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1.  Structure of the regulatory domain of the LysR family regulator NMB2055 (MetR-like protein) from Neisseria meningitidis  
The crystal structure of the regulatory domain of NMB2055, a putative MetR regulator, was solved at 2.5 Å resolution.
The crystal structure of the regulatory domain of NMB2055, a putative MetR regulator from Neisseria meningitidis, is reported at 2.5 Å resolution. The structure revealed that there is a disulfide bond inside the predicted effector-binding pocket of the regulatory domain. Mutation of the cysteines (Cys103 and Cys106) that form the disulfide bond to serines resulted in significant changes to the structure of the effector pocket. Taken together with the high degree of conservation of these cysteine residues within MetR-related transcription factors, it is suggested that the Cys103 and Cys106 residues play an important role in the function of MetR regulators.
doi:10.1107/S1744309112010603
PMCID: PMC3388910  PMID: 22750853
MetR; Neisseria meningitidis; LysR-type regulator
2.  Structural basis of initial RNA polymerase II transcription 
The EMBO Journal  2011;30(23):4755-4763.
Structural basis of initial RNA polymerase II transcription
Several RNA polymerase II–nucleic acid crystal structures reveal the transition of the initiating polymerase from the open complex (OC) state to the initially transcribing complex (ITC) containing several RNA nucleotides.
During transcription initiation by RNA polymerase (Pol) II, a transient open promoter complex (OC) is converted to an initially transcribing complex (ITC) containing short RNAs, and to a stable elongation complex (EC). We report structures of a Pol II–DNA complex mimicking part of the OC, and of complexes representing minimal ITCs with 2, 4, 5, 6, and 7 nucleotide (nt) RNAs, with and without a non-hydrolyzable nucleoside triphosphate (NTP) in the insertion site +1. The partial OC structure reveals that Pol II positions the melted template strand opposite the active site. The ITC-mimicking structures show that two invariant lysine residues anchor the 3′-proximal phosphate of short RNAs. Short DNA–RNA hybrids adopt a tilted conformation that excludes the +1 template nt from the active site. NTP binding induces complete DNA translocation and the standard hybrid conformation. Conserved NTP contacts indicate a universal mechanism of NTP selection. The essential residue Q1078 in the closed trigger loop binds the NTP 2′-OH group, explaining how the trigger loop couples catalysis to NTP selection, suppressing dNTP binding and DNA synthesis.
doi:10.1038/emboj.2011.396
PMCID: PMC3243610  PMID: 22056778
gene transcription; NTP binding; RNA polymerase; transcription initiation
3.  Architecture of the RNA polymerase–Spt4/5 complex and basis of universal transcription processivity 
The EMBO Journal  2011;30(7):1302-1310.
Architecture of the RNA polymerase–Spt4/5 complex and basis of universal transcription processivity
Spt5 and NusG play a conserved role in stimulating RNA polymerase II transcription elongation and processivity. Here, the crystal structure of Spt4/5 bound to the RNA polymerase clamp domain reveals that the factor binds above DNA and RNA in the active centre cleft preventing premature dissociation of the polymerase.
Related RNA polymerases (RNAPs) carry out cellular gene transcription in all three kingdoms of life. The universal conservation of the transcription machinery extends to a single RNAP-associated factor, Spt5 (or NusG in bacteria), which renders RNAP processive and may have arisen early to permit evolution of long genes. Spt5 associates with Spt4 to form the Spt4/5 heterodimer. Here, we present the crystal structure of archaeal Spt4/5 bound to the RNAP clamp domain, which forms one side of the RNAP active centre cleft. The structure revealed a conserved Spt5–RNAP interface and enabled modelling of complexes of Spt4/5 counterparts with RNAPs from all kingdoms of life, and of the complete yeast RNAP II elongation complex with bound Spt4/5. The N-terminal NGN domain of Spt5/NusG closes the RNAP active centre cleft to lock nucleic acids and render the elongation complex stable and processive. The C-terminal KOW1 domain is mobile, but its location is restricted to a region between the RNAP clamp and wall above the RNA exit tunnel, where it may interact with RNA and/or other factors.
doi:10.1038/emboj.2011.64
PMCID: PMC3094117  PMID: 21386817
gene regulation; gene transcription; multiprotein complex structure; RNA polymerase elongation; transcription elongation factor
4.  Crystal Structures of Penicillin-Binding Protein 3 from Pseudomonas aeruginosa: Comparison of Native and Antibiotic-Bound Forms 
Journal of Molecular Biology  2011;405(1-3):173-184.
