The crystal structure of Rv2991 from Mycobacterium tuberculosis : An F 420 binding protein with unknown function

The crystal structure of the conserved hypothetical protein Rv2991 from Mycobacterium tuberculosis has been solved by SAD using seleno-methionine substituted protein. The dimeric biological assembly and the sequence and fold conservation are typical of F 420 cofactor binding enzymes. Despite Rv2991 still being of unknown function, sequence and structural comparison with similar proteins enable a role to be proposed for its C-terminal stretch of residues in recognizing and orienting the substrate. In addition, the C-terminus is involved in both protein folding and determining the size of the active site cavity

Eukaryotic-like gephyrin and cognate membrane receptor coordinate corynebacterial cell division and polar elongation

The order Corynebacteriales includes major industrial and pathogenic Actinobacteria such as Corynebacterium glutamicum or Mycobacterium tuberculosis. These bacteria have multi-layered cell walls composed of the mycolyl-arabinogalactan-peptidoglycan complex and a polar growth mode, thus requiring tight coordination between the septal divisome, organized around the tubulin-like protein FtsZ, and the polar elongasome, assembled around the coiled-coil protein Wag31. Here, using C. glutamicum, we report the discovery of two divisome members: a gephyrin-like repurposed molybdotransferase (Glp) and its membrane receptor (GlpR). Our results show how cell cycle progression requires interplay between Glp/GlpR, FtsZ and Wag31, showcasing a crucial crosstalk between the divisome and elongasome machineries that might be targeted for anti-mycobacterial drug discovery. Further, our work reveals that Corynebacteriales have evolved a protein scaffold to control cell division and morphogenesis, similar to the gephyrin/GlyR system that mediates synaptic signalling in higher eukaryotes through network organization of membrane receptors and the microtubule cytoskeleton.Agencia Nacional de Investigación e InnovaciónECOS-Sud France-Uruguay U20B02FOCEM-COF 03/11Agence Nationale de la Recherche ANR-18-CE11-0017/ANR-21-CE11-000

Structural and Functional Characterization of VanG d-Ala:d-Ser Ligase Associated with Vancomycin Resistance in Enterococcus faecalis

International audienced-Alanyl:d-lactate (d-Ala:d-Lac) and d-alanyl:d-serine ligases are key enzymes in vancomycin resistance of Gram-positive cocci. They catalyze a critical step in the synthesis of modified peptidoglycan precursors that are low binding affinity targets for vancomycin. The structure of the d-Ala:d-Lac ligase VanA led to the understanding of the molecular basis for its specificity, but that of d-Ala:d-Ser ligases had not been determined. We have investigated the enzymatic kinetics of the d-Ala:d-Ser ligase VanG from Enterococcus faecalis and solved its crystal structure in complex with ADP. The overall structure of VanG is similar to that of VanA but has significant differences mainly in the N-terminal and central domains. Based on reported mutagenesis data and comparison of the VanG and VanA structures, we show that residues Asp-243, Phe-252, and Arg-324 are molecular determinants for d-Ser selectivity. These residues are conserved in both enzymes and explain why VanA also displays d-Ala:d-Ser ligase activity, albeit with low catalytic efficiency in comparison with VanG. These observations suggest that d-Ala:d-Lac and d-Ala:d-Ser enzymes have evolved from a common ancestral d-Ala:d-X ligase. The crystal structure of VanG showed an unusual interaction between two dimers involving residues of the omega loop that are deeply anchored in the active site. We constructed an octapeptide mimicking the omega loop and found that it selectively inhibits VanG and VanA but not Staphylococcus aureus d-Ala:d-Ala ligase. This study provides additional insight into the molecular evolution of d-Ala:d-X ligases and could contribute to the development of new structure-based inhibitors of vancomycin resistance enzymes

Crystallographic studies of two variants of Pseudomonas aeruginosa IMPDH with impaired allosteric regulation

International audienceInosine-5'-monophosphate dehydrogenases (IMPDHs), which are the rate-limiting enzymes in guanosine-nucleotide biosynthesis, are important therapeutic targets. Despite in-depth functional and structural characterizations of various IMPDHs, the role of the Bateman domain containing two CBS motifs remains controversial. Their involvement in the allosteric regulation of Pseudomonas aeruginosa IMPDH by Mg-ATP has recently been reported. To better understand the function of IMPDH and the importance of the CBS motifs, the structure of a variant devoid of these modules (ΔCBS) was solved at high resolution in the apo form and in complex with IMP. In addition, a single amino-acid substitution variant, D199N, was also structurally characterized: the mutation corresponds to the autosomal dominant mutant D226N of human IMPDH1, which is responsible for the onset of the retinopathy adRP10. These new structures shed light onto the possible mechanism of regulation of the IMPDH enzymatic activity. In particular, three conserved loops seem to be key players in this regulation as they connect the tetramer-tetramer interface with the active site and show significant modification upon substrate binding

