Hans Christian Thøgersen

Amino acid sequence of the factor XIIIa acceptor site in bovine plasma fibronectin

Blood coagulation factor XIII is a proenzyme which can be activated by thrombin [I] to the transglutaminase factor XIII, [2%4]. Factor XIII, catalyzes the formation of e(y-glutamyl)lysyl amide bonds between pairs of y-chains in aggregated fibrin, resulting in its transformation to a highly stable and insoluble covalently cross-linked clot (reviewed in [S-7]). Two other plasma proteins cYz-macroglobulin and fibronectin contain acceptor sites for factor XIII, as shown by incorporation of dansylcadaverine [8]. Only fibronectin, but not aa-macroglobulin, was shown to be crosslinked to fibrin [8]. Cross-linking of fibronectin to collagen [9 ,I 0] and to StaphyZoccocus aureus cells [ 1 l] has been demonstrated. It has also been suggested that rYa-antiplasmin could be covalently linked to. fibrin in a Ca’+dependent reaction probably catalyzed by factor XIII, [ 121. Here we report the ammo acid sequence in bovine fibronectin which contains the glutamine residue labelled with radioactive putrescine by factor XIII,. This glutamine is located at position 3 from the N-terminus of fibronectin.

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

Tetranectin is a plasminogen kringle 4‐binding protein. The crystal structure has been determined at 2.8 Å resolution using molecular replacement. Human tetranectin is a homotrimer forming a triple α‐helical coiled coil. Each monomer consists of a carbohydrate recognition domain (CRD) connected to a long α‐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.

EF-hands at atomic resolution: the structure of human psoriasin (S100A7) solved by MAD phasing

BACKGROUND The S100 family consists of small acidic proteins, belonging to the EF-hand class of calcium-binding proteins. They are primarily regulatory proteins, involved in cell growth, cell structure regulation and signal transduction. Psoriasin (S100A7) is an 11.7 kDa protein that is highly upregulated in the epidermis of patients suffering from the chronic skin disease psoriasis. Although its exact function is not known, psoriasin is believed to participate in the biochemical response which follows transient changes in the cellular Ca2+ concentration. RESULTS The three-dimensional structure of holmium-substituted psoriasin has been determined by multiple anomalous wavelength dispersion (MAD) phasing and refined to atomic resolution (1.05 A). The structure represents the most accurately determined structure of a calcium-binding protein. Although the overall structure of psoriasin is similar to those of other S100 proteins, several important differences exist, mainly in the N-terminal EF-hand motif that contains a distorted loop and lacks a crucial calcium-binding residue. It is these minor differences that may account for the different specificities among members of this family. CONCLUSIONS The structure of human psoriasin reveals that this protein, in contrast to other S100 proteins with known structure, is not likely to strongly bind more than one calcium ion per monomer. The present study contradicts the idea that calcium binding induces large changes in conformation, as suggested by previously determined structures of apo forms of S100 proteins. The substitution of Ca2+ ions in EF-hands by lanthanide ions may provide a general vehicle for structure determination of S100 proteins by means of MAD phasing.

Structural Organization of Spider Silk

Spider silks are protein polymers with outstanding physical properties. A benchmark for spider silks is the dragline thread of the golden silk spider Nephila. Our study using urea super-contraction suggests a new model for the microstructure of this silk. According to this model, which challenges the general view, this silk consists of a structured fibril tube surrounding a thin core. This hypothetical structural organization could explain the supreme tensile strength of this interesting biopolymer.

Sequence-specific binding of the N-terminal three-finger fragment of Xenopus transcription factor IIIA to the internal control region of a 5S RNA gene

An N‐terminal fragment of Xenopus TFIIIA, containing domains 1–3, (TF 3), was expressed in E. coli. High yields of recombinant zinc finger protein was isolated, and its DNA binding activity for the internal control region (ICR) of the Xenopus 5S RNA gene, was demonstrated by bandshift experiments and DNase I footprinting analysis. TF 3 protects 20 bp of ICR against DNase I digestion. The limits of protection are from +77 to +96 on both coding and noncoding strand. This protection pattern is identical to the protection pattern obtained with TFIIIA in the overlapping region, showing that the 3‐finger fragment accounts fully for the protein‐DNA interactions in TFIIIA‐5S RNA gene over this region.

