To find new principles for inhibiting serine proteases, we screened phage-displayed random peptide repertoires with urokinase-type plasminogen activator (uPA) as the target. The most frequent of the isolated phage clones contained the disulfide bridgeconstrained sequence CSWRGLENHRMC, which we designated upain-1. When expressed recombinantly with a protein fusion partner, upain-1 inhibited the enzymatic activity of uPA competitively with a temperature and pH-dependent K i , which at 25°C and pH 7.4 was ϳ500 nM. At the same conditions, the equilibrium dissociation constant K D , monitored by displacement of p-aminobenzamidine from the specificity pocket of uPA, was ϳ400 nM. By an inhibitory screen against other serine proteases, including trypsin, upain-1 was found to be highly selective for uPA. The cyclical structure of upain-1 was indispensable for uPA binding. Alanine-scanning mutagenesis identified Arg 4 of upain-1 as the P 1 residue and indicated an extended binding interaction including the specificity pocket and the 37-, 60-, and 97-loops of uPA and the P 1 , P 2 , P 3 , P 4 , and the P 5 residues of upain-1. Substitution with alanine of the P 2 residue, Trp 3 , converted upain-1 into a distinct, although poor, uPA substrate. Upain-1 represents a new type of uPA inhibitor that achieves selectivity by targeting uPA-specific surface loops. Most likely, the inhibitory activity depends on its cyclical structure and the unusual P 2 residue preventing the scissile bond from assuming a tetrahedral geometry and thus from undergoing hydrolysis. Peptide-derived inhibitors such as upain-1 may provide novel mechanistic information about enzyme-inhibitor interactions and alternative methodologies for designing effective protease inhibitors.Serine proteases of the trypsin family (clan SA) have many physiological and pathophysiological functions. There is therefore extensive interest in generating specific inhibitors to be used for pharmacological interference with their enzymatic activity. Moreover, serine proteases are classical subjects for studies of catalytic and inhibitory mechanisms.Serine protease-catalyzed peptide bond hydrolysis proceeds through a tetrahedral transition state formed by a nucleophilic attack on the carbonyl group of the substrate P 1 amino acid by the hydroxyl group of Ser 195 (using the chymotrypsin template numbering (1)), with His 57 and Asp 102 acting as a charge relay system. The protonated His 57 functions as a general acid to facilitate collapse of the tetrahedral intermediate that is stabilized through interactions at the oxyanion hole and main chain ␤-strand-type hydrogen bonds between the P 1 -P 3 and P 2 Ј amino acids of the substrate and residues within the polypeptide binding cleft, as well as specific contacts within the S 1 , S 2 , S 3 , S 1 Ј, and S 2 Ј pockets, which bind respective side chains of the P 1 , P 2 , P 3 , P 1 Ј, and P 2 Ј residues (for reviews, see Refs. 2 and 3). Substrate specificity is governed by the fit of the P residues into their corresponding S-pockets as ...

Variants of the receptor binding domain of both human alpha2-macroglobulin and the corresponding domain of hen egg white ovomacroglobulin have been expressed in Escherichia coli and refolded in vitro. Competition experiments with methylamine-treated alpha2-macroglobulin for binding to the multifunctional alpha2-macroglobulin receptor identify two Lys residues (residues 1370 and 1374 in human alpha2-macroglobulin) spaced by three amino acid residues as crucial for receptor binding. From this result and mutational evidence from other ligands for the alpha2-macroglobulin receptor, a tentative sequence motif for receptor binding is proposed.

