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ToxR, a Vibrio cholerae transmembrane one-component signal transduction factor, lies within a regulatory cascade that results in the expression of ToxT, toxin coregulated pilus, and cholera toxin. While ToxR has been extensively studied for its ability to activate or repress various genes in V. cholerae , here we present the crystal structures of the ToxR cytoplasmic domain bound to DNA at the toxT and ompU promoters. The structures confirm some predicted interactions, yet reveal other unexpected promoter interactions with implications for other potential regulatory roles for ToxR. We show that ToxR is a versatile virulence regulator that recognizes diverse and extensive, eukaryotic-like regulatory DNA sequences, that relies more on DNA structural elements than specific sequences for binding. Using this topological DNA recognition mechanism, ToxR can bind both in tandem and in a twofold inverted-repeat-driven manner. Its regulatory action is based on coordinated multiple binding to promoter regions near the transcription start site, which can remove the repressing H-NS proteins and prepares the DNA for optimal interaction with the RNA polymerase.

Medium-resolution cryo-electron microscopy maps, in particular when they include a significant number of α-helices, may allow the building of partial models that are useful for molecular-replacement searches in large crystallographic structures when the structures of homologs are not available and experimental phasing has failed. Here, as an example, the solution of the structure of a bacteriophage portal using a partial 30% model built into a 7.8 Å resolution cryo-EM map is shown. Inspection of the self-rotation function allowed the correct oligomerization state to be determined, and density-modification procedures using rotation matrices and a mask based on the cryo-EM structure were critical for solving the structure. A workflow is described that may be applicable to similar cases and this strategy is compared with direct use of the cryo-EM map for molecular replacement., (open access.)

The human pathogen Mycoplasma genitalium is responsible for urethritis in men, and for cervicitis and pelvic inflammatory disease in women. The adherence of M. genitalium to host target epithelial cells is mediated through an adhesion complex called Nap, which is essential for infectivity. Nap is a transmembrane dimer of heterodimers of the immunodominant proteins P110 and P140. The M. genitalium genome contains multiple copies of portions that share homology with the extracellular regions of P140 and P110 encoded by the genes mg191 and mg192, respectively. Homologous recombination between the genes and the copies allows the generation of a large diversity of P140 and P110 variants to overcome surveillance by the host immune system. Interestingly, the C-terminal domain (C-domain) of the extracellular region of P140, which is essential for the function of Nap by acting as a flexible stalk anchoring the protein to the mycoplasma membrane, presents a low degree of sequence variability. In the present work, the X-ray crystal structures of two crystal forms of a construct of the P140 C-domain are reported. In both crystal forms, the construct forms a compact octamer with D4 point-group symmetry. The structure of the C-domain determined in this work presents significant differences with respect to the structure of the C-domain found recently in intact P140. The structural plasticity of the C-domain appears to be a possible mechanism that may help in the functioning of the mycoplasma adhesion complex.

Mycoplasma pneumoniae is a bacterial human pathogen that causes primary atypical pneumonia. M. pneumoniae motility and infectivity are mediated by the immunodominant proteins P1 and P40/P90, which form a transmembrane adhesion complex. Here we report the structure of P1, determined by X-ray crystallography and cryo-electron microscopy, and the X-ray structure of P40/P90. Contrary to what had been suggested, the binding site for sialic acid was found in P40/P90 and not in P1. Genetic and clinical variability concentrates on the N-terminal domain surfaces of P1 and P40/P90. Polyclonal antibodies generated against the mostly conserved C-terminal domain of P1 inhibited adhesion of M. pneumoniae, and serology assays with sera from infected patients were positive when tested against this C-terminal domain. P40/P90 also showed strong reactivity against human infected sera. The architectural elements determined for P1 and P40/P90 open new possibilities in vaccine development against M. pneumoniae infections.

Double-stranded DNA bacteriophages package their genome at high pressure inside a procapsid through the portal, an oligomeric ring protein located at a unique capsid vertex. Once the DNA has been packaged, the tail components assemble on the portal to render the mature infective virion. The tail tightly seals the ejection conduit until infection, when its interaction with the host membrane triggers the opening of the channel and the viral genome is delivered to the host cell. Using high-resolution cryo-electron microscopy and X-ray crystallography, here we describe various structures of the T7 bacteriophage portal and fiber-less tail complex, which suggest a possible mechanism for DNA retention and ejection: a portal closed conformation temporarily retains the genome before the tail is assembled, whereas an open portal is found in the tail. Moreover, a fold including a seven-bladed β-propeller domain is described for the nozzle tail protein.

Split inteins are expressed as two separated subunits (N-intein and C-intein) fused to the corresponding exteins. The specific association of both intein subunits precedes protein splicing, which results in excision of the intein subunits and in ligation, by a peptide bond, of the concomitant exteins. Catalytically active intein precursors are typically too reactive for crystallization or even isolation. Neq pol is the trans-intein of the B-type DNA polymerase I split gene from hyperthermophile Nanoarchaeum equitans. We have determined the crystal structures of both the isolated NeqN and the complex of NeqN and NeqC subunits carrying the wild-type sequences, including the essential catalytic residues Ser1 and Thr+1, in addition to seven and three residues of the N- and C-exteins, respectively. These structures provide detailed information on the unique oxyester chemistry of the splicing mechanism of Neq pol and of the extensive rearrangements that occur in NeqN during the association step., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOB V family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOB V relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterized histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. We discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOB V histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria., Competing Interests: The authors declare no conflict of interest.

A limited repertoire of PPP family of serine/threonine phosphatases with a highly conserved catalytic domain acts on thousands of protein targets to orchestrate myriad central biological roles. A major structural reorganization of human calcineurin, a ubiquitous Ser/Thr PPP regulated by calcium and calmodulin and targeted by immunosuppressant drugs cyclosporin A and FK506, is unveiled here. The new conformation involves trans- to cis-isomerization of proline in the SAPNY sequence, highly conserved across PPPs, and remodels the main regulatory site where NFATc transcription factors bind. Transitions between cis- and trans-conformations may involve peptidyl prolyl isomerases such as cyclophilin A and FKBP12, which are known to physically interact with and modulate calcineurin even in the absence of immunosuppressant drugs. Alternative conformations in PPPs provide a new perspective on interactions with substrates and other protein partners and may foster development of more specific inhibitors as drug candidates.

The MobM relaxase (494 amino acids) encoded by the promiscuous streptococcal plasmid pMV158 recognizes the plasmid origin of transfer, oriTpMV158, and converts supercoiled pMV158 DNA into relaxed molecules by cleavage of the phosphodiester bond of a specific dinucleotide within the sequence 5'-GTGTG/TT-3' ("/" being the nick site). After cleavage, the protein remains stably bound to the 5'-end of the nick site. Band-shift assays with single-stranded oligonucleotides and size-exclusion chromatography allowed us to show that MobM was able to generate specific complexes with one of the inverted repeats of the oriTpMV158, presumably extruded as stem-loop structure. A number of tests have been performed to attain a better characterization of the nicking activity of MobM and its linkage with its target DNA. The optimal pH for DNA relaxation was found to be 6.5. Upon nicking, gel retardation assays showed that MobM formed stable complexes with its target DNA. Moreover, MobM bound to relaxed pMV158 molecules were visualized by electron microscopy. The staphylococcal plasmids pUB110 and pE194, and the streptococcal plasmid pDL287 harbour putative oriTs and may encode Mob proteins homologous to MobM. The oriTpUB110, oriTpDL287, and oriTpE194 sequences share 100%, 70%, and 67% (in a 43-nucleotide stretch and allowing a 3-bp gap) identity to oriTpMV158, respectively. Nicking assays using supercoiled DNAs from pUB110, pDL287, and pE194 showed that MobM was able to relax, to differing degrees, all plasmid DNAs. Our results suggest that cross-recognition of heterologous oriTs by Mob proteins could play an important role in the plasmid spreading between bacteria., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A crucial element in the horizontal transfer of mobilizable and conjugative plasmids is the relaxase, a single-stranded endonuclease that nicks the origin of transfer (oriT) of the plasmid DNA. The relaxase of the pMV158 mobilizable plasmid is MobM (494 residues). In solution, MobM forms a dimer through its C-terminal domain, which is proposed to anchor the protein to the cell membrane and to participate in type 4 secretion system (T4SS) protein-protein interactions. In order to gain a deeper insight into the structural MobM requirements for efficient DNA catalysis, we studied two endonuclease domain variants that include the first 199 or 243 amino acid residues (MobMN199 and MobMN243, respectively). Our results confirmed that the two proteins behaved as monomers in solution. Interestingly, MobMN243 relaxed supercoiled DNA and cleaved single-stranded oligonucleotides harboring oriTpMV158, whereas MobMN199 was active only on supercoiled DNA. Protein stability studies using gel electrophoresis and mass spectrometry showed increased susceptibility to degradation at the domain boundary between the N- and C-terminal domains, suggesting that the domains change their relative orientation upon DNA binding. Overall, these results demonstrate that MobMN243 is capable of nicking the DNA substrate independently of its topology and that the amino acids 200 to 243 modulate substrate specificity but not the nicking activity per se. These findings suggest that these amino acids are involved in positioning the DNA for the nuclease reaction rather than in the nicking mechanism itself.

Decarboxylation of malonyl-CoA to acetyl-CoA by malonyl-CoA decarboxylase (MCD; EC 4.1.1.9) is an essential facet in the regulation of fatty acid metabolism. The structure of human peroxisomal MCD reveals a molecular tetramer that is best described as a dimer of structural heterodimers, in which the two subunits present markedly different conformations. This molecular organization is consistent with half-of-the-sites reactivity. Each subunit has an all-helix N-terminal domain and a catalytic C-terminal domain with an acetyltransferase fold (GNAT superfamily). Intersubunit disulfide bridges, Cys-206-Cys-206 and Cys-243-Cys-243, can link the four subunits of the tetramer, imparting positive cooperativity to the catalytic process. The combination of a half-of-the-sites mechanism within each structural heterodimer and positive cooperativity in the tetramer produces a complex regulatory picture that is further complicated by the multiple intracellular locations of the enzyme. Transport into the peroxisome has been investigated by docking human MCD onto the peroxisomal import protein peroxin 5, which revealed interactions that extend beyond the C-terminal targeting motif.

Resistance of viruses to mutagenic agents is an important problem for the development of lethal mutagenesis as an antiviral strategy. Previous studies with RNA viruses have documented that resistance to the mutagenic nucleoside analogue ribavirin (1-β-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is mediated by amino acid substitutions in the viral polymerase that either increase the general template copying fidelity of the enzyme or decrease the incorporation of ribavirin into RNA. Here we describe experiments that show that replication of the important picornavirus pathogen foot-and-mouth disease virus (FMDV) in the presence of increasing concentrations of ribavirin results in the sequential incorporation of three amino acid substitutions (M296I, P44S and P169S) in the viral polymerase (3D). The main biological effect of these substitutions is to attenuate the consequences of the mutagenic activity of ribavirin -by avoiding the biased repertoire of transition mutations produced by this purine analogue-and to maintain the replicative fitness of the virus which is able to escape extinction by ribavirin. This is achieved through alteration of the pairing behavior of ribavirin-triphosphate (RTP), as evidenced by in vitro polymerization assays with purified mutant 3Ds. Comparison of the three-dimensional structure of wild type and mutant polymerases suggests that the amino acid substitutions alter the position of the template RNA in the entry channel of the enzyme, thereby affecting nucleotide recognition. The results provide evidence of a new mechanism of resistance to a mutagenic nucleoside analogue which allows the virus to maintain a balance among mutation types introduced into progeny genomes during replication under strong mutagenic pressure.

Passage of poliovirus (PV) or foot-and-mouth disease virus (FMDV) in the presence of ribavirin selected for viruses with decreased sensitivity to R, which included different mutations in their polymerase (3D): G64S located in the finger subdomain in the case of PV and M296I located within loop beta9-alpha11 at the active site in the case of FMDV. To investigate why disparate substitutions were selected in two closely related 3Ds, we constructed FMDVs with a 3D that included either G62S (the equivalent replacement in FMDV of PV G64S), M296I, or both substitutions. G62S, but not M296I, inflicts upon FMDV a strong selective disadvantage which is partially compensated for by the substitution M296I. The corresponding mutant polymerases, 3D(G62S), 3D(M296I), and 3D(G62S-M296I), were analyzed functionally and structurally. G62S in 3D impairs RNA-binding, polymerization, and R monophosphate incorporation activities. The X-ray structures of the 3D(G62S)-RNA, 3D(M296I)-RNA, and 3D(G62S-M296I)-RNA complexes show that although the two positions are separated by 13.1 A, the loops where the replacements reside are tightly connected through an extensive network of interactions that reach the polymerase active site. In particular, G62S seems to restrict the flexibility of loop beta9-alpha11 and, as a consequence, the flexibility of the active site and its ability to bind the RNA template. Thus, a localized change in the finger subdomain of 3D may affect the catalytic domain. The results provide a structural interpretation of why different amino acid substitutions were selected to confer R resistance in closely related viruses and reveal a complex network of intra-3D interactions that can affect the recognition of both the RNA template and incoming nucleotide.

