Search

Cells are continuously facing the risk of taking up foreign DNA that can compromise genomic and cellular integrity. Therefore, bacteria are in a constant arms race with mobile genetic elements such as phages, transposons and plasmids. They have developed several active strategies against invading DNA molecules that can be seen as a bacterial ‘innate immune system’. Anti-phage systems are usually organized in ‘defense islands’ and can consist of restriction-modification (R-M) systems, CRISPR-Cas, and abortive infection (Abi) systems. Despite recent advances in the field, much less is known about plasmid defense systems. We have recently identified the MksBEFG system in Corynebacterium glutamicum as a novel plasmid defense system which comprises homologues of the condensin system MukFEB. Here, we investigated the molecular arrangement of the MksBEFG complex. Importantly, we identified MksG as a novel nuclease that degrades plasmid DNA and is, thus, the executing part of the system. The crystal structure of MksG revealed a dimeric assembly through its DUF2220 C-terminal domains. This domain is homologous to the TOPRIM domain of the topoisomerase II family of enzymes and contains the corresponding divalent ion binding site that is essential for DNA cleavage in topoisomerases, explaining the in vitro nuclease activity of MksG. We further show that the MksBEF subunits exhibit an ATPase cycle similar to MukBEF in vitro and we reason that this reaction cycle, in combination with the nuclease activity provided by MksG, allows for processive degradation of invading plasmids. Super-resolution localization microscopy revealed that the Mks system is spatially regulated via to the polar scaffold protein DivIVA. Introduction of plasmids increases the diffusion rate and alters the localization of MksG, indicating an activation of the system in vivo.

International audience; Rap1 is an essential DNA-binding factor from the yeast Saccharomyces cerevisiae involved in transcription and telomere maintenance. Its binding to DNA targets Rap1 at particular loci, and may optimize its ability to form functional macromolecular assemblies. It is a modular protein, rich in large potentially unfolded regions, and comprising BRCT, Myb and RCT well-structured domains. Here, we present the architectures of Rap1 and a Rap1/DNA complex, built through a step-by-step integration of small angle X-ray scattering, X-ray crystallography and nuclear magnetic resonance data. Our results reveal Rap1 structural adjustment upon DNA binding that involves a specific orientation of the C-terminal (RCT) domain with regard to the DNA binding domain (DBD). Crystal structure of DBD in complex with a long DNA identifies an essential wrapping loop, which constrains the orientation of the RCT and affects Rap1 affinity to DNA. Based on our structural information, we propose a model for Rap1 assembly at telomere.

International audience; The Plasmodium subtilase SUB1 plays a pivotal role during the egress of malaria parasites from host hepatocytes and erythrocytes. Here we report the crystal structure of full-length SUB1 from the human-infecting parasite Plasmodium vivax, revealing a bacterial-like catalytic domain in complex with a Plasmodium-specific prodomain. The latter displays a novel architecture with an amino-terminal insertion that functions as a 'belt', embracing the catalytic domain to further stabilize the quaternary structure of the pre-protease, and undergoes calcium-dependent autoprocessing during subsequent activation. Although dispensable for recombinant enzymatic activity, the SUB1 'belt' could not be deleted in Plasmodium berghei, suggesting an essential role of this domain for parasite development in vivo. The SUB1 structure not only provides a valuable platform to develop new anti-malarial candidates against this promising drug target, but also defines the Plasmodium-specific 'belt' domain as a key calcium-dependent regulator of SUB1 during parasite egress from host cells.

