Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine α-thrombin·boophilin complex, refined at 2.35 Å resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S1 pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9° and is displaced by 6 Å, while the C-terminal domain rotates almost 6° accompanied by a 3 Å displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P1 residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin·boophilin·trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo.

In heme enzymes belonging to the peroxidase-cyclooxygenase superfamily the proximal histidine is in close interaction with a fully conserved asparagine. The crystal structure of a mixture of glycoforms of myeloperoxidase (MPO) purified from granules of human leukocytes prompted us to revise the orientation of this asparagine and the protonation status of the proximal histidine. The data we present contrast with previous MPO structures, but are strongly supported by molecular dynamics simulations. Moreover, comprehensive analysis of published lactoperoxidase structures suggest that the described proximal heme architecture is a general structural feature of animal heme peroxidases. Its importance is underlined by the fact that the MPO variant N421D, recombinantly expressed in mammalian cell lines, exhibited modified spectral properties and diminished catalytic activity compared with wild-type recombinant MPO. It completely lost its ability to oxidize chloride to hypochlorous acid, which is a characteristic feature of MPO and essential for its role in host defense. The presented crystal structure of MPO revealed further important differences compared with the published structures including the extent of glycosylation, interaction between light and heavy polypeptides, as well as heme to protein covalent bonds. These data are discussed with respect to biosynthesis and post-translational maturation of MPO as well as to its peculiar biochemical and biophysical properties.

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SummaryMycoplasma genitalium is an emerging human pathogen with the smallest genome found among self-replicating organisms. M. genitalium presents a complex cytoskeleton with a differentiated protrusion known as the terminal organelle. This polar structure plays a central role in functions essential for the virulence of the microorganism, such as motility and cellhost adhesion. A well-conserved Enriched in Aromatic and Glycine Residues motif, the EAGR box, is present in many of the proteins found in the terminal organelle. We determined the crystal structure of the globular domain from M. genitalium MG200 that contains an EAGR box. This structural information is the first at near atomic resolution for the components of the terminal organelle. The structure revealed a dimer stabilized by a compact hydrophobic core that extends throughout the dimer interface. Monomers present a new fold that contains an accurate intra-subunit symmetry relating two conspicuous hairpins. Some features, such as the domain plasticity and the presence and organization of the intra-and inter-subunit symmetry axes, support a role for the EAGR box in protein-protein interactions. Genetic, biochemical and microcinematography analyses of MG200 variants lacking the EAGR box containing domain confirm the relevant and specific association of this domain with cell motility.

The X-ray crystal structure of the 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS) from Arabidopsis thaliana has been solved at 2.3 Å resolution in complex with a cytidine-5-monophosphate (CMP) molecule. This is the first structure determined of an MCS enzyme from a plant. Major differences between the A. thaliana and bacterial MCS structures are found in the large molecular cavity that forms between subunits and involve residues that are highly conserved among plants. In some bacterial enzymes, the corresponding cavity has been shown to be an isoprenoid diphosphate-like binding pocket, with a proposed feedback-regulatory role. Instead, in the structure from A. thaliana the cavity is unsuited for binding a diphosphate moiety, which suggests a different regulatory mechanism of MCS enzymes between bacteria and plants.Keywords: plant enzymes; MEP pathway; isoprenoid-binding proteins; cytidine-5-monophosphate; zinc ions Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) are the universal five-carbon precursors of isoprenoids, one of the largest family of natural products accounting for more than 30,000 molecules of both primary and secondary metabolism (McGarvey and Croteau 1995;Sacchettini and Poulter 1997). After the discovery of the mevalonic acid (MVA) pathway in yeast and animals, it was assumed that IPP was synthesized from acetyl-CoA via MVA and then isomerized to DMAPP in all organisms (McGarvey and Croteau 1995;Chappell 2002). However, an alternative MVA-independent pathway for the biosynthesis of IPP was later identified in bacteria (Flesch and Rohmer 1988;Rohmer et al. 1993) and plants (Lichtenthaler et al. 1997;Lichtenthaler 1999) and was named the MEP pathway according to its first committed precursor, 2C-methyl-D-erythritol 4-phosphate (Rodriguez-Concepcion and Boronat 2002;Testa and Brown 2003).In the synthesis of isoprenoids, the MEP pathway is the only one present in most eubacteria, including the causal agents for diverse and serious human diseases like leprosy, bacterial meningitis, various gastrointestinal and sexually transmitted infections, tuberculosis, and certain types of pneumonia. The MEP pathway is the only one present in some protozoans, such as in the Reprint requests to: Ignacio Fita, Institut de Biologia Molecular de Barcelona-CSIC and Institut de Recerca Biomedica, Parc Cientific de Barcelona, Josep Samitier 1-5, 08028 Barcelona, Spain; e-mail: ifrcri@ibmb.csic.es; fax: +34-904-034-979.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

Background:The current antibiotic resistance epidemic demands new drugs specifically targeting infective agents. Results: The crystal structure of the Brucella DRL enzyme shows major differences compared with DXR, which catalyzes the same reaction in most other bacteria. Conclusion: Structural information will allow development of inhibitors targeting only DRL. Significance: Drugs against DRL could function as highly specific, narrow-range antibiotics.

The emergent human pathogen Mycoplasma genitalium, with one of the smallest genomes among cells capable of growing in axenic cultures, presents a flask-shaped morphology due to a protrusion of the cell membrane, known as the terminal organelle, that is involved in cell adhesion and motility and is an important virulence factor of this microorganism. The terminal organelle is supported by a cytoskeleton complex of about 300 nm in length that includes three substructures: the terminal button, the rod and the wheel complex. The crystal structure of the MG491 protein, a proposed component of the wheel complex, has been determined at ~3 Å resolution. MG491 subunits are composed of a 60-residue N-terminus, a central three-helix-bundle spanning about 150 residues and a C-terminal region that appears to be quite flexible and contains the region that interacts with MG200, another key protein of the terminal organelle. The MG491 molecule is a tetramer presenting a unique organization as a dimer of asymmetric pairs of subunits. The asymmetric arrangement results in two very different intersubunit interfaces between the central three-helix-bundle domains, which correlates with the formation of only ~50% of the intersubunit disulfide bridges of the single cysteine residue found in MG491 (Cys87). Moreover, M. genitalium cells with a point mutation in the MG491 gene causing the change of Cys87 to Ser present a drastic reduction in motility (as determined by microcinematography) and important alterations in morphology (as determined by electron microscopy), while preserving normal levels of the terminal organelle proteins. Other variants of MG491, designed also according to the structural information, altered significantly the motility and/or the cell morphology. Together, these results indicate that MG491 plays a key role in the functioning, organization and stabilization of the terminal organelle.