Crystal structure of tetranectin, a trimeric plasminogen‐binding protein with an α‐helical coiled coil

Abstract

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.

Mapping the Region of the α-Type Phospholipase A2 Inhibitor Responsible for Its Inhibitory Activity

“…Residues 13-36 of GbPLI␣, which were found to be critical for PLA 2 binding and inhibition in the present study, are located at the helical neck region in this model. This ␣-helical coiled-coil neck region is known to mediate the trimerization of CTLD in many C-type lectins, including human MBL (24), rat MBP-A (25), human SP-D (26), rat SP-A (23), and tetranectin (27). This is likely to apply to GbPLI␣ as well because the truncated recombinant protein GbCTLD lacking the N-terminal 48 residues of GbPLI␣ was in monomeric form.…”

Selection of a Novel and Highly Specific Tumor Necrosis Factor α (TNFα) Antagonist

“…This protein binds the lysine-binding kringle domains from apolipoprotein A (2), plasminogen (3), and angiostatin (4). Tetranectin is a 60-kDa protein built from a structural unit composed of three identical chains, each with a CTLD domain located C-terminally to a trimerizing coil-coil region (5). The CTLD domains retain their structural integrity as separate protein domains, (6, 7) and, moreover, it was shown that their binding to the known tetranectin ligand, plasminogen kringle-4, exhibits the same thermodynamic parameters, irrespective of whether it was analyzed in the form of free monomeric domains or as tethered domains in the complete homotrimeric protein (3).…”

The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain

“…The crystallographic analysis of TN [15] showed no electron density corresponding to the 26 N-terminal residues, probably owing to accidental proteolytic cleavage during crystallization. The crystal structure does, however, show that residues 26-52 form an α-helical coiled-coil trimerization module.…”

“…The Plg kringle-4-binding site is located within the domain encoded by exon 3 [13], which encodes a domain similar with the carbohydrate recognition domains (CRDs) of the C-type lectin superfamily [14]. TN exhibits pronounced structural similarity to both the neck region and the CRD of the collectins, and binds Ca# + in a similar way [15,16]. The binding to Plg kringle 4 has been shown to involve amino acid residues near and within the Ca# + -binding site 2 ; accordingly, Plg kringle 4 and Ca# + are competitive ligands for the CRD domain of TN [13].…”