Mycobacterium abscessusis the most pathogenic rapid-growing mycobacterium and is one of the most resistant organisms to chemotherapeutic agents. However, structural and functional studies ofM. abscessusproteins that could modify/inactivate antibiotics remain nonexistent. Here, the structural and functional characterization of an arylamineN-acetyltransferase (NAT) fromM. abscessus[(MYCAB)NAT1] are reported. This novel prokaryotic NAT displays significantN-acetyltransferase activity towards aromatic substrates, including antibiotics such as isoniazid andp-aminosalicylate. The enzyme is endogenously expressed and functional in both the rough and smoothM. abscessusmorphotypes. The crystal structure of (MYCAB)NAT1 at 1.8 Å resolution reveals that it is more closely related toNocardia farcinicaNAT than to mycobacterial isoforms. In particular, structural and physicochemical differences from other mycobacterial NATs were found in the active site. Peculiarities of (MYCAB)NAT1 were further supported by kinetic and docking studies showing that the enzyme was poorly inhibited by the piperidinol inhibitor of mycobacterial NATs. This study describes the first structure of an antibiotic-modifying enzyme fromM. abscessusand provides bases to better understand the substrate/inhibitor-binding specificities among mycobacterial NATs and to identify/optimize specific inhibitors. These data should also contribute to the understanding of the mechanisms that are responsible for the pathogenicity and extensive chemotherapeutic resistance ofM. abscessus.

International audience; Pentameric ligand-gated ion channels mediate fast chemical transmission of nerve signals. The structure of a bacterial proton-gated homolog has been established in its open and locally closed conformations at acidic pH. Here we report its crystal structure at neutral pH, thereby providing the X-ray structures of the two end-points of the gating mechanism in the same pentameric ligand-gated ion channel. The large structural variability in the neutral pH structure observed in the four copies of the pentamer present in the asymmetric unit has been used to analyze the intrinsic fluctuations in this state, which are found to prefigure the transition to the open state. In the extracellular domain (ECD), a marked quaternary change is observed, involving both a twist and a blooming motion, and the pore in the transmembrane domain (TMD) is closed by an upper bend of helix M2 (as in locally closed form) and a kink of helix M1, both helices no longer interacting across adjacent subunits. On the tertiary level, detachment of inner and outer β sheets in the ECD reshapes two essential cavities at the ECD-ECD and ECD-TMD interfaces. The first one is the ligand-binding cavity; the other is close to a known divalent cation binding site in other pentameric ligand-gated ion channels. In addition, a different crystal form reveals that the locally closed and open conformations coexist as discrete ones at acidic pH. These structural results, together with site-directed mutagenesis, physiological recordings, and coarse-grained modeling, have been integrated to propose a model of the gating transition pathway.

International audience; d-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.

International audience; a b s t r a c t This review will focus on a description of the three-dimensional structures of two b-neurotoxins, the monomeric PLA 2 ammodytoxin from Vipera ammodytes ammodytes, and heterodimeric crotoxin from Crotalus durissus terrificus, and a detailed structural analysis of their multiple functional sites. We have recently determined at high resolution the crystal structures of two natural isoforms of ammodytoxin (AtxA and AtxC) (Saul et al., 2010) which exhibit different toxicity profiles and different anticoagulant properties. Compara-tive structural analysis of these two PLA 2 isoforms, which differ only by two amino acid residues, allowed us to detect local conformational changes and delineate the role of critical residues in the anticoagulant and neurotoxic functions of these PLA 2 (Saul et al., 2010). We have also determined, at 1.35 Å resolution, the crystal structure of heterodimeric crotoxin (Faure et al., 2011). The three-dimensional structure of crotoxin revealed details of the binding interface between its acidic (CA) and basic (CB) subunits and allowed us to identify key residues involved in the stability and toxicity of this potent heterodimeric b-neurotoxin (Faure et al., 2011). The precise spatial orientation of the three covalently linked polypeptide chains in the mature CA subunit complexed with CB helps us to understand the role played by critical residues of the CA subunit in the increased toxicity of the cro-toxin complex. Since the CA subunit is a natural inhibitor of the catalytic and anticoagulant activities of CB, identification of the CA–CB binding interface describes residues involved in this inhibition. We propose future research directions based on knowledge of the recently reported 3D structures of crotoxin and ammodytoxin.

