Uwe Jacob

The 3.2-A crystal structure of the human IgG1 Fc fragment-Fc gammaRIII complex.

The immune response depends on the binding of opsonized antigens to cellular Fc receptors and the subsequent initiation of various cellular effector functions of the immune system. Here we describe the crystal structures of a soluble Fcγ receptor (sFcγRIII, CD16), an Fc fragment from human IgG1 (hFc1) and their complex. In the 1:1 complex the receptor binds to the two halves of the Fc fragment in contact with residues of the Cγ2 domains and the hinge region. Upon complex formation the angle between the two sFcγRIII domains increases significantly and the Fc fragment opens asymmetrically. The high degree of amino acid conservation between sFCγRIII and other Fc receptors, and similarly between hFc1 and related immunoglobulins, suggest similar structures and modes of association. Thus the described structure is a model for immune complex recognition and helps to explain the vastly differing affinities of other FcγR–IgG complexes and the FcεRIα–IgE complex.

Crystal structure of the soluble form of the human fcgamma-receptor IIb: a new member of the immunoglobulin superfamily at 1.7 A resolution.

Fcγ‐receptors (FcγRs) represent the link between the humoral and cellular immune responses. Via the binding to FcγR‐positive cells, immunocomplexes trigger several functions such as endocytosis, antibody‐dependent cell‐mediated cytotoxity (ADCC) and the release of mediators, making them a valuable target for the modulation of the immune system. We solved the crystal structure of the soluble human Fcγ‐receptor IIb (sFcγRIIb) to 1.7 Å resolution. The structure reveals two typical immunoglobulin (Ig)‐like domains enclosing an angle of ∼70°, leading to a heart‐shaped overall structure. In contrast to the observed flexible arrangement of the domains in other members of the Ig superfamily, the two domains are anchored by several hydrogen bonds. The structure reveals that the residues relevant for IgG binding, which were already partially characterized by mutagenesis studies, are located within the BC, C′E and FG loops between the β‐strands of the second domain. Moreover, we discuss a model for the sFcγRIIb:IgG complex. In this model, two FcγR molecules bind one IgG molecule with their second domains, while the first domain points away from the complex and is therefore available for binding other cell surface molecules, by which potential immunosuppressing functions could be mediated.

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.

Structural insights into the antigenicity of myelin oligodendrocyte glycoprotein

Multiple sclerosis is a chronic disease of the central nervous system (CNS) characterized by inflammation, demyelination, and axonal loss. The immunopathogenesis of demyelination in multiple sclerosis involves an autoantibody response to myelin oligodendrocyte glycoprotein (MOG), a type I transmembrane protein located at the surface of CNS myelin. Here we present the crystal structures of the extracellular domain of MOG (MOGIgd) at 1.45-Å resolution and the complex of MOGIgd with the antigen-binding fragment (Fab) of the MOG-specific demyelinating monoclonal antibody 8-18C5 at 3.0-Å resolution. MOGIgd adopts an IgV like fold with the A′GFCC′C″ sheet harboring a cavity similar to the one used by the costimulatory molecule B7-2 to bind its ligand CTLA4. The antibody 8-18C5 binds to three loops located at the membrane-distal side of MOG with a surprisingly dominant contribution made by MOG residues 101–108 containing a strained loop that forms the upper edge of the putative ligand binding site. The sequence R101DHSYQEE108 is unique for MOG, whereas large parts of the remaining sequence are conserved in potentially tolerogenic MOG homologues expressed outside the immuno-privileged environment of the CNS. Strikingly, the only sequence identical to DHSYQEE was found in a Chlamydia trachomatis protein of unknown function, raising the possibility that Chlamydia infections may play a role in the MOG-specific autoimmune response in man. Our data provide the structural basis for the development of diagnostic and therapeutic strategies targeting the pathogenic autoantibody response to MOG.

The crystal structure of the novel snake venom plasminogen activator TSV-PA: a prototype structure for snake venom serine proteinases.

BACKGROUND Trimeresurus stejnejeri venom plasminogen activator (TSV-PA) is a snake venom serine proteinase that specifically activates plasminogen. Snake venom serine proteinases form a subfamily of trypsin-like proteinases that are characterised by a high substrate specificity and resistance to inhibition. Many of these venom enzymes specifically interfere with haemostatic mechanisms and display a long circulating half-life. For these reasons several of them have commercial applications and are potentially attractive pharmacological tools. RESULTS The crystal structure of TSV-PA has been determined to 2.5 A resolution and refined to an R factor of 17.8 (R free, 24.4). The enzyme, showing the overall polypeptide fold of trypsin-like serine proteinases, displays unique structural elements such as the presence of a phenylalanine at position 193, a C-terminal tail clamped via a disulphide bridge to the 99-loop, and a structurally conserved Asp97 residue. The presence of a cis proline at position 218 is in agreement with evolutionary relationships to glandular kallikrein. CONCLUSIONS We postulate that Phe 193 accounts for the high substrate specificity of TSV-PA and renders it incapable of forming a stable complex with bovine pancreatic trypsin inhibitor and other extended substrates and inhibitors. Mutational studies previously showed that Asp97 is crucial for the plasminogenolytic activity of TSV-PA, here we identify the conservation of Asp97 in both types of mammalian plasminogen activator - tissue-type (tPA) and urokinase-type (uPA). It seems likely that Asp97 of tPA and uPA will have a similar role in plasminogen recognition. The C-terminal extension of TSV-PA is conserved among snake venom serine proteinases, although its function is unknown. The three-dimensional structure presented here is the first of a snake venom serine proteinase and provides an excellent template for modelling other homologous family members.

Crystal structure of the APC10/DOC1 subunit of the human anaphase-promoting complex.

The anaphase-promoting complex (APC), or cyclosome, is a cell cycle-regulated ubiquitin ligase that controls progression through mitosis and the G1 phase of the cell cycle. The APC is composed of at least 11 subunits; no structure has been determined for any of these subunits. The subunit APC10/DOC1, a one-domain protein consisting of 185 amino acids, has a conserved core (residues 22–161) that is homologous to domains found in several other putative ubiquitin ligases and, therefore, may play a role in ubiquitination reactions. Here we report the crystal structure of human APC10 at 1.6 Å resolution. The core of the protein is formed by a β-sandwich that adopts a jellyroll fold. Unexpectedly, this structure is highly similar to ligand-binding domains of several bacterial and eukaryotic proteins, such as galactose oxidase and coagulation factor Va, raising the possibility that APC10 may function by binding a yet unidentified ligand. We further provide biochemical evidence that the C-terminus of APC10 binds to CDC27/APC3, an APC subunit that contains multiple tetratrico peptide repeats.

The 1.25 A crystal structure of sepiapterin reductase reveals its binding mode to pterins and brain neurotransmitters.

Sepiapterin reductase catalyses the last steps in the biosynthesis of tetrahydrobiopterin, the essential co‐factor of aromatic amino acid hydroxylases and nitric oxide synthases. We have determined the crystal structure of mouse sepiapterin reductase by multiple isomorphous replacement at a resolution of 1.25 Å in its ternary complex with oxaloacetate and NADP. The homodimeric structure reveals a single‐domain α/β‐fold with a central four‐helix bundle connecting two seven‐stranded parallel β‐sheets, each sandwiched between two arrays of three helices. Ternary complexes with the substrate sepiapterin or the product tetrahydrobiopterin were studied. Each subunit contains a specific aspartate anchor (Asp258) for pterin‐substrates, which positions the substrate side chain C1′‐carbonyl group near Tyr171 OH and NADP C4′N. The catalytic mechanism of SR appears to consist of a NADPH‐dependent proton transfer from Tyr171 to the substrate C1′ and C2′ carbonyl functions accompanied by stereospecific side chain isomerization. Complex structures with the inhibitor N‐acetyl serotonin show the indoleamine bound such that both reductase and isomerase activity for pterins is inhibited, but reaction with a variety of carbonyl compounds is possible. The complex structure with N‐acetyl serotonin suggests the possibility for a highly specific feedback regulatory mechanism between the formation of indoleamines and pteridines in vivo.

Glutaconate CoA-transferase from Acidaminococcus fermentans: the crystal structure reveals homology with other CoA-transferases