We report the first crystal structures of a penicillin-binding protein (PBP), PBP3, from Pseudomonas aeruginosa in native form and covalently linked to two important β-lactam antibiotics, carbenicillin and ceftazidime. Overall, the structures of apo and acyl complexes are very similar; however, variations in the orientation of the amino-terminal membrane-proximal domain relative to that of the carboxy-terminal transpeptidase domain indicate interdomain flexibility. Binding of either carbenicillin or ceftazidime to purified PBP3 increases the thermostability of the enzyme significantly and is associated with local conformational changes, which lead to a narrowing of the substrate-binding cleft. The orientations of the two β-lactams in the active site and the key interactions formed between the ligands and PBP3 are similar despite differences in the two drugs, indicating a degree of flexibility in the binding site. The conserved binding mode of β-lactam-based inhibitors appears to extend to other PBPs, as suggested by a comparison of the PBP3/ceftazidime complex and the Escherichia coli PBP1b/ceftoxamine complex. Since P. aeruginosa is an important human pathogen, the structural data reveal the mode of action of the frontline antibiotic ceftazidime at the molecular level. Improved drugs to combat infections by P. aeruginosa and related Gram-negative bacteria are sought and our study provides templates to assist that process and allows us to discuss new ways of inhibiting PBPs.
doi:10.1016/j.jmb.2010.10.024
PMCID: PMC3025346  PMID: 20974151
PBP, penicillin-binding protein; HMM, high molecular mass; LMM, low molecular mass; PDB, Protein Data Bank; ESRF, European Synchrotron Radiation Facility; anti-bacterial; Pseudomonas aeruginosa; carbenicillin; ceftazidime; enzyme structure
5.  Crystallization and preliminary X-ray analysis of CrgA, a LysR-type transcriptional regulator from pathogenic Neisseria meningitidis MC58 
The full length and the regulatory domain of the LysR-type transcriptional regulator CrgA have been crystallized. Diffraction data were collected from two crystal forms of full-length CrgA to 3.0 and 3.8 Å resolution, respectively. Crystals of the selenomethionine derivative of the C-terminal regulatory domain of CrgA diffracted to 2.3 Å resolution.
Although LysR-type regulators (LTTRs) represent the largest family of transcriptional regulators in bacteria, the full-length structure of only one annotated LTTR (CbnR) has been deposited in the PDB. CrgA, a LTTR from pathogenic Neisseria meningitidis MC58, which is up-regulated upon bacterial cell contact with human epithelial cells, has been cloned, purified and crystallized. Crystals of full-length CrgA were obtained after buffer screening with a thermal shift assay and concentration with 0.2 M NDSB-256. Data were collected from two crystal forms of full-length CrgA belonging to space groups P212121 and P21, diffracting to 3.0 and 3.8 Å resolution and consistent with the presence of between six and ten and between ten and 20 copies of CrgA in the asymmetric unit, respectively. In addition, diffraction data were collected to 2.3 Å resolution from the selenomethionine derivative of the regulatory domain of CrgA. The crystals belonged to space group P21 and contained two molecules in the asymmetric unit.
doi:10.1107/S1744309108024068
PMCID: PMC2531262  PMID: 18765907
CrgA; Neisseria meningitidis; LysR-type regulators
6.  The structure of a reduced form of OxyR from Neisseria meningitidis 
Background
Survival of the human pathogen, Neisseria meningitidis, requires an effective response to oxidative stress resulting from the release of hydrogen peroxide by cells of the human immune system. In N. meningitidis, expression of catalase, which is responsible for detoxifying hydrogen peroxide, is controlled by OxyR, a redox responsive LysR-type regulator. OxyR responds directly to intracellular hydrogen peroxide through the reversible formation of a disulphide bond between C199 and C208 in the regulatory domain of the protein.
Results
We report the first crystal structure of the regulatory domain of an OxyR protein (NMB0173 from N. meningitidis) in the reduced state i.e. with cysteines at positions 199 and 208. The protein was crystallized under reducing conditions and the structure determined to a resolution of 2.4 Å. The overall fold of the Neisseria OxyR shows a high degree of similarity to the structure of a C199S mutant OxyR from E. coli, which cannot form the redox sensitive disulphide. In the neisserial structure, C199 is located at the start of helix α3, separated by 18 Å from C208, which is positioned between helices α3 and α4. In common with other LysR-type regulators, full length OxyR proteins are known to assemble into tetramers. Modelling of the full length neisserial OxyR as a tetramer indicated that C199 and C208 are located close to the dimer-dimer interface in the assembled tetramer. The formation of the C199-C208 disulphide may thus affect the quaternary structure of the protein.
Conclusion
Given the high level of structural similarity between OxyR from N. meningitidis and E. coli, we conclude that the redox response mechanism is likely to be similar in both species, involving the reversible formation of a disulphide between C199-C208. Modelling suggests that disulphide formation would directly affect the interface between regulatory domains in an OxyR tetramer which in turn may lead to an alteration in the spacing/orientation of the DNA-binding domains and hence the interaction of OxyR with its DNA binding sites.
doi:10.1186/1472-6807-10-10
PMCID: PMC2881104  PMID: 20478059
7.  Structure of the cold-shock domain protein from Neisseria meningitidis reveals a strand-exchanged dimer 
The X-ray crystal structure of the cold-shock domain protein from N. meningitidis reveals a strand-exchanged dimer.
The structure of the cold-shock domain protein from Neisseria meningitidis has been solved to 2.6 Å resolution and shown to comprise a dimer formed by the exchange of two β-strands between protein monomers. The overall fold of the monomer closely resembles those of other bacterial cold-shock proteins. The neisserial protein behaved as a monomer in solution and was shown to bind to a hexathymidine oligonucleotide with a stoichiometry of 1:1 and a K d of 1.25 µM.
doi:10.1107/S1744309108005411
PMCID: PMC2374261  PMID: 18391418
cold-shock domain proteins; Neisseria meningitidis; domain-exchanged dimers
8.  Semi-automated microseeding of nanolitre crystallization experiments 
A procedure for microseeding into nanolitre crystallization drops is described with selected successful examples.
A simple semi-automated microseeding procedure for nanolitre crystallization experiments is described. Firstly, a microseed stock solution is made from microcrystals using a Teflon bead. A dilution series of this microseed stock is then prepared and dispensed as 100 nl droplets into 96-well crystallization plates, facilitating the incorporation of seeding into high-throughput crystallization pipelines. This basic microseeding procedure has been modified to include additive-screening and cross-seeding methods. Five examples in which these techniques have been used successfully are described.
doi:10.1107/S1744309107057260
PMCID: PMC2373990  PMID: 18097093
crystallization; crystal optimization; microseeding; additives
9.  The structure of CrgA from Neisseria meningitidis reveals a new octameric assembly state for LysR transcriptional regulators 
Nucleic Acids Research  2009;37(14):4545-4558.
LysR-type transcriptional regulators (LTTRs) form the largest family of bacterial regulators acting as both auto-repressors and activators of target promoters, controlling operons involved in a wide variety of cellular processes. The LTTR, CrgA, from the human pathogen Neisseria meningitidis, is upregulated during bacterial–host cell contact. Here, we report the crystal structures of both regulatory domain and full-length CrgA, the first of a novel subclass of LTTRs that form octameric rings. Non-denaturing mass spectrometry analysis and analytical ultracentrifugation established that the octameric form of CrgA is the predominant species in solution in both the presence and absence of an oligonucleotide encompassing the CrgA-binding sequence. Furthermore, analysis of the isolated CrgA–DNA complex by mass spectrometry showed stabilization of a double octamer species upon DNA binding. Based on the observed structure and the mass spectrometry findings, a model is proposed in which a hexadecameric array of two CrgA oligomers binds to its DNA target site.
doi:10.1093/nar/gkp445
PMCID: PMC2724274  PMID: 19474343
10.  The structure of NMB1585, a MarR-family regulator from Neisseria meningitidis  
The structure of the MarR-family regulator NMB1585 from N. meningitidis has been solved using data extending to 2.1 Å resolution.