Comment in : PDZ-peptide complexes: as exciting as ever./ Trave G. Structure. 2011 Oct 12;19(10):1350-1. doi: 10.1016/j.str.2011.09.008.PMID: 22000506; International audience; PTPN4, a human tyrosine phosphatase, protects cells against apoptosis. This protection could be abrogated by targeting the PDZ domain of this phosphatase with a peptide mimicking the C-terminal sequence of the G protein of an attenuated rabies virus strain. Here, we demonstrate that glioblastoma death is triggered upon intracellular delivery of peptides, either from viral origin or from known endogenous ligands of PTPN4-PDZ, such as the C terminus sequence of the glutamate receptor subunit GluN2A. The killing efficiency of peptides closely reflects their affinities for the PTPN4-PDZ. The crystal structures of two PTPN4-PDZ/peptide complexes allow us to pinpoint the main structural determinants of binding and to synthesize a peptide of high affinity for PTPN4-PDZ enhancing markedly its cell death capacity. These results allow us to propose a potential mechanism for the efficiency of peptides and provide a target and a robust framework for the design of new pro-death compounds.

International audience; Tuberculosis is still a leading cause of death in developing countries, for which there is an urgent need for new pharmacological agents. The synthesis of the novel antimycobacterial drug class of benzothiazinones (BTZs) and the identification of their cellular target as DprE1 (Rv3790), a component of the decaprenylphosphoryl-β-d-ribose 2'-epimerase complex, have been reported recently. Here, we describe the identification and characterization of a novel resistance mechanism to BTZ in Mycobacterium smegmatis. The overexpression of the nitroreductase NfnB leads to the inactivation of the drug by reduction of a critical nitro-group to an amino-group. The direct involvement of NfnB in the inactivation of the lead compound BTZ043 was demonstrated by enzymology, microbiological assays and gene knockout experiments. We also report the crystal structure of NfnB in complex with the essential cofactor flavin mononucleotide, and show that a common amino acid stretch between NfnB and DprE1 is likely to be essential for the interaction with BTZ. We performed docking analysis of NfnB-BTZ in order to understand their interaction and the mechanism of nitroreduction. Although Mycobacterium tuberculosis seems to lack nitroreductases able to inactivate these drugs, our findings are valuable for the design of new BTZ molecules, which may be more effective in vivo.

The vancomycin-resistant Staphylococcus aureus VRSA-9 clinical isolate was partially dependent on glycopeptide for growth. The responsible vanA operon had the same organization as that of Tn 1546 and was located on a plasmid. The chromosomal d -Ala: d -Ala ligase ( ddl ) gene had two point mutations that led to Q260K and A283E substitutions, resulting in a 200-fold decrease in enzymatic activity compared to that of the wild-type strain VRSA-6. To gain insight into the mechanism of enzyme impairment, we determined the crystal structure of VRSA-9 Ddl and showed that the A283E mutation induces new ion pair/hydrogen bond interactions, leading to an asymmetric rearrangement of side chains in the dimer interface. The Q260K substitution is located in an exposed external loop and did not induce any significant conformational change. The VRSA-9 strain was susceptible to oxacillin due to synthesis of pentadepsipeptide precursors ending in d -alanyl- d -lactate which are not substrates for the β-lactam-resistant penicillin binding protein PBP2′. Comparison with the partially vancomycin-dependent VRSA-7, whose Ddl is 5-fold less efficient than that of VRSA-9, indicated that the levels of vancomycin dependence and susceptibility to β-lactams correlate with the degree of Ddl impairment. Ddl drug targeting could therefore be an effective strategy against vancomycin-resistant S. aureus .

International audience; The crystal structure of the extracellular domain (ECD) of the pentameric ligand-gated ion-channel from Gloeobacter violaceus (GLIC) was solved at neutral pH at 2.3 A resolution in two crystal forms, showing a surprising hexameric quaternary structure with a 6-fold axis replacing the expected 5-fold axis. While each subunit retains the usual beta-sandwich immunoglobulin-like fold, small deviations from the whole GLIC structure indicate zones of differential flexibility. The changes in interface between two adjacent subunits in the pentamer and the hexamer can be described in a downward translation by one inter-strand distance and a global rotation of the second subunit, using the first one for superposition. While global characteristics of the interface, such as the buried accessible surface area, do not change very much, most of the atom-atom interactions are rearranged. It thus appears that the transmembrane domain is necessary for the proper oligomeric assembly of GLIC and that there is an intrinsic plasticity or polymorphism in possible subunit-subunit interfaces at the ECD level, the latter behaving as a monomer in solution. Possible functional implications of these novel structural data are discussed in the context of the allosteric transition of this family of proteins. In addition, we propose a novel way to quantify elastic energy stored in the interface between subunits, which indicates a tenser interface for the open form than for the closed form (rest state). The hexameric or pentameric forms of the ECD have a similar negative curvature in their subunit-subunit interface, while acetylcholine binding proteins have a smaller and positive curvature that increases from the apo to the holo form.

