent. We propose that the Ca(2+)-induced exposure of hydrophobic side chains is crucial for membrane binding of Gla-containing coagulation proteins.

Integrin ␣ 2 subunit forms in the complex with the ␤ 1 subunit a cell surface receptor binding extracellular matrix molecules, such as collagens and laminin-1. It is a receptor for echovirus-1, as well. Ligands are recognized by the special "inserted" domain (I domain) in the integrin ␣ 2 subunit. Venom from a pit viper, Bothrops jararaca, has been shown to inhibit the interaction of platelet ␣ 2 ␤ 1 integrin with collagen because of the action of a disintegrin/metalloproteinase named jararhagin. The finding that crude B. jararaca venom could prevent the binding of human recombinant r␣ 2 I domain to type I collagen led us to study jararhagin further. Synthetic peptides representing hydrophilic and charged sequences of jararhagin, including the RSECD sequence replacing the well known RGD motif in the disintegrinlike domain, were synthesized. Although the disintegrin-like domain derived peptides failed to inhibit r␣ 2 I domain binding to collagen, a basic peptide from the metalloproteinase domain proved to be functional. In an in vitro assay, the cyclic peptide, CTRKKHDNAQC, was shown to bind strongly to human recombinant ␣ 2 I domain and to prevent its binding to type I and IV collagens and to laminin-1. Mutational analysis indicated that a sequence of three amino acids, arginine-lysinelysine (RKK), is essential for r␣ 2 I domain binding, whereas the mutation of the other amino acids in the peptide had little if any effect on its binding function. Importantly, the peptide was functional only in the cyclic conformation and its affinity was strictly dependent on the size of the cysteine-constrained loop. Furthermore, the peptide could not bind to ␣ 2 I domain in the absence of Mg 2؉ , suggesting that the conformation of the I domain was critical, as well. Cells could attach to the peptide only if they expressed ␣ 2 ␤ 1 integrin, and the attachment was inhibited by anti-integrin antibodies.Integrins ␣ 1 ␤ 1 and ␣ 2 ␤ 1 are the major cellular receptors for native collagens (for review, see Refs. 1 and 2). Like all integrins their interaction with ligands is dependent on divalent cations (3). The ␣ 1 and ␣ 2 subunits contain an special inserted domain, the I domain, resembling the A domain found e.g. in von Willenbrand factor (4). It is evident that ␣ 1 I and ␣ 2 I domains are responsible for the primary recognition of collagen by the corresponding integrins (5, 6). Two other ligands for ␣ 2 ␤ 1 integrin, namely laminin-1 and echovirus-1, each bind to ␣ 2 I domain, as well. However, echovirus-1 seems to recognize a different site on the ␣ 2 I domain than the matrix proteins do (7).The binding sites of ␣ 1 ␤ 1 and ␣ 2 ␤ 1 integrins in collagens have been localized to the triple helical areas of the molecules (8, 9). One peptide sequence derived from the collagen ␣ chain has been reported to block integrin-collagen interaction (10), but in many studies it has been ineffective and it probably does not represent the actual binding site in collagen (11-13). More likely, collagen-receptor integrins recognize amino acid resi...

A method is presented for the isolation of genes encoding hydrolytic enzymes without any knowledge of the corresponding proteins. cDNA made from the organism of interest is cloned into a yeast vector to construct an expression library in the yeast Saccharomyces cerevisiae. Colonies producing hydrolytic enzymes are screened by activity plate assays. In this work, we constructed a yeast expression library from the filamentous fungus Trichoderma reesei and isolated a new beta-1,4-endoglucanase gene on plates containing beta-glucan. This gene, egl5, codes for a previously unknown small protein of 242 amino acids. Despite its small size, the protein contains two conservative domains found in Trichoderma cellulases, namely the cellulose-binding domain (CBD) and the linker region that connects the CBD to the catalytic core domain. Molecular modelling of the EGV CBD revealed some interesting structural differences compared to the CBD of the major cellulase CBHI from T. reesei. The catalytic core of EGV is unusually small for a cellulase and represents a new family of cellulases (Family K) and of glycosyl hydrolases (Family 45) together with the endoglucanase B of Pseudomonas fluorescens and the endoglucanase V of Humicola insolens on the basis of hydrophobic cluster analysis.

We present a mechanism for agonist-promoted alpha(2A)-adrenergic receptor (alpha(2A)-AR) activation based on structural, pharmacological, and theoretical evidence of the interactions between phenethylamine ligands and alpha(2A)-AR. In this study, we have: 1) isolated enantiomerically pure phenethylamines that differ both in their chirality about the beta-carbon, and in the presence/absence of one or more hydroxyl groups: the beta-OH and the catecholic meta- and para-OH groups; 2) used [(3)H]UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; agonist] and [(3)H]RX821002 [2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline; antagonist] competition binding assays to determine binding affinities of these ligands to the high- and low-affinity forms of alpha(2A)-AR; 3) tested the ability of the ligands to promote receptor activation by measuring agonist-induced stimulation of [(35)S]GTPgammaS binding in isolated cell membranes; and 4) used automated docking methods and our alpha(2A)-AR model to predict the binding modes of the ligands inside the alpha(2A)-AR binding site. The ligand molecules are sequentially missing different functional groups, and we have correlated the structural features of the ligands and ligand-receptor interactions with experimental ligand binding and receptor activation data. Based on the analysis, we show that structural rearrangements in transmembrane helix (TM) 5 could take place upon binding and subsequent activation of alpha(2A)-AR by phenethylamine agonists. We suggest that the following residues are important in phenethylamine interactions with alpha(2A)-AR: Asp113 (D(3.32)), Val114 (V(3.33)), and Thr118 (T(3.37)) in TM3; Ser200 (S(5.42)), Cys201 (C(5.43)), and Ser204 (S(5.46)) in TM5; Phe391 (F(6.52)) and Tyr394 (Y(6.55)) in TM6; and Phe411 (F(7.38)) and Phe412 (F(7.39)) in TM7.