The structure of the MarR-family transcription factor NMB1585 from Neisseria meningitidis has been solved using data extending to a resolution of 2.1 Å. Overall, the dimeric structure resembles those of other MarR proteins, with each subunit comprising a winged helix–turn–helix (wHtH) domain connected to an α-helical dimerization domain. The spacing of the recognition helices of the wHtH domain indicates that NMB1585 is pre-configured for DNA binding, with a putative inducer pocket that is largely occluded by the side chains of two aromatic residues (Tyr29 and Trp53). NMB1585 was shown to bind to its own promoter region in a gel-shift assay, indicating that the protein acts as an auto-repressor.
doi:10.1107/S174430910900414X
PMCID: PMC2650471  PMID: 19255465
MarR; Neisseria meningitidis; transcription factors
11.  A versatile ligation-independent cloning method suitable for high-throughput expression screening applications 
Nucleic Acids Research  2007;35(6):e45.
This article describes the construction of a set of versatile expression vectors based on the In-Fusion™ cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion™ has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His6-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification.
doi:10.1093/nar/gkm047
PMCID: PMC1874605  PMID: 17317681
12.  Structure of the PII signal transduction protein of Neisseria meningitidis at 1.85 Å resolution 
The structure of the PII signal transduction protein of N. meningitidis at 1.85 Å resolution is described.
The PII signal transduction proteins GlnB and GlnK are implicated in the regulation of nitrogen assimilation in Escherichia coli and other enteric bacteria. PII-like proteins are widely distributed in bacteria, archaea and plants. In contrast to other bacteria, Neisseria are limited to a single PII protein (NMB 1995), which shows a high level of sequence identity to GlnB and GlnK from Escherichia coli (73 and 62%, respectively). The structure of the PII protein from N. meningitidis (serotype B) has been solved by molecular replacement to a resolution of 1.85 Å. Comparison of the structure with those of other PII proteins shows that the overall fold is tightly conserved across the whole population of related proteins, in particular the positions of the residues implicated in ATP binding. It is proposed that the Neisseria PII protein shares functions with GlnB/GlnK of enteric bacteria.
doi:10.1107/S1744309106015430
PMCID: PMC2243107  PMID: 16754965
PII signal transduction proteins; Neisseria meningitidis
13.  Crystal structure of nitrogen regulatory protein IIANtr from Neisseria meningitidis 
Background
The NMB0736 gene of Neisseria meningitidis serogroup B strain MC58 encodes the putative nitrogen regulatory protein, IIANtr (abbreviated to NM-IIANtr). The homologous protein present in Escherichia coli is implicated in the control of nitrogen assimilation. As part of a structural proteomics approach to the study of pathogenic Neisseria spp., we have selected this protein for structure determination by X-ray crystallography.
Results
The NM-IIANtr was over-expressed in E. coli and was shown to be partially mono-phosphorylated, as assessed by mass spectrometry of the purified protein.
Crystals of un-phosphorylated protein were obtained and diffraction data collected to 2.5 Å resolution. The structure of NM-IIANtr was solved by molecular replacement using the coordinates of the E. coli nitrogen regulatory protein IIAntr [PDB: 1A6J] as the starting model. The overall fold of the Neisseria enzyme shows a high degree of similarity to the IIANtr from E. coli, and the position of the phosphoryl acceptor histidine residue (H67) is conserved. The orientation of an adjacent arginine residue (R69) suggests that it may also be involved in coordinating the phosphate group. Comparison of the structure with that of E. coli IIAmtl complexed with HPr [PDB: 1J6T] indicates that NM-IIANtr binds in a similar way to the HPr-like enzyme in Neisseria.
Conclusion
The structure of NM-IIANtr confirms its assignment as a homologue of the IIANtr proteins found in a range of other Gram-negative bacteria. We conclude that the NM- IIANtr protein functions as part of a phosphorylation cascade which, in contrast to E. coli, shares the upstream phosphotransfer protein with the sugar uptake phosphoenolpyruvate:sugar phosphotransferase system (PTS), but in common with E. coli has a distinct downstream effector mechanism.
doi:10.1186/1472-6807-5-13
PMCID: PMC1201152  PMID: 16092953

Results 1-13 (13)