International audience; NEMO is an integral part of the IkappaB kinase complex and serves as a molecular switch by which the NF-kappaB signaling pathway can be regulated. Oligomerization and polyubiquitin (poly-Ub) binding, mediated through the regulatory CC2-LZ domain, were shown to be key features governing NEMO function, but the relationship between these two activities remains unclear. In this study, we solved the structure of this domain in complex with a designed ankyrin repeat protein, which helps its crystallization. We generated several NEMO mutants in this domain, including those associated with human diseases incontinentia pigmenti and immunodeficiency with or without anhidrotic ectodermal dysplasia. Analytical ultracentrifugation and thermal denaturation experiments were used to evaluate the dimerization properties of these mutants. A fluorescence-based assay was developed, as well, to quantify the interaction to monoubiquitin and poly-Ub chains. Moreover, the effect of these mutations was investigated for the full-length protein. We show that a proper folding of the ubiquitin-binding domain, termed NOA/UBAN/NUB, into a stable coiled-coil dimer is required but not sufficient for efficient interaction with poly-Ub. In addition, we show that binding to poly-Ub and, to a lesser extent, to monoubiquitin increases the stability of the NOA coiled-coil dimer. Collectively, these data provide structural insights into how several pathological mutations within and outside of the CC2-LZ's NOA ubiquitin binding site affect IkappaB kinase activation in the NF-kappaB signaling pathway.

The gene Rv2714 from Mycobacterium tuberculosis, which codes for a hypothetical protein of unknown function, is a representative member of a gene family that is largely confined to the order Actinomycetales of Actinobacteria. Sequence analysis indicates the presence of two paralogous genes in most mycobacterial genomes and suggests that gene duplication was an ancient event in bacterial evolution. The crystal structure of Rv2714 has been determined at 2.6 A resolution, revealing a trimer in which the topology of the protomer core is similar to that observed in a functionally diverse set of enzymes, including purine nucleoside phosphorylases, some carboxypeptidases, bacterial peptidyl-tRNA hydrolases and even the plastidic form of an intron splicing factor. However, some structural elements, such as a beta-hairpin insertion involved in protein oligomerization and a C-terminal alpha-helical domain that serves as a lid to the putative substrate-binding (or ligand-binding) site, are only found in Rv2714 bacterial homologues and represent specific signatures of this protein family.

International audience; Acquired VanG-type resistance to vancomycin in Enterococcus faecalis BM4518 arises from inducible synthesis of peptidoglycan precursors ending in d-alanyld-serine, to which vancomycin exhibits low binding affinity. VanG, a d-alanine: d-serine ligase, catalyzes the ATP-dependent synthesis of the d-Ala-d-Ser dipeptide, which is incorporated into the peptidoglycan synthesis of VanG-type vancomycin-resistant strains. Here, the purification, crystallization and preliminary crystallographic analysis of VanG in complex with ADP are reported. The crystal belonged to space group P3 1 21, with unit-cell parameters a = b = 116.1, c = 177.2 Å , and contained two molecules in the asymmetric unit. A complete data set has been collected to 2.35 Å resolution from a single crystal under cryogenic conditions using synchrotron radiation.