BACKGROUND Coenzyme A-transferases are a family of enzymes with a diverse substrate specificity and subunit composition. Members of this group of enzymes are found in anaerobic fermenting bacteria, aerobic bacteria and in the mitochondria of humans and other mammals, but so far none have been crystallized. A defect in the human gene encoding succinyl-CoA: 3-oxoacid CoA-transferase causes a metabolic disease which leads to severe ketoacidosis, thus reflecting the importance of this family of enzymes. All CoA-transferases share a common mechanism in which the CoA moiety is transferred from a donor (e.g. acetyl CoA) to an acceptor, (R)-2-hydroxyglutarate, whereby acetate is formed. The transfer has been described by a ping-pong mechanism in which CoA is bound to the active-site residue of the enzyme as a covalent thiol ester intermediate. We describe here the crystal structure of glutaconate CoA-transferase (GCT) from the strictly anaerobic bacterium Acidaminococcus fermentans. This enzyme activates (R)-2-hydroxyglutarate to (R)-2-hydroxyglutaryl-CoA in the pathway of glutamate fermentation. We initiated this project to gain further insight into the function of this enzyme and the structural basis for the characteristics of CoA-transferases. RESULTS The crystal structure of GCT was solved by multiple isomorphous replacement to 2.55 A resolution. The enzyme is a heterooctamer and its overall arrangement of subunits can be regarded as an (AB)4tetramer obeying 222 symmetry. Both subunits A and B belong to the open alpha/beta-protein class and can be described as a four-layered alpha/alpha/beta/alpha type with a novel composition and connectivity of the secondary structure elements. The core of subunit A consists of seven alpha/beta repeats resulting in an all parallel central beta sheet, against which helices pack from both sides. In contrast, the centre of subunit B is formed by a ninefold mixed beta sheet. In both subunits the helical C terminus is folded back onto the N-terminal domain to form the third layer of helices. CONCLUSIONS The active site of GCT is located at the interface of subunits A and B and is formed by loops of both subunits. The funnel-shaped opening to the active site has a depth and diameter of about 20 A with the catalytic residue, Glu54 of subunit B, at the bottom. The active-site glutamate residue is stabilized by hydrogen bonds. Despite very low amino acid sequence similarity, subunits A and B reveal a similar overall fold. Large parts of their structures can be spatially superimposed, suggesting that both subunits have evolved from a common ancestor.

Kinetics and inhibition of recombinant human cystathionine gamma-lyase. Toward the rational control of transsulfuration.

The gene encoding human cystathionine γ-lyase was cloned from total cellular Hep G2 RNA. Fusion to a T7 promoter allowed expression in Escherichia coli, representing the first mammalian cystathionine γ-lyase overproduced in a bacterial system. About 90% of the heterologous gene product was insoluble, and renaturation experiments from purified inclusion bodies met with limited success. About 5 mg/liter culture of human cystathionine γ-lyase could also be extracted from the soluble lysis fraction, employing a three-step native procedure. While the enzyme showed high γ-lyase activity toward l-cystathionine (K m = 0.5 mm,V max = 2.5 units/mg) with an optimum pH of 8.2, no residual cystathionine β-lyase behavior and only marginal reactivity toward l-cystine and l-cysteine were detected. Inhibition studies were performed with the mechanism-based inactivators propargylglycine, trifluoroalanine, and aminoethoxyvinylglycine. Propargylglycine inactivated human cystathionine γ-lyase much more strongly than trifluoroalanine, in agreement with the enzyme’s preference for C-γ–S bonds. Aminoethoxyvinylglycine showed slow and tight binding characteristics with a K i of 10.5 μm, comparable with its effect on cystathionine β-lyase. The results have important implications for the design of specific inhibitors for transsulfuration components.

HUMAN FCGAMMA RECEPTOR IIB EXPRESSED IN ESCHERICHIA COLI REVEALS IGG BINDING CAPABILITY

Abstract Fcγ receptors (FcγR) are expressed on immunologically active cells where they trigger B and T cell responses and are responsible for the clearance of immunocomplexes. They occur as type I transmembrane proteins and also in soluble forms (sFcR) comprising only the ecto domains of the receptors. State-of-the-art research has generated demand for highly pure and homogeneous sFcγR preparations: first, studies of the immunoregulative potential of the soluble FcγRs have been hampered by co-purified growth factors. Second, they are needed for crystallographic analyses to solve questions such as the exact location of the binding site for IgG on the receptor, and the graded affinities of the receptors for different IgG subclasses. This has been unsuccessful due to limitations in availability and homogeneity of sFcγR expressed in eukaryotic cells. In order to address these problems we expressed the extracellular part of the human Fc-γRIIb in E. coli. The protein was refolded, purified in a three-step procedure and characterized by SDS-PAGE, mass spectrometry as well as N-terminal sequencing. The unglycosylated FcγRIIb is active because it binds immobilized antibody as well as the IgG Fc-fragment in solution. Finally, the receptor was crystallized in orthorhombic, tetragonal and hexagonal crystal forms that diffracted X-rays to resolutions of 1.7 Å, 2.7 Å and 3.8 Å respectively.