RNA virus replication is an error-prone event caused by the low fidelity of viral RNA-dependent RNA polymerases. Replication fidelity can be decreased further by the use of mutagenic ribonucleoside analogs to a point where viral genetic information can no longer be maintained. For foot-and-mouth disease virus, the antiviral analogs ribavirin and 5-fluorouracil have been shown to be mutagenic, contributing to virus extinction through lethal mutagenesis. Here, we report the x-ray structure of four elongation complexes of foot-and-mouth disease virus polymerase 3D obtained in presence of natural substrates, ATP and UTP, or mutagenic nucleotides, ribavirin triphosphate and 5-fluorouridine triphosphate with different RNAs as template-primer molecules. The ability of these complexes to synthesize RNA in crystals allowed us to capture different successive replication events and to define the critical amino acids involved in (i) the recognition and positioning of the incoming nucleotide or analog; (ii) the positioning of the acceptor base of the template strand; and (iii) the positioning of the 3'-OH group of the primer nucleotide during RNA replication. The structures identify key interactions involved in viral RNA replication and provide insights into the molecular basis of the low fidelity of viral RNA polymerases.

TrwC is a DNA strand transferase that catalyzes the initial and final stages of conjugative DNA transfer. We have solved the crystal structure of the N-terminal relaxase domain of TrwC in complex with a 27 base-long DNA oligonucleotide that contains both the recognition hairpin and the scissile phosphate. In addition, a series of ternary structures of protein-DNA complexes with different divalent cations at the active site have been solved. Systematic anomalous difference analysis allowed us to determine unambiguously the nature of the metal bound. Zn2+, Ni2+ and Cu2+ were found to bind the histidine-triad metal binding site. Comparison of the structures of the different complexes suggests two pathways for the DNA to exit the active pocket. They are probably used at different steps of the conjugative DNA-processing reaction. The structural information allows us to propose (i) an enzyme mechanism where the scissile phosphate is polarized by the metal ion facilitating the nucleophilic attack of the catalytic tyrosine, and (ii) a probable sequence of events during conjugative DNA processing that explains the biological function of the relaxase.

Picornavirus RNA replication is initiated by the covalent attachment of a UMP molecule to the hydroxyl group of a tyrosine in the terminal protein VPg. This reaction is carried out by the viral RNA-dependent RNA polymerase (3D). Here, we report the X-ray structure of two complexes between foot-and-mouth disease virus 3D, VPg1, the substrate UTP and divalent cations, in the absence and in the presence of an oligoadenylate of 10 residues. In both complexes, VPg fits the RNA binding cleft of the polymerase and projects the key residue Tyr3 into the active site of 3D. This is achieved by multiple interactions with residues of motif F and helix alpha8 of the fingers domain and helix alpha13 of the thumb domain of the polymerase. The complex obtained in the presence of the oligoadenylate showed the product of the VPg uridylylation (VPg-UMP). Two metal ions and the catalytic aspartic acids of the polymerase active site, together with the basic residues of motif F, have been identified as participating in the priming reaction.

A foot-and-mouth disease virus (FMDV) polymerase (3D) with amino acid replacements G118D, V239M and G373D (triple DMD mutant) was obtained from a molecular clone derived from a virus population treated with ribavirin, in the transition to error catastrophe (virus extinction through lethal mutagenesis). DMD 3D was expressed in Escherichia coli, purified, and its activity compared with that of wild-type enzyme and mutant enzymes with either replacement G118D, G118A or D338A (the latter affecting the catalytic motif YGDD), generated by site-directed mutagenesis. No differences among the enzymes were noted in their interaction with monoclonal antibodies specific for the FMDV polymerase. Mutant enzymes with G118D or G118A showed a 100-fold decrease in polymerization activity relative to wild-type 3D, using poly(A)/oligo(dT)15 and poly(A)/VPg as template-primers, under several reaction conditions. As expected, the activity of 3D with D338A was undetectable (<0.01 times the value for wild-type 3D). DMD and the G118 mutants showed impaired binding to template-primer RNA whereas the D338A mutant showed a binding similar to wild-type 3D. Transfection of cells with FMDV RNA encoding DMD 3D resulted in selection of revertant viruses that maintained only substitutions V239M and G373D. Consistently, when infectious transcripts encoded 3D with either G118D, G118A or D338A, viruses with reversions to the wild-type sequence were isolated. The implication of G118 in template-primer binding is supported by the location of this residue in the template-binding groove of the FMDV polymerase. In addition to identifying an amino acid residue that is critical for the binding of polymerase to RNA, the results document the presence of defective genomes in the transition of virus to error catastrophe.