Temperature sensing is essential for the survival of living cells. A major challenge is to understand how a biological thermometer processes thermal information to optimize cellular functions. Using structural and biochemical approaches, we show that the thermosensitive histidine kinase, DesK, from Bacillus subtilis is cold-activated through specific interhelical rearrangements in its central four-helix bundle domain. As revealed by the crystal structures of DesK in different functional states, the plasticity of this helical domain influences the catalytic activities of the protein, either by modifying the mobility of the ATP-binding domains for autokinase activity or by modulating binding of the cognate response regulator to sustain the phosphotransferase and phosphatase activities. The structural and biochemical data suggest a model in which the transmembrane sensor domain of DesK promotes these structural changes through conformational signals transmitted by the membrane-connecting two-helical coiled-coil, ultimately controlling the alternation between output autokinase and phosphatase activities. The structural comparison of the different DesK variants indicates that incoming signals can take the form of helix rotations and asymmetric helical bends similar to those reported for other sensing systems, suggesting that a similar switching mechanism could be operational in a wide range of sensor histidine kinases.

International audience

International audience; Mycobacterium leprae protein ML2640c belongs to a large family of conserved hypothetical proteins predominantly found in mycobacteria, some of them predicted as putative S-adenosylmethionine (AdoMet)-dependent methyltransferases (MTase). As part of a Structural Genomics initiative on conserved hypothetical proteins in pathogenic mycobacteria, we have determined the structure of ML2640c in two distinct crystal forms. As expected, ML2640c has a typical MTase core domain and binds the methyl donor substrate AdoMet in a manner consistent with other known members of this structural family. The putative acceptor substrate-binding site of ML2640c is a large internal cavity, mostly lined by aromatic and aliphatic side-chain residues, suggesting that a lipid-like molecule might be targeted for catalysis. A flap segment (residues 222-256), which isolates the binding site from the bulk solvent and is highly mobile in the crystal structures, could serve as a gateway to allow substrate entry and product release. The multiple sequence alignment of ML2640c-like proteins revealed that the central alpha/beta core and the AdoMet-binding site are very well conserved within the family. However, the amino acid positions defining the binding site for the acceptor substrate display a higher variability, suggestive of distinct acceptor substrate specificities. The ML2640c crystal structures offer the first structural glimpses at this important family of mycobacterial proteins and lend strong support to their functional assignment as AdoMet-dependent methyltransferases.

The gene Rv0813c from Mycobacterium tuberculosis , which codes for a hypothetical protein of unknown function, is conserved within the order Actinomycetales but absent elsewhere. The crystal structure of Rv0813c reveals a new family of proteins that resemble the fatty acid-binding proteins (FABPs) found in eukaryotes. Rv0813c adopts the 10-stranded β-barrel fold typical of FABPs but lacks the double-helix insert that covers the entry to the binding site in the eukaryotic proteins. The barrel encloses a deep cavity, at the bottom of which a small cyclic ligand was found to bind to the hydroxyl group of Tyr192. This residue is part of a conserved Arg-X-Tyr motif much like the triad that binds the carboxylate group of fatty acids in FABPs. Most of the residues forming the internal surface of the cavity are conserved in homologous protein sequences found in CG-rich prokaryotes, strongly suggesting that Rv0813c is a member of a new family of bacterial FABP-like proteins that may have roles in the recognition, transport, and/or storage of small molecules in the bacterial cytosol.

Gamma carbonic anhydrases (gCA) are widespread in Prokaryotes. In Eukaryotes, homologous genes were found only in plant genomes. In Arabidopsis and maize, the corresponding gene products are subunits of mitochondrial Complex I.At present, only gCA homotrimers of Methanosarcina thermophila (CAM) show reversible carbon dioxide (CO2) hydration activity. In the present work, it is shown that recombinant plant Gamma -CA2 could form homotrimers and bind H14CO-3 . However, they are unable to catalyse the reversible hydration of CO2. These results suggest that plant gCAs do not act as carbonic anhydrases but with a related activity possibly contributing to recycle CO2 in the context of photorespiration. Fil: Martin, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Villarreal, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Miras, Isabelle. Instituto Pasteur; Francia Fil: Navaza, Alda. Universite de Paris; Francia Fil: Haouz, Ahmed. Instituto Pasteur; Francia Fil: Gonzalez-Lebrero, Rodolfo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Kaufman, Sergio Benjamín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Zabaleta, Eduardo Julian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina