Structural analysis of a novel class of R-M controller proteins: C.Csp231I from Citrobacter sp. RFL231

Controller proteins play a key role in the temporal regulation of gene expression in bacterial restriction-modification (R-M) systems and are important mediators of horizontal gene transfer. They form the basis of a highly cooperative, concentration-dependent genetic switch involved in both activation and repression of R-M genes. Here we present biophysical, biochemical, and high-resolution structural analysis of a novel class of controller proteins, exemplified by C.Csp231I. In contrast to all previously solved C-protein structures, each protein subunit has two extra helices at the C-terminus, which play a large part in maintaining the dimer interface. The DNA binding site of the protein is also novel, having largely AAAA tracts between the palindromic recognition half-sites, suggesting tight bending of the DNA. The protein structure shows an unusual positively charged surface that could form the basis for wrapping the DNA completely around the C-protein dimer.     

High-resolution crystal structure of peptidyl-tRNA hydrolase from Thermus thermophilus

Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are the products of defective translation, to recycle the tRNA for further rounds of protein synthesis. Pth is ubiquitous in nature, and its activity is essential for bacterial viability. Here, we have determined the crystal structure of Pth from Thermus thermophilus (TtPth) at 1.00 Å resolution. This is the first structure of a Pth from a thermophilic bacterium and the highest resolution Pth structure reported so far. The present atomic resolution data enabled the calculation of anisotropic displacement parameters for all atoms, which revealed the directionality of the fluctuations of key regions for the substrate recognition. Comparisons between TtPth and mesophilic bacterial Pths revealed that their structures are similar overall. However, the structures of the N- and C-terminal, loop-helix α4, and helix α6 regions are different. In addition, the helix α1 to strand β4 region of TtPth is remarkably different from those of the mesophilic bacterial Pths, because this region is 9 or 10 amino acid residues shorter than those of the mesophilic bacterial Pths. This shortening seems to contribute to the thermostability of TtPth. To further understand the determinants for the thermostability of TtPth, we compared various structural factors of TtPth with those of mesophilic bacterial Pths. The data suggest that the decreases in accessible surface area and thermolabile amino acid residues, and the increases in ion pairs, hydrogen bonds, and proline residues cooperatively contribute to the thermostability of TtPth.     

Disulfide engineering at the dimer interface of Lactobacillus casei thymidylate synthase: crystal structure of the T155C/E188C/C244T mutant

The crystal structure of a covalently cross-linked Lactobacillus casei thymidylate synthase has been determined at 2.8 A resolution. The sites for mutation to achieve the bis-disulfide linked dimer were identified using the disulfide modeling program MODIP. The mutant so obtained was found to be remarkably thermostable. This increase in stability has been reasoned to be entirely a consequence of the covalent gluing between the two subunits.     

Solution structure and dynamics of a designed monomeric variant of the lambda Cro repressor.

The solution structure of a monomeric variant of the lambda Cro repressor has been determined by multidimensional NMR. Cro K56[DGEVK] differs from wild-type Cro by the insertion of five amino acids at the center of the dimer interface. 1H and 15N resonances for 70 of the 71 residues have been assigned. Thirty-two structures were calculated by hybrid distance geometry/simulated annealing methods using 463 NOE-distance restraints, 26 hydrogen-bond, and 39 dihedral-angle restraints. The root-mean-square deviation (RMSD) from the average structure for atoms in residues 3-60 is 1.03 +/- 0.44 A for the peptide backbone and 1.6 +/- 0.73 A for all nonhydrogen atoms. The overall structure conforms very well to the original design. Although the five inserted residues form a beta hairpin as expected, this engineered turn as well as other turns in the structure are not well defined by the NMR data. Dynamics studies of backbone amides reveal T1/T2 ratios of residues in the alpha2-alpha3, beta2-beta3, and engineered turn that are reflective of chemical exchange or internal motion. The solution structure and dynamics are discussed in light of the conformational variation that has been observed in other Cro structures, and the importance of flexibility in DNA recognition.     

An ancestral nuclear protein assembly: crystal structure of the Methanopyrus kandleri histone

Eukaryotic histone proteins condense DNA into compact structures called nucleosomes. Nucleosomes were viewed as a distinguishing feature of eukaryotes prior to identification of histone orthologs in methanogens. Although evolutionarily distinct from methanogens, the methane-producing hyperthermophile Methanopyrus kandleri produces a novel, 154-residue histone (HMk). Amino acid sequence comparisons show that HMk differs from both methanogenic and eukaryotic histones, in that it contains two histone-fold ms within a single chain. The two HMk histone-fold ms, N and C terminal, are 28% identical in amino acid sequence to each other and approximately 21% identical in amino acid sequence to other histone proteins. Here we present the 1.37-A-resolution crystal structure of HMk and report that the HMk monomer structure is homologous to the eukaryotic histone heterodimers. In the crystal, HMk forms a dimer homologous to [H3-H4](2) in the eukaryotic nucleosome. Based on the spatial similarities to structural ms found in the eukaryotic nucleosome that are important for DNA-binding, we infer that the Methanopyrus histone binds DNA in a manner similar to the eukaryotic histone tetramer [H3-H4](2).     