STRUCTURE OF THE C-TYPE LECTIN CARBOHYDRATE RECOGNITION DOMAIN OF HUMAN TETRANECTIN

Tetranectin (TN) is a C-type lectin involved in fibrinolysis, being the only endogenous ligand known to bind specifically to the kringle 4 domain of plasminogen. TN was originally isolated from plasma, but shows a wide tissue distribution. Furthermore, TN has been found in the extracellular matrix of certain human carcinomas, whereas none or little is present in the corresponding normal tissue. The crystal structure of full-length trimeric TN (2.8 A resolution) has recently been published [Nielsen et al. (1997). FEBS Lett. 412, 388-396]. The crystal structure of the carbohydrate recognition domain (CRD) of human TN (TN3) has been determined separately at 2.0 A resolution in order to obtain detailed information on the two calcium binding sites. This information is essential for the elucidation of the specificity of TN towards oligosaccharides. TN3 crystallizes as a dimer, whereas it appears as a monomer in solution. The overall fold of TN3 is similar to other known CRDs. Each monomer is built of two distinct regions, one region consisting of six beta-strands and two alpha-helices, and the other region is composed of four loops harboring two calcium ions. The calcium ion at site 1 forms an eightfold coordinated complex and has Asp116, Glu120, Gly147, Glu150, Asn151, and one water molecule as ligands. The calcium ion at site 2, which is believed to be involved in recognition and binding of oligosaccharides, is sevenfold coordinated with ligands Gln143, Asp145, Glu150, Asp165, and two water molecules. One sulfate ion has been located at the surface of TN3, forming contacts to Glu120, Lys148, Asn106 of a symmetry-related molecule, and to an ethanol molecule.

The plasminogen binding site of the C-type lectin tetranectin is located in the carbohydrate recognition domain, and binding is sensitive to both calcium and lysine.

Tetranectin, a homotrimeric protein belonging to the family of C-type lectins and structurally highly related to corresponding regions of the mannose-binding proteins, is known specifically to bind the plasminogen kringle 4 protein domain, an interaction sensitive to lysine. Surface plasmon resonance and isothermal calorimetry binding analyses using single-residue and deletion mutant tetranectin derivatives produced in Escherichia coli showed that the kringle 4 binding site resides in the carbohydrate recognition domain and includes residues of the putative carbohydrate binding site. Furthermore, the binding analysis revealed that the interaction is sensitive to calcium in addition to lysine.

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

Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca(2+)-binding C-type lectins. Tetranectin is encoded in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca(2+). Exon 2 encodes an alpha-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6-15) are shown to be of primary importance in heparin binding.

The carboxy-terminal domain of the receptor-associated protein binds to the Vps10p domain of sortilin

Binding of the receptor‐associated protein (RAP) to the newly identified putative sorting receptor, sortilin, was analyzed by surface plasmon resonance analysis of recombinant RAP and sortilin domains and compared with binding to megalin and low density lipoprotein receptor‐related protein (LRP). The data show that the RAP‐binding site in sortilin is localized in the cysteine‐rich lumenal part homologous to yeast vacuolar protein‐sorting 10 protein (Vps10p), and the sortilin‐binding site in RAP is localized in the carboxy‐terminal domain III of the three homologous domains in RAP. Whereas sortilin bound only RAP domain III, megalin and LRP bound all RAP domains with the functional affinity order: domain III>domain I>domain II.

Receptor‐binding domain of human α2‐macroglobulin Expression, folding and biochemical characterization of a high‐affinity recombinant derivative

A recombinant version of the receptor binding domain (RBDv) of human α2‐macroglobulin (α2M) has been expressed in E. coli and refolded using a novel iterative procedure. RBDv (Val1299‐Ala1451) is extended by 15 residues at the N‐terminal side of the Lys1313‐Glu papain cleavage site in human α2M. RBDv contains the intra‐chain bridge Cys1329‐Cys1444 and is soluble and monomeric. Competition experiments with 125I‐labelled methylamine‐treated α2M reveal that RBDv binds to the placental receptor for transformed α2M with a K d of 8 nM, i.e. the binding affinity of RBDv is of the same order of magnitude as the intrinsic affinity for binding of one domain in transformed α2M to one receptor molecule.