The crystal structure of Trypanosoma cruzi arginine kinase

International audienc

Overproduction, purification, crystallization and preliminary X-ray analysis of the peroxiredoxin domain of a larger natural hybrid protein from Thermotoga maritima

Crystals of the peroxiredoxin domain of a larger natural hybrid protein from T. maritima were obtained which diffracted to 2.9 Å resolution on a synchrotron source

Structural plasticity of the HHD 2 domain of whirlin

International audienceWhirlin is a protein essential to sensory neurons. Its defects are responsible for nonsyndromic deafness or for the Usher syndrome, a condition associating congenital deafness and progressive blindness. This large multidomain scaffolding protein is expressed in three isoforms with different functions and localizations in stereocilia bundles of hearing hair cells or in the connecting cilia of photoreceptor cells. The HHD2 domain of whirlin is the only domain shared by all isoforms, but its function remains unknown. In this article, we report its crystal structure in two distinct conformations, a monomeric five‐helix bundle, similar to the known structure of other HHD domains, and a three‐helix bundle organized as a swapped dimer. Most of the hydrophobic contacts and electrostatic interactions that maintain the globular monomeric form are conserved at the protomer interface of the dimer. NMR experiments revealed that the five‐helix conformation is predominant in solution, but exhibits increased dynamics on one face encompassing the hinge loops. Using NMR and SAXS, we also show that HHD2 does not interact with its preceding domains. Our findings suggest that structural plasticity might play a role in the function of the HHD2 domai

Crystallization and preliminary X-ray analysis of a d-­Ala:d-Ser ligase associated with VanG-type vancomycin resistance

The VanG d-alanine:d-serine ligase was crystallized in complex with ADP and diffraction data were collected at 2.35 Å resolution

Insights into the Rrf2 repressor family - the structure of CymR, the global cysteine regulator of Bacillus subtilis

International audienceThe global regulator CymR represses the transcription of a large set of genes involved in cystine uptake and cysteine biosynthesis in Bacillus subtilis and Staphylococcus aureus. This repressor belongs to the widespread and poorly characterized Rrf2 family of regulators. The crystal structure of CymR from B. subtilis reveals a biologically active dimer, where each monomer folds into two tightly packed domains: a DNA-binding domain, which houses a winged helix-turn-helix (wHTH) motif; and a long dimerization domain, which places the wHTH motifs at the extremes. This architecture explains how these small regulators can span 23-27-bp DNA targets. The wHTH motif of CymR resembles those of the GntR superfamily of regulators, such as FadR and HutC. Superimposing the FadR wHTH motifs bound to their DNA fragments onto the wHTH motifs of the CymR dimer structure suggests that the DNA target and/or the protein must undergo some conformational changes upon binding. The CymR structure also hints at a possible location of the Fe-S centre associated with several Rrf2-type regulators

Structural characterization of a novel subfamily of leucine-rich repeat proteins from the human pathogen Leptospira interrogans.

International audiencePathogenic Leptospira spp. are the agents of leptospirosis, an emerging zoonotic disease. Analyses of Leptospira genomes have shown that the pathogenic leptospires (but not the saprophytes) possess a large number of genes encoding proteins containing leucine-rich repeat (LRR) domains. In other pathogenic bacteria, proteins with LRR domains have been shown to be involved in mediating host-cell attachment and invasion, but their functions remain unknown in Leptospira. To gain insight into the potential function of leptospiral LRR proteins, the crystal structures of four LRR proteins that represent a novel subfamily with consecutive stretches of a 23-amino-acid LRR repeat motif have been solved. The four proteins analyzed adopt the characteristic α/β-solenoid horseshoe fold. The exposed residues of the inner concave surfaces of the solenoid, which constitute a putative functional binding site, are not conserved. The various leptospiral LRR proteins could therefore recognize distinct structural motifs of different host proteins and thus serve separate and complementary functions in the physiology of these bacteria