Margit Bauer

Two heads are better than one: crystal structure of the insect derived double domain Kazal inhibitor rhodniin in complex with thrombin.

Rhodniin is a highly specific inhibitor of thrombin isolated from the assassin bug Rhodnius prolixus. The 2.6 Angstrum crystal structure of the non‐covalent complex between recombinant rhodniin and bovine alpha‐thrombin reveals that the two Kazal‐type domains of rhodniin bind to different sites of thrombin. The amino‐terminal domain binds in a substrate‐like manner to the narrow active‐site cleft of thrombin; the imidazole group of the P1 His residue extends into the S1 pocket to form favourable hydrogen/ionic bonds with Asp189 at its bottom, and additionally with Glu192 at its entrance. The carboxy‐terminal domain, whose distorted reactive‐site loop cannot adopt the canonical conformation, docks to the fibrinogen recognition exosite via extensive electrostatic interactions. The rather acidic polypeptide linking the two domains is displaced from the thrombin surface, with none of its residues involved in direct salt bridges with thrombin. The tight (Ki = 2 × 10(‐13) M) binding of rhodniin to thrombin is the result of the sum of steric and charge complementarity of the amino‐terminal domain towards the active‐site cleft, and of the electrostatic interactions between the carboxy‐terminal domain and the exosite.

The Three-dimensional Structure of Recombinant Leech-derived Tryptase Inhibitor in Complex with Trypsin IMPLICATIONS FOR THE STRUCTURE OF HUMAN MAST CELL TRYPTASE AND ITS INHIBITION

The x-ray crystal structure of recombinant leech-derived tryptase inhibitor (rLDTI) has been solved to a resolution of 1.9 Å in complex with porcine trypsin. The nonclassical Kazal-type inhibitor exhibits the same overall architecture as that observed in solution and in rhodniin. The complex reveals structural aspects of the mast cell proteinase tryptase. The conformation of the binding region of rLDTI suggests that tryptase has a restricted active site cleft. The basic amino terminus of rLDTI, apparently flexible from previous NMR measurements, approaches the 148-loop of trypsin. This loop has an acidic equivalent in tryptase, suggesting that the basic amino terminus could make favorable electrostatic interactions with the tryptase molecule. A series of rLDTI variants constructed to probe this hypothesis confirmed that the amino-terminal Lys-Lys sequence plays a role in inhibition of human lung tryptase but not of trypsin or chymotrypsin. The location of such an acidic surface patch is in accordance with the known low molecular weight inhibitors of tryptase.

Structural basis for inhibition promiscuity of dual specific thrombin and factor Xa blood coagulation inhibitors.

BACKGROUND A major current focus of pharmaceutical research is the development of selective inhibitors of the blood coagulation enzymes thrombin or factor Xa to be used as orally bioavailable anticoagulant drugs in thromboembolic disorders and in the prevention of venous and arterial thrombosis. Simultaneous direct inhibition of thrombin and factor Xa by synthetic proteinase inhibitors as a novel approach to antithrombotic therapy could result in potent anticoagulants with improved pharmacological properties. RESULTS The binding mode of such dual specific inhibitors of thrombin and factor Xa was determined for the first time by comparative crystallography using human alpha-thrombin, human des-Gla (1--44) factor Xa and bovine trypsin as the ligand receptors. The benzamidine-based inhibitors utilize two different conformations for the interaction with thrombin and factor Xa/trypsin, which are evoked by the steric requirements of the topologically different S2 subsites of the enzymes. Compared to the unliganded forms of the proteinases, ligand binding induces conformational adjustments of thrombin and factor Xa active site residues indicative of a pronounced induced fit mechanism. CONCLUSION The structural data reveal the molecular basis for a desired unselective inhibition of the two key components of the blood coagulation cascade. The 4-(1-methyl-benzimidazole-2-yl)-methylamino-benzamidine moieties of the inhibitors are able to fill both the small solvent accessible as well as the larger hydrophobic S2 pockets of factor Xa and thrombin, respectively. Distal fragments of the inhibitors are identified which fit into both the cation hole/aromatic box of factor Xa and the hydrophobic aryl binding site of thrombin. Thus, binding constants in the medium-to-low nanomolar range are obtained against both enzymes.

One Target—Two Different Binding Modes: Structural Insights into Gevokizumab and Canakinumab Interactions to Interleukin-1β

Interleukin-1β (IL-1β) is a key orchestrator in inflammatory and several immune responses. IL-1β exerts its effects through interleukin-1 receptor type I (IL-1RI) and interleukin-1 receptor accessory protein (IL-1RAcP), which together form a heterotrimeric signaling-competent complex. Canakinumab and gevokizumab are highly specific IL-1β monoclonal antibodies. Canakinumab is known to neutralize IL-1β by competing for binding to IL-1R and therefore blocking signaling by the antigen:antibody complex. Gevokizumab is claimed to be a regulatory therapeutic antibody that modulates IL-1β bioactivity by reducing the affinity for its IL-1RI:IL-1RAcP signaling complex. How IL-1β signaling is affected by both canakinumab and gevokizumab was not yet experimentally determined. We have analyzed the crystal structures of canakinumab and gevokizumab antibody binding fragment (Fab) as well as of their binary complexes with IL-1β. Furthermore, we characterized the epitopes on IL-1β employed by the antibodies by NMR epitope mapping studies. The direct comparison of NMR and X-ray data shows that the epitope defined by the crystal structure encompasses predominantly those residues whose NMR resonances are severely perturbed upon complex formation. The antigen:Fab co-structures confirm the previously identified key contact residues on IL-1β and provide insight into the mechanisms leading to their distinct modulation of IL-1β signaling. A significant steric overlap of the binding interfaces of IL-1R and canakinumab on IL-1β causes competitive inhibition of the association of IL-1β and its receptor. In contrast, gevokizumab occupies an allosteric site on IL-1β and complex formation results in a minor reduction of binding affinity to IL-1RI. This suggests two different mechanisms of IL-1β pathway attenuation.