Identification of the dimer interface of the lactose transport protein from Streptococcus thermophilus

The lactose transporter from Streptococcus thermophilus catalyses the symport of galactosides and protons. The carrier domain of the protein harbours the contact sites for dimerization, and the individual subunits in the dimer interact functionally during the transport reaction. As a first step towards the elucidation of the mechanism behind the cooperation between the subunits, regions involved in the dimer interface were determined by oxidative and chemical cross-linking of 12 cysteine substitution mutants. Four positions in the protein were found to be susceptible to intermolecular cross-linking. To ensure that the observed cross-links were not the result of randomly colliding particles, the cross-linking was studied in samples in which either the concentration of LacS in the membrane was varied or the oligomeric state was manipulated. These experiments showed that the cross-links were formed specifically within the dimer. The four regions of the protein located at the dimer interface are close to the extracellular ends of transmembrane segments V and VIII and the intracellular ends of transmembrane segments VI and VII.     

Lipidic cubic phase crystal structure of the photosynthetic reaction centre from Rhodobacter sphaeroides at 2.35A resolution

Well-ordered crystals of the bacterial photosynthetic reaction centre from Rhodobacter sphaeroides were grown from a lipidic cubic phase. Here, we report the type I crystal packing that results from this crystallisation medium, for which 3D crystals grow as stacked 2D crystals, and the reaction centre X-ray structure is refined to 2.35A resolution. In this crystal form, the location of the membrane bilayer could be assigned with confidence. A cardiolipin-binding site is found at the protein-protein interface within the membrane-spanning region, shedding light on the formation of crystal contacts within the membrane. A chloride-binding site was identified in the membrane-spanning region, which suggests a putative site for interaction with the light-harvesting complex I, the cytochrome bc(1) complex or PufX. Comparisons with the X-ray structures of this reaction centre deriving from detergent-based crystals are drawn, indicating that a slight compression occurs in this lipid-rich environment.     

Shape and subunit organisation of the DNA methyltransferase M.AhdI by small-angle neutron scattering

Type I restriction-modification (R-M) systems encode multisubunit/multidomain enzymes. Two genes (M and S) are required to form the methyltransferase (MTase) that methylates a specific base within the recognition sequence and protects DNA from cleavage by the endonuclease. The DNA methyltransferase M.AhdI is a 170 kDa tetramer with the stoichiometry M(2)S(2) and has properties typical of a type I MTase. The M.AhdI enzyme has been prepared with deuterated S subunits, to allow contrast variation using small-angle neutron scattering (SANS) methods. The SANS data were collected in a number of (1)H:(2)H solvent contrasts to allow matching of one or other of the subunits in the multisubunit enzyme. The radius of gyration (R(g)) and maximum dimensions (D(max)) of the M subunits in situ in the multisubunit enzyme (50 A and 190 A, respectively) are close of those of the entire MTase (51 A and 190 A). In contrast, the S subunits in situ have experimentally determined values of R(g)=35 A and D(max)=110 A, indicating their more central location in the enzyme. Ab initio reconstruction methods yield a low-resolution structural model of the shape and subunit organization of M.AhdI, in which the Z-shaped structure of the S subunit dimer can be discerned. In contrast, the M subunits form a much more elongated and extended structure. The core of the MTase comprises the two S subunits and the globular regions of the two M subunits, with the extended portion of the M subunits most probably forming highly mobile regions at the outer extremities, which collapse around the DNA when the MTase binds.     

Degradation of benzyldimethylalkylammonium chloride by Aeromonas hydrophila sp. K

AIMS: The aim of this work was to study the biodegradation of benzyldimethylalkylammonium chloride (BAC) by Aeromonas hydrophila sp. K, an organism isolated from polluted soil and capable of utilizing BAC as sole source of carbon and energy. METHODS AND RESULTS: High performance liquid chromatography and gas chromatography-mass spectrometry (GC-MS) analysis was used to study BAC degradation pathway. It was shown that during BAC biodegradation, formation of benzyldimethylamine, benzylmethylamine, benzylamine, benzaldehyde and benzoic acid occurred. Formation of benzyldimethylamine as the initial metabolite suggested that the cleavage of Calkyl-N bond occurred as the first step of BAC catabolism. Liberation of benzylmethylamine and benzylamine likely resulted from subsequent demethylation reactions, followed by deamination with formation of benzaldehyde. Benzaldehyde was rapidly converted into benzoic acid, which was further degraded. CONCLUSIONS: Aer. hydrophila sp. K is able to degrade BAC. A degradation pathway for BAC and related compounds is proposed. SIGNIFICANCE AND IMPACT OF STUDY: These findings are significant for understanding biodegradation pathways of benzyl-containing quaternary ammonium compounds.     

High-resolution structure of the photosynthetic Mn4Ca catalyst from X-ray spectroscopy

The application of high-resolution X-ray spectroscopy methods to study the photosynthetic water oxidizing complex, which contains a unique hetero-nuclear catalytic Mn4Ca cluster, is described. Issues of X-ray damage, especially at the metal sites in the Mn4Ca cluster, are discussed. The structure of the Mn4Ca catalyst at high resolution, which has so far eluded attempts of determination by X-ray diffraction, X-ray absorption fine structure (EXAFS) and other spectroscopic techniques, has been addressed using polarized EXAFS techniques applied to oriented photosystem II (PSII) membrane preparations and PSII single crystals. A review of how the resolution of traditional EXAFS techniques can be improved, using methods such as range-extended EXAFS, is presented, and the changes that occur in the structure of the cluster as it advances through the catalytic cycle are described. X-ray absorption and emission techniques (XANES and Kbeta emission) have been used earlier to determine the oxidation states of the Mn4Ca cluster, and in this report we review the use of X-ray resonant Raman spectroscopy to understand the electronic structure of the Mn4Ca cluster as it cycles through the intermediate S-states.     

High-resolution crystal structure of the Fab-fragments of a family of mouse catalytic antibodies with esterase activity

The crystal structures of four related Fab fragments of a family of catalytic antibodies displaying differential levels of esterase activity have been solved in the presence and in the absence of the transition-state analogue (TSA) that was used to elicit the immune response. The electron density maps show that the TSA conformation is essentially identical, with limited changes on hapten binding. Interactions with the TSA explain the specificity for the D rather than the L-isomer of the substrate. Differences in the residues in the hapten-binding pocket, which increase hydrophobicity, appear to correlate with an increase in the affinity of the antibodies for their substrate. Analysis of the structures at the active site reveals a network of conserved hydrogen bond contacts between the TSA and the antibodies, and points to a critical role of two conserved residues, HisL91 and LysH95, in catalysis. However, these two key residues are set into very different contexts in their respective structures, with an apparent direct correlation between the catalytic power of the antibodies and the complexity of their interactions with the rest of the protein. This suggests that the catalytic efficiency may be controlled by contacts arising from a second sphere of residues at the periphery of the active site.     

Bactericidal activity of grass carp Ctenopharyngodon idella C9-deficient serum against Aeromonas hydrophila

Neutralizing complement C9 in grass carp Ctenopharyngodon idella sera with rabbit anti-C9 sera against fish complement C9, demonstrated that bactericidal activity against Aeromonas hydrophila of the C9-deficient fish sera was greatly impaired. These results indicated that the fish complement C9 plays a key role in pathogen killing through the lytic pathway.     

The crystal structure of the globular domain of sheep prion protein

The prion protein PrP is a naturally occurring polypeptide that becomes transformed from a normal conformation to that of an aggregated form, characteristic of pathological states in fatal transmissible spongiform conditions such as Creutzfeld-Jacob Disease and Bovine Spongiform Encephalopathy. We report the crystal structure, at 2 A resolution, of residues 123-230 of the C-terminal globular domain of the ARQ allele of sheep prion protein (PrP). The asymmetric unit contains a single molecule whose secondary structure and overall organisation correspond to those structures of PrPs from various mammalian species determined by NMR. The globular domain shows a close association of helix-1, the C-terminal portion of helix-2 and the N-terminal portion of helix-3, bounded by the intramolecular disulphide bond, 179-214. The loop 164-177, between beta2 and helix-2 is relatively well structured compared to the human PrP NMR structure. Analysis of the sheep PrP structure identifies two possible loci for the initiation of beta-sheet mediated polymerisation. One of these comprises the beta-strand, residues 129-131 that forms an intra-molecular beta-sheet with residues 161-163. This strand is involved in lattice contacts about a crystal dyad to generate a four-stranded intermolecular beta-sheet between neighbouring molecules. The second locus involves the region 188-204, which modelling suggests is able to undergo a partial alpha-->beta switch within the monomer. These loci provide sites within the PrPc monomer that could readily give rise to early intermediate species on the pathway to the formation of aggregated PrPSc containing additional intermolecular beta-structure.     

Crystal structure of the N-terminal dimerisation domain of VicH, the H-NS-like protein of Vibrio cholerae

The histone-like nucleoid structuring (H-NS) protein is a global modulator of gene expression in Gram-negative bacteria. VicH, the H-NS protein of Vibrio cholerae, regulates the expression of certain major virulence determinants implicated in the pathogenesis of cholera. We present here the 2.5A crystal structure of the N-terminal oligomerisation domain of VicH (VicH_Nt). VicH_Nt adopts the same fold and dimeric assembly as the NMR structure of Escherichia coli H-NS_Nt, thus validating this fold against conflicting data. The structural similarity of V.cholerae VicH_Nt and E.coli H-NS_Nt, despite differences in origin, system of expression, experimental conditions and techniques used, indicates that the fold determined in our studies is robust to experimental conditions. Structural analysis and homology modelling were carried out to further elucidate the molecular basis of the functional polyvalence of the N-terminal domain. Our analysis of members of the H-NS superfamily supports the suggestion that the oligomerisation function of H-NS_Nt is conserved even in more distantly related proteins.     

Isolation and biochemical characterization of the S-layer protein from a pathogenic Aeromonas hydrophila strain.

The regular surface protein array (S layer) present on Aeromonas hydrophila TF7 is composed of a single species of protein of apparent molecular weight 52,000. This protein was extracted from whole cells by treatment with 0.2 M glycine hydrochloride (pH 3.0). The protein was purified to homogeneity by ion-exchange chromatography and reverse-phase high-performance liquid chromatography. Amino acid composition analysis showed that the protein contained 520 residues per molecule, 41% of which were hydrophobic. Cysteine was absent. A pI of 4.6 was determined for the protein, and only a single isoelectric form was detected. The purified protein displayed the hydrophobic characteristic of binding to octyl-Sepharose gels, but the salt aggregation test showed that it did not confer hydrophobicity to the cell surface when present as an intact S layer. The molecule aggregated strongly in aqueous solution as determined by sedimentation equilibrium studies. Circular dichroism spectra showed that the S-layer protein was composed of a large amount of beta-sheet (approximately 44%), a limited amount of alpha-helix (19%), and 12% beta-turn, with the remainder of the molecule being aperiodic. No significant difference in secondary structure content was measured in the presence of the metal chelator EDTA. The N-terminal amino acid sequence was determined for the first 30 residues. No sequence homology with other S-layer proteins was found.     

Amine free crystal structure: the crystal structure of d(CGCGCG)2 and methylamine complex crystal

We succeeded in the crystallization of d(CGCGCG)2 and methylamine Complex. The crystal was clear and of sufficient size to collect the X-ray crystallographic data up to 1.0 A resolution using synchrotron radiation. As a result of X-ray crystallographic analysis of 2Fo-Fc map was much clear and easily traced. It is the first time monoamine co-crystallizes with d(CGCGCG)2. However, methylamine was not found from the complex crystal of d(CGCGCG)2 and methylamine. Five Mg ions were found around d(CGCGCG)2 molecules. These Mg ions neutralized the anion of 10 values of the phosphate group of DNA with five Mg2+. DNA stabilized only by a metallic ion and there is no example of analyzing the X-ray crystal structure like this. Mg ion stabilizes the conformation of Z-DNA. To use monoamine for crystallization of DNA, we found that we can get only d(CGCGCG)2 and Mg cation crystal. Only Mg cation can stabilize the conformation of Z-DNA. The method of using the monoamine for the crystallization of DNA can be applied to the crystallization of DNA of long chain of length in the future like this.     

Comparison of the NMR solution structure with the X-ray crystal structure of the activation domain from procarboxypeptidase B

Summary The NMR solution structure of the activation domain isolated from porcine procarboxypeptidase B is compared with the X-ray crystal structure of the corresponding segment in the intact proenzyme. For the region of the polypeptide chain that has a well-defined three-dimensional structure in solution, i.e., the backbone atoms of residues 11–76 and 25 amino acid side chains in this segment that form a hydrophobic core in the activation domain, the root-mean-square distance between the two structures is 1.1 Å. There are no significant differences in average atom positions between the two structures, but only the NMR structure shows increased structural disorder in three outlying loops located along the same edge of the activation domain. These regions of increased structural disorder in the free domain coincide only partially with the interface to the enzyme domain in the proenzyme.