Structural and mutational analyses of the Leptospira interrogans virulence-related heme oxygenase provide insights into its catalytic mechanism

© 2017 Soldano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Heme oxygenase from Leptospira interrogans is an important virulence factor. During catalysis, redox equivalents are provided to this enzyme by the plastidic-type ferredoxin-NADP+ reductase also found in L. interrogans. This process may have evolved to aid this bacterial pathogen to obtain heme-iron from their host and enable successful colonization. Herein we report the crystal structure of the heme oxygenase-heme complex at 1.73 Å resolution. The structure reveals several distinctive features related to its function. A hydrogen bonded network of structural water molecules that extends from the catalytic site to the protein surface was cleared observed. A depression on the surface appears to be the H+ network entrance from the aqueous environment to the catalytic site for O2 activation, a key step in the heme oxygenase reaction. We have performed a mutational analysis of the F157, located at the above-mentioned depression. The mutant enzymes were unable to carry out the complete degradation of heme to biliverdin since the reaction was arrested at the verdoheme stage. We also observed that the stability of the oxyferrous complex, the efficiency of heme hydroxylation and the subsequent conversion to verdoheme was adversely affected. These findings underscore a long-range communication between the outer fringes of the hydrogen-bonded network of structural waters and the heme active site during catalysis. Finally, by analyzing the crystal structures of ferredoxin-NADP+ reductase and heme oxygenase, we propose a model for the productive association of these proteins

An Atypical Riboflavin Pathway Is Essential for Brucella abortus Virulence

Brucellosis is a worldwide zoonosis that affects livestock and humans and is caused by closely related Brucella spp., which are adapted to intracellular life within cells of a large variety of mammals. Brucella can be considered a furtive pathogen that infects professional and non-professional phagocytes. In these cells Brucella survives in a replicative niche, which is characterized for having a very low oxygen tension and being deprived from nutrients such as amino acids and vitamins. Among these vitamins, we have focused on riboflavin (vitamin B2). Flavin metabolism has been barely implicated in bacterial virulence. We have recently described that Brucella and other Rhizobiales bear an atypical riboflavin metabolic pathway. In the present work we analyze the role of the flavin metabolism on Brucella virulence. Mutants on the two lumazine synthases (LS) isoenzymes RibH1 and RibH2 and a double RibH mutant were generated. These mutants and different complemented strains were tested for viability and virulence in cells and in mice. In this fashion we have established that at least one LS must be present for B. abortus survival and that RibH2 and not RibH1 is essential for intracellular survival due to its LS activity in vivo. In summary, we show that riboflavin biosynthesis is essential for Brucella survival inside cells or in mice. These results highlight the potential use of flavin biosynthetic pathway enzymes as targets for the chemotherapy of brucellosis

A disordered region retains the full protease inhibitor activity and the capacity to induce CD8+ T cells in vivo of the oral vaccine adjuvant U-Omp19

U-Omp19 is a bacterial protease inhibitor from Brucella abortus that inhibits gastrointestinal and lysosomal proteases, enhancing the half-life and immunogenicity of co-delivered antigens. U-Omp19 is a novel adjuvant that is in preclinical development with various vaccine candidates. However, the molecular mechanisms by which it exerts these functions and the structural elements responsible for these activities remain unknown. In this work, a structural, biochemical, and functional characterization of U-Omp19 is presented. Dynamic features of U-Omp19 in solution by NMR and the crystal structure of its C-terminal domain are described. The protein consists of a compact C-terminal beta-barrel domain and a flexible N-terminal domain. The latter domain behaves as an intrinsically disordered protein and retains the full protease inhibitor activity against pancreatic elastase, papain and pepsin. This domain also retains the capacity to induce CD8+ T cells in vivo of U-Omp19. This information may lead to future rationale vaccine designs using U-Omp19 as an adjuvant to deliver other proteins or peptides in oral formulations against infectious diseases, as well as to design strategies to incorporate modifications in its structure that may improve its adjuvanticity.Fil: Darriba, María Laura. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Pueblas Castro, Celeste. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Coria, Lorena M. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Bruno, Laura. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Cerutti, María Laura. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Chemes, Lucía B. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Cassataro, Juliana. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Pasquevich, Karina A. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Darriba, María Laura. Universidad Nacional de San Martín. Escuela de Bio y Nanotecnologías (EByN); Argentina.Fil: Pueblas Castro, Celeste. Universidad Nacional de San Martín. Escuela de Bio y Nanotecnologías (EByN); Argentina.Fil: Coria, Lorena M. Universidad Nacional de San Martín. Escuela de Bio y Nanotecnologías (EByN); Argentina.Fil: Bruno, Laura. Universidad Nacional de San Martín. Escuela de Bio y Nanotecnologías (EByN); Argentina.Fil: Cerutti, María Laura. Universidad Nacional de San Martín. Escuela de Bio y Nanotecnologías (EByN); Argentina.Fil: Chemes, Lucía B. Universidad Nacional de San Martín. Escuela de Bio y Nanotecnologías (EByN); Argentina.Fil: Cassataro, Juliana. Universidad Nacional de San Martín. Escuela de Bio y Nanotecnologías (EByN); Argentina.Fil: Pasquevich, Karina A. Universidad Nacional de San Martín. Escuela de Bio y Nanotecnologías (EByN); Argentina.Fil: Otero, Lisandro H. Fundación Instituto Leloir. Plataforma Argentina de Biología Estructural y Metabolómica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Klinke, Sebastián. Fundación Instituto Leloir. Plataforma Argentina de Biología Estructural y Metabolómica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Rasia, Rodolfo M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario. Plataforma Argentina de Biología Estructural y Metabolómica; Argentina