The X-Ray Crystal Structure of Thrombin in Complex with Nα-2-Naphthylsulfonyl-L-3-Amidino-Phenylalanyl-4-Methylpiperidide: The Beneficial Effect of Filling Out an Empty Cavity

The 2.5 Angstrum structure of bovine epsilon-thrombin in complex with N alpha-2-naphthyl-sulfonyl-L-3-amidinophenylalanyl-4-methylpiper idide (L-NAPAMP) was solved and crystallographically refined to an R-value of 0.19. The L-NAPAMP moiety is completely and unambiguosly defined in the electron density. NAPAMP binds almost identical to the related 4-methyl deficient 3-amidino-phenylalanyl derivative TAPAP. The overall binding geometry appears dominated by the fixation of the 3-amidinophenyl ring in thrombins S1-pocket and the hydrogen bonds to Gly 216, irrespective of the presence or absence of a substituent in the 4-position of the piperidine ring. The additional 4-methyl group gives rise to a 17-fold better binding. The more complete spatial occupancy of the hydrophobic S2-cavity therefore accounts for a decrease in free energy of binding of 15 kcal/mol, a value comparable with that anticipated for filling up a stable empty cavity of similar size by a methyl group.

Catheter-based Intraluminal Optical Coherence Tomography Versus Endoluminal Ultrasonography of Porcine Ureter Ex Vivo

OBJECTIVES To compare the distinction of tissue layers of porcine ureters ex vivo between optical coherence tomography (OCT) and endoluminal ultrasonography (ELUS). Catheter-guided OCT is a new method of intraluminal microstructural imaging, with a spatial resolution of 10-20 mum. METHODS Porcine ureters and kidneys were obtained fresh from the municipal slaughtery, cannulated with a 7F catheter sheath, flushed with normal saline solution, and marked on the outside with surgical suture. Between the marked positions, images were obtained from within the ureter lumen using OCT (M1, Lightlab, Westport, MA) and ELUS at 40 MHz. The distinction of the urothelium, lamina propria, and inner and outer muscle layers was rated as possible (1) or impossible (0) by 2 independent observers (O1, O2). The rates of distinction were compared between OCT and ELUS image quadrants using the chi(2) test. RESULTS Of the 224 OCT image quadrants and 144 ELUS image quadrants, OCT was superior to ELUS in the distinction of any wall layers (O1, chi(2)P = 68.1051, P < .001; O2, chi(2)P = 66.1630, P < .001), urothelium and lamina propria (O1, chi(2)P = 200.0750, P < .001; O2, chi(2)P = 240.0024, P < .001), and lamina propria and muscle layer (O1, chi(2)P = 38.8411, P < .001; O2, chi(2)P = 24.7536, P < .001) but was inconclusive for the inner and outer muscle layer (O1, chi(2)P = 260.3004, P < .001; O2, chi(2)P = 0.4992, P > .25). CONCLUSIONS OCT was able to distinguish significantly better than ELUS between different wall layers of porcine ureter ex vivo. The feasibility of OCT in vivo and in the presence of pathologic wall thickening of the ureter remains to be demonstrated.

Molecular structure of human GM-CSF in complex with a disease-associated anti-human GM-CSF autoantibody and its potential biological implications

Polyclonal autoantibodies against human GM-CSF (granulocyte/macrophage colony-stimulating factor) are a hallmark of PAP (pulmonary alveolar proteinosis) and several other reported autoimmune diseases. MB007 is a high-affinity anti-(human GM-CSF) autoantibody isolated from a patient suffering from PAP which shows only modest neutralization of GM-CSF bioactivity. We describe the first crystal structure of a cytokine-directed human IgG1λ autoantibody-binding fragment (Fab) at 1.9 Å (1 Å=0.1 nm) resolution. Its CDR3-H substantially differs from all VH7 germline IgG1 structures reported previously. We derive a reliable model of the antigen-autoantibody complex by using NMR chemical shift perturbation data in combination with computational methods. Superposition of the modelled complex structure with the human GM-CSF-GM-CSF ternary receptor complex reveals only little overlap between receptor and Fab when bound to GM-CSF. Our model provides a structural basis for understanding the mode of action of the MB007 autoantibody.

Crystallization of Proteinases

The three dimensional structure of an enzyme is a rich source of information for the modern biochemist. It can give valuable insights into the function and specificity of your proteinase, providing a framework on which to focus previous biochemical data and a sound basis for the design of new experiments. Structural data on your proteinase can have a profound influence on the search and design of novel specific inhibitors (“rational drug design”). The role played by proteinases in a wide range of pathological conditions has provided a spur to this latter theme.