Hans-Georg Beisel

Tachylectin-2: crystal structure of a specific GlcNAc/GalNAc-binding lectin involved in the innate immunity host defense of the Japanese horseshoe crab Tachypleus tridentatus.

Tachylectin‐2, isolated from large granules of the hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus), is a 236 amino acid protein belonging to the lectins. It binds specifically to N‐acetylglucosamine and N‐acetylgalactosamine and is a part of the innate immunity host defense system of the horseshoe crab. The X‐ray structure of tachylectin‐2 was solved at 2.0 Å resolution by the multiple isomorphous replacement method and this molecular model was employed to solve the X‐ray structure of the complex with N‐acetylglucosamine. Tachylectin‐2 is the first protein displaying a five‐bladed β‐propeller structure. Five four‐stranded antiparallel β‐sheets of W‐like topology are arranged around a central water‐filled tunnel, with the water molecules arranged as a pentagonal dodecahedron. Tachylectin‐2 exhibits five virtually identical binding sites, one in each β‐sheet. The binding sites are located between adjacent β‐sheets and are made by a large loop between the outermost strands of the β‐sheets and the connecting segment from the previous β‐sheet. The high number of five binding sites within the single polypeptide chain strongly suggests the recognition of carbohydrate surface structures of pathogens with a fairly high ligand density. Thus, tachylectin‐2 employs strict specificity for certain N‐acetyl sugars as well as the surface ligand density for self/non‐self recognition.

Crystal structure of gingipain R: an Arg-specific bacterial cysteine proteinase with a caspase-like fold

Gingipains are cysteine proteinases acting as key virulence factors of the bacterium Porphyromonas gingivalis, the major pathogen in periodontal disease. The 1.5 and 2.0 Å crystal structures of free and D‐Phe‐Phe‐Arg‐chloromethylketone‐inhibited gingipain R reveal a 435‐residue, single‐polypeptide chain organized into a catalytic and an immunoglobulin‐like domain. The catalytic domain is subdivided into two subdomains comprising four‐ and six‐stranded β‐sheets sandwiched by α‐helices. Each subdomain bears topological similarities to the p20‐p10 heterodimer of caspase‐1. The second subdomain harbours the Cys‐His catalytic diad and a nearby Glu arranged around the S1 specificity pocket, which carries an Asp residue to enforce preference for Arg‐P1 residues. This gingipain R structure is an excellent template for the rational design of drugs with a potential to cure and prevent periodontitis. Here we show the binding mode of an arginine‐containing inhibitor in the active‐site, thus identifying major interaction sites defining a suitable pharmacophor.

The 2.0-A crystal structure of tachylectin 5A provides evidence for the common origin of the innate immunity and the blood coagulation systems.

Because invertebrates lack an adaptive immune system, they had to evolve effective intrinsic defense strategies against a variety of microbial pathogens. This ancient form of host defense, the innate immunity, is present in all multicellular organisms including humans. The innate immune system of the Japanese horseshoe crab Tachypleus tridentatus, serving as a model organism, includes a hemolymph coagulation system, which participates both in defense against microbes and in hemostasis. Early work on the evolution of vertebrate fibrinogen suggested a common origin of the arthropod hemolymph coagulation and the vertebrate blood coagulation systems. However, this conjecture could not be verified by comparing the structures of coagulogen, the clotting protein of the horseshoe crab, and of mammalian fibrinogen. Here we report the crystal structure of tachylectin 5A (TL5A), a nonself-recognizing lectin from the hemolymph plasma of T. tridentatus. TL5A shares not only a common fold but also related functional sites with the γ fragment of mammalian fibrinogen. Our observations provide the first structural evidence of a common ancestor for the innate immunity and the blood coagulation systems.

The interaction of insulin-like growth factor-I with the N-terminal domain of IGFBP-5

Insulin‐like growth factors (IGFs) are key regulators of cell proliferation, differentiation and transformation, and are thus pivotal in cancer, especially breast, prostate and colon neoplasms. They are also important in many neurological and bone disorders. Their potent mitogenic and anti‐apoptotic actions depend primarily on their availability to bind to the cell surface IGF‐I receptor. In circulation and interstitial fluids, IGFs are largely unavailable as they are tightly associated with IGF‐binding proteins (IGFBPs) and are released after IGFBP proteolysis. Here we report the 2.1 Å crystal structure of the complex of IGF‐I bound to the N‐terminal IGF‐binding domain of IGFBP‐5 (mini‐IGFBP‐5), a prototype interaction for all N‐terminal domains of the IGFBP family. The principal interactions in the complex comprise interlaced hydrophobic side chains that protrude from both IGF‐I and the IGFBP‐5 fragment and a surrounding network of polar interactions. A solvent‐exposed hydrophobic patch is located on the IGF‐I pole opposite to the mini‐IGFBP‐5 binding region and marks the IGF‐I receptor binding site.

HORSESHOE CRAB COAGULOGEN IS AN INVERTEBRATE PROTEIN WITH A NERVE GROWTH FACTOR-LIKE DOMAIN

The rapid clotting of the horseshoe crab hemolymph is essential for both its host defense and hemostasis. It is mediated by the clotting cascade system which consists of four serine proteinase zymogens and the clottable protein coagulogen. Coagulogen, the target protein of the cascade, is converted to an insoluble gel upon activation of the cascade, giving rise to clot formation. Thus this cascade is reminiscent of the mammalian blood coagulation leading to fibrin clot. The structural analysis of coagulogen revealed a polypeptide fold and disulfide bridge pattern in the C-terminal half of the molecule very similar to nerve growth factor (NGF). This finding assigns coagulogen as the first structurally characterized invertebrate protein which belongs to the cystine knot superfamily. The putative structural similarity of coagulogen and the Drosophila morphogen Spaetzle as well as the homology of its processing proteinases suggests a common origin of the two functionally different cascades. This would exemplify a divergent evolution of two proteinase cascades having totally different functions from common ancestors in a long history of evolution.

Role of Tachylectins in Host Defense of the Japanese Horseshoe Crab Tachypleus Tridentatus

Immunity to pathogens is mediated by two general systems, innate and acquired immunity. Acquired immunity is found only in vertebrates and it is the system of B and T lymphocytes, which produces a multitude of specific antigen receptors and antibodies by somatic gene rearrangement. Innate immunity is phylogenetically older than acquired immunity and is present in all multicellular organisms. These proteins pre-exist in hosts, or are rapidly induced within hours of infection. With this immunity polysaccharides or other substances on pathogens are detected through pattern recognition (1,2). The innate immunity of invertebrates is triggered by polysaccharide derivatives, such as lipopolysaccharides (LPS), proteoglycans, and (β-1,3-glucans. Examples are hemolymph coagulation in horseshoe crabs (3–5) and phenoloxidasemediated melanization in crustaceans and insects (6).