The function of major histocompatibility complex (MHC) class I molecules is to sample peptides derived from intracellular proteins and to present these peptides to CD8+ cytotoxic T lymphocytes. In this paper, biochemical assays addressing MHC class I binding of both peptide and beta 2-microglobulin (beta 2m) have been used to examine the assembly of the trimolecular MHC class I/beta 2m/peptide complex. Recombinant human beta 2m and mouse beta 2ma have been generated to compare the binding of the two beta 2m to mouse class I. It is frequently assumed that human beta 2m binds to mouse class I heavy chain with a much higher affinity than mouse beta 2m itself. We find that human beta 2m only binds to mouse class I heavy chain with slightly (about 3-fold) higher affinity than mouse beta 2m. In addition, we compared the effect of the two beta 2m upon peptide binding to mouse class I. The ability of human beta 2m to support peptide binding correlated well with its ability to saturate mouse class I heavy chains. Surprisingly, mouse beta 2m only facilitated peptide binding when mouse beta 2m was used in excess (about 20-fold) of what was needed to saturate the class I heavy chains. The inefficiency of mouse beta 2m to support peptide binding could not be attributed to a reduced affinity of mouse beta 2m/MHC class I complexes for peptides or to a reduction in the fraction of mouse beta 2m/MHC class I molecules participating in peptide binding. We have previously shown that only a minor fraction of class I molecules are involved in peptide binding, whereas most of class I molecules are involved in beta 2m binding. We propose that mouse beta 2m interacts with the minor peptide binding (i.e. the "empty") fraction with a lower affinity than human beta 2m does, whereas mouse and human beta 2m interact with the major peptide-occupied fraction with almost similar affinities. This would explain why mouse beta 2m is less efficient than human beta 2m in generating the peptide binding moiety, and identifies the empty MHC class I heavy chain as the molecule that binds human beta 2m preferentially.

The a,macroglobulin-receptor-associated protein (RAP) binds to the a,macroglobulin receptor/lowdensity lipoprotein receptor-related protein (a,MR/LRP), a multi-functional cell surface receptor known to bind and internalize several macromolecular ligands. RAP has been shown to inhibit binding of all known a,MR/LRP ligands. Mutational studies have implicated distinct parts of RAP as specifically involved in inhibition of binding of a multitude of ligands.In the present paper we provide experimental evidence allowing assignment of elements of triplicate internal sequence similarity in RAP, noted previously [Warshawsky, I., Sites within the 39-kDa protein important for regulating ligand binding to the low-density lipoprotein receptor-related protein, Biochemistry 34, 3404-34151, to three structural domains, 1, 2 and 3, comprising residues 18-112, 113-218 and 219-323 of RAP, respectively. Structural analysis by 'H-NMR spectroscopy shows that domains 1 and 2 as separate domains have similar secondary structures, consisting almost exclusively of a-helices, whereas domain 3 as a separate domain appears only to be marginally stable. Ligand competition titration of recombinant RAP domains 1, 2 and 3 and double domains 1 +2 and 2+ 3 against '"1-RAP and 'ZSI-a,M* (methylamine-activated a,M) for binding to a,MR/LRP demonstrated (a) that functional integrity in single domains is largely preserved, and (b) that important determinants for the inhibition of test ligands reside in the C-terminal regions of domains 1 and 3.

Tetranectin is a plasminogen kringle 4-binding protein. The crystal structure has been determined at 2.8 A resolution using molecular replacement. Human tetranectin is a homotrimer forming a triple oc-helical coiled coil. Each monomer consists of a carbohydrate recognition domain (CRD) connected to a long a-helix. Tetranectin has been classified in a distinct group of the C-type lectin superfamily but has structural similarity to the proteins in the group of collectins. Tetranectin has three intramolecular disulfide bridges. Two of these are conserved in the C-type lectin superfamily, whereas the third is present only in long-form CRDs. Tetranectin represents the first structure of a long-form CRD with intact calcium-binding sites. In tetranectin, the third disulfide bridge tethers the CRD to the long helix in the coiled coil. The trimerization of tetranectin as well as the fixation of the CRDs relative to the helices in the coiled coil indicate a demand for high specificity in the recognition and binding of ligands.© 1997 Federation of European Biochemical Societies.Key words: C-type lectin; X-ray crystal structure; Carbohydrate recognition domain; Plasminogen; Kringle 4; a-Helical coiled coilHuman TN is a homotrimeric protein [17], each polypeptide chain consisting of 181 amino acid residues encoded by three exons [18]. TN contains a carbohydrate recognition domain (CRD) and, according to sequence homology studies, belongs to a distinct group of the C-type (calcium dependent) lectin superfamily, which also includes pancreatic stone protein (lithostathine), sea raven antifreeze protein, and snake venom botrocetin, all isolated CRDs without additional domains in contrast to TN [19,20]. The X-ray structure of human lithostathine, which is a C-type lectin homolog without calcium-binding sites, has recently been published [21]. In addition, structure determinations of proteins of two other groups of C-type lectins have been reported: the group of collectins (three mannose-binding proteins, rat MBP from serum (MBP-A), rat MBP from liver (MBP-C), human MBP) [22][23][24][25][26], and of selectins (human E-selectin) [27].We present here the X-ray structure of recombinant human TN. Knowledge on the structure of TN is essential for unraveling the molecular mechanisms of action determining the biological functions of this protein and for the development of drugs interfering with its function.