Structural basis for the Pr-Pfr long-range signaling mechanism of a full-length bacterial phytochrome at the atomic level

Phytochromes constitute a widespread photoreceptor family that typically interconverts between two photostates called Pr (red light–absorbing) and Pfr (far-red light–absorbing). The lack of full-length structures solved at the (near-)atomic level in both pure Pr and Pfr states leaves gaps in the structural mechanisms involved in the signal transmission pathways during the photoconversion. Here, we present the crystallographic structures of three versions from the plant pathogen Xanthomonas campestris virulence regulator XccBphP bacteriophytochrome, including two full-length proteins, in the Pr and Pfr states. The structures show a reorganization of the interaction networks within and around the chromophore-binding pocket, an α-helix/β-sheet tongue transition, and specific domain reorientations, along with interchanging kinks and breaks at the helical spine as a result of the photoswitching, which subsequently affect the quaternary assembly. These structural findings, combined with multidisciplinary studies, allow us to describe the signaling mechanism of a full-length bacterial phytochrome at the atomic level.DFG, 221545957, SFB 1078: Proteinfunktion durch ProtonierungsdynamikEC/H2020/664726/EU/EMBL Interdisciplinary, International and Intersectorial Postdocs/EI3PO

Cristalografía en el colegio

El año 2014 fue declarado por UNESCO "Año Internacional de Cristalografía". Esto motivó a la Asociación Argentina de Cristalografía (AACr) a crear el Concurso Nacional de Crecimiento de Cristales para Colegios Secundarios como estrategia de acercamiento a la educación científica escolar y visibilizar la cristalografía como disciplina científica en diversos públicos.Facultad de Informátic

Cristalografía en el colegio

El año 2014 fue declarado por UNESCO "Año Internacional de Cristalografía". Esto motivó a la Asociación Argentina de Cristalografía (AACr) a crear el Concurso Nacional de Crecimiento de Cristales para Colegios Secundarios como estrategia de acercamiento a la educación científica escolar y visibilizar la cristalografía como disciplina científica en diversos públicos.Facultad de Informátic

Crystallization and initial X-ray diffraction analysis of the multi-domain Brucella blue light-activated histidine kinase LOV-HK in its illuminated state

The pathogenic bacterium Brucella abortus codes for a multi-domain dimeric cytoplasmic histidine kinase called LOV-HK, which is a key blue light-activated virulence factor in this microorganism. The structural basis of the light activation mechanism of this protein remains unclear. In this work, full-length LOV-HK was cloned, expressed and purified. The protein was activated by light and crystallized under a controlled illumination environment. The merge of 14 individual native data sets collected on a single crystal resulted in a complete X-ray diffraction data set to a resolution of 3.70 Å with over 2 million reflections. Crystals belong to space group P212121, with unit-cell parameters a = 95.96, b = 105.30, c = 164.49 Å with a dimer in the asymmetric unit. Molecular replacement with Phaser using the individual domains as search models allowed for the reconstruction of almost the whole protein. Very recently, improved LOV-HK crystals led to a 3.25-Å resolution dataset. Refinement and model building is underway. This crystal model will represent one of the very few examples of a multi-domain histidine kinase with known structure. Keywords: Multi-domain protein, Light activation, Signal transduction, Two-component system, Histidine kinas

The Reaction Mechanism of Metallo-β-Lactamases Is Tuned by the Conformation of an Active-Site Mobile Loop

International audienceCarbapenems are "last resort" β-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-β-lactamases (MβLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all β-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MβLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MβLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different β-lactams in all MβLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MβLs

Reduction of the ferric LepHO-heme complex in anaerobic conditions and spontaneous reoxidation.

<p>Time dependent formation (A) and autoxidation (B) of the ferrous heme complex of wild type LepHO (●) and F157I mutant (○) as monitored by variations in absorbance at 426 and 403 nm, respectively. Data extracted from the spectra shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182535#pone.0182535.g005" target="_blank">Fig 5</a>.</p