The unique structure of Haemophilus influenzae protein E reveals multiple binding sites for host factors.

Haemophilus influenzae protein E (PE) is a multifunctional adhesin, involved in direct interactions with lung epithelial cells and host proteins, including plasminogen and the extracellular matrix proteins vitronectin and laminin. We recently crystallized PE and successfully collected X-ray diffraction data to 1.8 Å. Here we solved the structure of a recombinant version of PE and analyzed different functional regions. It is a dimer in solution and in the asymmetric unit of the crystals. The dimer has a structure that resembles a flattened β-barrel. It is however not a true β-barrel as there are differences in both the hydrogen bonding pattern and the shape. Each monomer consisted of a 6-stranded antiparallel β-sheet with a rigid α-helix at the C-terminal tethered to the concave side of the sheet by a disulfide bridge. The laminin/plasminogen binding region (residues 41-68) is exposed, while the vitronectin binding region (residues 84-108) is partially accessible in the dimer. The dimerized PE explains the simultaneous interaction with laminin and vitronectin. In addition, we found this unique adhesin being present in many bacterial genera of the family Pasteurallaceae and also orthologues in other unrelated species (Enterobacter cloacae and Listeria monocytogenes). Peptides corresponding to the surface-exposed regions PE24-37, PE74-89, and PE134-156 were immunogenic in the mouse. Importantly, these peptide-based antibodies also recognised PE at the bacterial surface. Taken together, our detailed structure of PE explains how this important virulence factor of H. influenzae simultaneously interacts with host vitronectin, laminin or plasminogen promoting bacterial pathogenesis

Functional Properties and Molecular Architecture of Leukotriene A4 Hydrolase, a Pivotal Catalyst of Chemotactic Leukotriene Formation

The leukotrienes are a family of lipid mediators involved in inflammation and allergy. Leukotriene B4 is a classical chemoattractant, which triggers adherence and aggregation of leukocytes to the endothelium at only nM concentrations. In addition, leukotriene B4 modulates immune responses, participates in the host defense against infections, and is a key mediator of PAF-induced lethal shock. Because of these powerful biological effects, leukotriene B4 is implicated in a variety of acute and chronic inflammatory diseases, e.g., nephritis, arthritis, dermatitis, and chronic obstructive pulmonary disease. The final step in the biosynthesis of leukotriene B4 is catalyzed by leukotriene A4 hydrolase, a unique bifunctional zinc metalloenzyme with an anion-dependent aminopeptidase activity. Here we describe the most recent developments regarding our understanding of the function and molecular architecture of leukotriene A4 hydrolase

Structure of an Engineered Porcine Phospholipase A2 with Enhanced Activity at 2.1 Å Resolution. Comparison with the Wild-type Porcine and Crotalus atrox Phospholipase A2

The crystal structure of an engineered phospholipase A2 with enhanced activity has been refined to an R-factor of 18.6% at 2.1 Å resolution using a combination of molecular dynamics refinement by the GROMOS package and least-squares refinement by TNT. This mutant phospholipase was obtained previously by deleting residues 62 to 66 in porcine pancreatic phospholipase A2, and changing Asp59 to Ser, Ser60 to Gly and Asn67 to Tyr. The refined structure allowed a detailed comparison with wild-type porcine and Crotalus atrox phospholipase A2. The conformation of the deletion region appears to be intermediate between that in those two enzymes. The residues in the active center are virtually the same. An internal hydrophobic area occupied by Phe63 in the wild-type porcine phospholipase A2 is kept as conserved as possible by local rearrangement of neighboring atoms. In the mutant structure, this hydrophobic pocket is now occupied by the disulfide bond between residues 61 and 91. A detailed description of the second binding site for a calcium ion in this enzyme is given

Enhanced Activity and Altered Specificity of Phospholipase A2 by Deletion of a Surface Loop

Protein engineering and x-ray crystallography have been used to study the role of a surface loop that is present in pancreatic phospholipases but is absent in snake venom phospholipases. Removal of residues 62 to 66 from porcine pancreatic phospholipase A2 does not change the binding constant for micelles significantly, but it improves catalytic activity up to 16 times on micellar (zwitterionic) lecithin substrates. In contrast, the decrease in activity on negatively charged substrates is greater than fourfold. A crystallographic study of the mutant enzyme shows that the region of the deletion has a well-defined structure that differs from the structure of the wild-type enzyme. No structural changes in the active site of the enzyme were detected

Structure and pathogenicity of antibodies specific for citrullinated collagen type II in experimental arthritis

Antibodies to citrulline-modifi ed proteins have a high diagnostic value in rheumatoid arthritis (RA). However, their biological role in disease development is still unclear. To obtain insight into this question, a panel of mouse monoclonal antibodies was generated against a major triple helical collagen type II (CII) epitope (position 359 – 369; ARGLTGRPGDA) with or without arginines modifi ed by citrullination. These antibodies bind cartilage and synovial tissue, and mediate arthritis in mice. Detection of citrullinated CII from RA patients ’ synovial fl uid demonstrates that cartilage-derived CII is indeed citrullinated in vivo. The structure determination of a Fab fragment of one of these antibodies in complex with a citrullinated peptide showed a surprising beta -turn conformation of the peptide and provided information on citrulline recognition. Based on these findings, we propose that autoimmunity to CII, leading to the production of antibodies specific for both native and citrullinated CII, is an important pathogenic factor in the development of RA

Expression, purification, and characterization of the 4 zinc finger region of human tumor suppressor WT1

Wilm's Tumor gene 1 (WT1) encodes a zinc finger protein with four distinct splice isoforms. WT1 has a critical role in genesis of various cancer types both at the DNA/RNA and the protein level. The zinc-finger DNA-binding capacity of the protein is located in the C-terminal domain. Two recombinant proteins, 6HIS-ZN-(wt1) and 6HIS-ZN+(wt1), corresponding to two alternative splice variants of the C-terminal regions of human WT1 (-KTS) and WT1 (+KTS), respectively, were over-expressed with hexa-histidine fusion tags in inclusion bodies in Escherichia coli for crystallization studies. A combination of Ni2+-NTA affinity and size-exclusion chromatography was applied for purification of the proteins in denaturing conditions. The effects of various buffers, salts and other additives were scrutinized in a systematic screening to establish the optimal conditions for solubility and refolding of the recombinant WT1 proteins. Circular dichroism analysis revealed the expected beta beta alpha content for the refolded proteins, with a notable degradation of the alpha-helical segment in the DNA-free state. Electrophoretic mobility shift assay with double-stranded DNA containing the double Egr1 consensus site 5'-GCG-T GG-GCG-3' confirmed that 6HIS-ZN-(wt1) has higher DNA binding affinity than 6HIS-ZN+(wt1). (c) 2005 Elsevier Inc. All rights reserved

X-ray studies on porcine pancreatic phospholipase A2 Mutants.

Phospholipase A2 is a digestive enzyme that is produced in the pancreas of mammals as a proenzyme. This proenzyme is activated in the duodenum by tryptic cleavage of the first seven N-terminal amino acids. The enzyme can also be found in large quantities in the venom of snakes and bees. In much smaller amounts it occurs in almost every cell-type, where it plays a role in the turnover and maintenance of membranes and in the production of arachidonic acid. ... Zie: Summary

Crystallization and X-ray diffraction data analysis of leukotriene A4 hydrolase from Saccharomyces cerevisiae

The Saccharomyces cerevisiae leukotriene A4 (LTA4) hydrolase (scLTA4 hydrolase) has been crystallized in order to study the two activities of LTA4 hydrolase in an evolutionary perspective. Single well diffracting crystals are obtained after switching from the hanging-drop method to liquid-liquid diffusion in capillaries using PEG 8000 as precipitant. These crystals belong to space group P212121, with unit-cell parameters a = 70.8, b = 98.1, c = 99.2 Å. Intensity data to 2.3 Å resolution were collected from a native scLTA4 hydrolase crystal using synchrotron radiation. A molecular-replacement solution was obtained using the human LTA4 hydrolase structure and the program BEAST