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Tetranectin, a plasminogen-binding protein belonging to the family of C-type lectins, was expressed in E. coli and converted to its native form by in vitro refolding and proteolytic processing. Recombinant tetranectin-as well as natural tetranectin from human plasma-was shown by chemical cross-linking analysis and SDS-PAGE to be a homo-trimer in solution as are other known members of the collectin family of C-type lectins. Biochemical evidence is presented showing that an N-terminal domain encoded within exons 1 and 2 of the tetranectin gene is necessary and sufficient to govern subunit trimerization.Keywords: C-lectin; domain structure; refolding; trimerization Tetranectin (TN) is a homo-oligomeric Caz+-binding protein primarily found in plasma (10 mg/L) and in the extracellular matrix of certain human carcinomas, whereas little or no TN is found in the extracellular matrix of the corresponding normal tissues (Christensen & Clemmensen, 1989;Wewer &Albrechtsen, 1992). Although specific binding of TN to plasminogen kringle-4 domain (PLM K4), to complex sulfated polysaccharides (Clemmensen, 1989) and to proteins in the extracellular matrix have been shown (Kluft et al., 1989a(Kluft et al., , 1989b, indicating that TN may participate in processes involved in tissue remodeling, the physiological role of TN remains to be clarified.The mature TN polypeptide chain, 18 1 amino acid residues with three intra-chain disulfide bridges (Fuhlendorff et al., 1987), is encoded in three exons as shown by molecular cloning and characterization of the gene (Berglund & Petersen, 1992;Wewer & Albrechtsen, 1992). The posttranslational modification of threonine-4, originally reported by Fuhlendorff et al. (1987), has been identified as 0-linked glycosylation (Jaquinod et al., in prep.). On the basis of gel filtration data, natural human TN was originally proposed to occur as a homo-tetramer in solution (Clemmensen et al., 1986).Exon 3 of the human TN gene encodes a separate functional and structural unit, a single long-form carbohydrate recognition domain (CRD) clearly related to C-type lectins by sequence homology, conservation of disulfide topology (Fuhlendorff et al., 1987) and by presence of an almost complete suit of amino acid residues predicted to be involved in Ca2+ and sugar binding. Due to the Reprint requests to: Michael Etzerodt, Laboratory of Gene Expression, University of Aarhus, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark; e-mail: met@biobase.dk.'Deceased.reported tetrameric nature along with single residue differences TN is considered to belong to a distinct class of C-type lectins (Drickamer & Taylor, 1993;Day, 1994).In the present study, we report on cross-linking analysis of the homo-oligomeric organization of natural and recombinant TN in solution. ResultsA series of TN derivatives, rTN123 (El-V181, mature protein), rTN12 (El-V49, exon 1 and 2), rTN23 (V17-V181, exon 2 and 3), and rTN3 (A45-Vl8 1, exon 3), was expressed in E. coli and refolded in vitro with the purpose of analyzing the oligomeric nature...

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