Relaxases are DNA strand transferases that catalyze the initial and final stages of DNA processing during conjugative cell-to-cell DNA transfer. Upon binding to the origin of transfer (oriT) DNA, relaxase TrwC melts the double helix. The three-dimensional structure of the relaxase domain of TrwC in complex with its cognate DNA at oriT shows a fold built on a two-layer alpha/beta sandwich, with a deep narrow cleft that houses the active site. The DNA includes one arm of an extruded cruciform, an essential feature for specific recognition. This arm is firmly embraced by the protein through a beta-ribbon positioned in the DNA major groove and a loop occupying the minor groove. It is followed by a single-stranded DNA segment that enters the active site, after a sharp U-turn forming a hydrophobic cage that traps the N-terminal methionine. Structural analysis combined with site-directed mutagenesis defines the architecture of the active site.

Biliverdin IXbeta reductase (BVR-B) catalyzes the pyridine nucleotide-dependent production of bilirubin-IXbeta, the major heme catabolite during early fetal development. BVR-B displays a preference for biliverdin isomers without propionates straddling the C10 position, in contrast to biliverdin IXalpha reductase (BVR-A), the major form of BVR in adult human liver. In addition to its tetrapyrrole clearance role in the fetus, BVR-B has flavin and ferric reductase activities in the adult. We have solved the structure of human BVR-B in complex with NADP+ at 1.15 A resolution. Human BVR-B is a monomer displaying an alpha/beta dinucleotide binding fold. The structures of ternary complexes with mesobiliverdin IValpha, biliverdin IXalpha, FMN and lumichrome show that human BVR-B has a single substrate binding site, to which substrates and inhibitors bind primarily through hydrophobic interactions, explaining its broad specificity. The reducible atom of both biliverdin and flavin substrates lies above the reactive C4 of the cofactor, an appropriate position for direct hydride transfer. BVR-B discriminates against the biliverdin IXalpha isomer through steric hindrance at the bilatriene side chain binding pockets. The structure also explains the enzyme's preference for NADP(H) and its B-face stereospecificity.

The transfer of DNA across membranes and between cells is a central biological process; however, its molecular mechanism remains unknown. In prokaryotes, trans-membrane passage by bacterial conjugation, is the main route for horizontal gene transfer. It is the means for rapid acquisition of new genetic information, including antibiotic resistance by pathogens. Trans-kingdom gene transfer from bacteria to plants or fungi and even bacterial sporulation are special cases of conjugation. An integral membrane DNA-binding protein, called TrwB in the Escherichia coli R388 conjugative system, is essential for the conjugation process. This large multimeric protein is responsible for recruiting the relaxosome DNA-protein complex, and participates in the transfer of a single DNA strand during cell mating. Here we report the three-dimensional structure of a soluble variant of TrwB. The molecule consists of two domains: a nucleotide-binding domain of alpha/beta topology, reminiscent of RecA and DNA ring helicases, and an all-alpha domain. Six equivalent protein monomers associate to form an almost spherical quaternary structure that is strikingly similar to F1-ATPase. A central channel, 20 A in width, traverses the hexamer.

Plasmid pMV158 encodes a 45 amino acid transcriptional repressor, CopG, which is involved in copy number control. A new procedure for overproduction and purification of the protein has been developed. The CopG protein thus obtained retained its ability to specifically bind to DNA and to repress its own promoter. Purified CopG protein has been crystallized using the sitting-drop vapor diffusion method. The crystals, belonging to orthorhombic space group C222(1) (cell constants a = 67.2 A, b = 102.5 A, c = 40.2 A), were obtained from a solution containing methylpentanediol, benzamidine and sodium chloride, buffered to pH 6.7. Complete diffraction data up to 1.6 A resolution have been collected. Considerations about the Matthews parameter account for the most likely presence of three molecules in the asymmetric unit (2.27 A3/Da).