Stanley A. Moore

Molecular Basis of the Interaction between the Flagellar Export Proteins FliI and FliH from Helicobacter pylori

Bacterial flagellar protein export requires an ATPase, FliI, and presumptive inhibitor, FliH. We have explored the molecular basis for FliI/FliH interaction in the human gastric pathogen Helicobacter pylori. By using bioinformatic and biochemical analyses, we showed that residues 1–18 of FliI very likely form an amphipathic α-helix upon interaction with FliH, and that residues 21–91 of FliI resemble the N-terminal oligomerization domain of the F1-ATPase catalytic subunits. A truncated FliI-(2–91) protein was shown to be folded, although the N-terminal 18 residues were likely unstructured. Deletion and scanning mutagenesis showed that residues 1–18 of FliI were essential for the FliI/FliH interaction. Scanning mutation of amino acids in the N-terminal 10 residues of FliI indicated that a cluster of hydrophobic residues in this segment was critical for the interaction with FliH. The interaction between FliI and FliH has similarities to the interaction between the N-terminal α-helix of the F1-ATPase α-subunit and the globular domain of the F1-ATPase δ-subunit, respectively. This similarity suggests that FliH may function as a molecular stator.

Structural and Biochemical Studies on the Chromo-barrel Domain of Male Specific Lethal 3 (MSL3) Reveal a Binding Preference for Mono- or Dimethyllysine 20 on Histone H4

We have determined the human male specific lethal 3 (hMSL3) chromo-barrel domain structure by x-ray crystallography to a resolution of 2.5 Å (r = 0.226, Rfree = 0.270). hMSL3 contains a canonical methyllysine binding pocket made up of residues Tyr-31, Phe-56, Trp-59, and Trp-63. A six-residue insertion between strands β1 and β2 of the hMSL3 chromo-barrel domain directs the side chain of Glu-21 into the methyllysine binding pocket where it hydrogen bonds to the NH group of a bound cyclohexylamino ethanesulfonate buffer molecule, likely mimicking interactions with a histone tail dimethyllysine residue. In vitro binding studies revealed that both the human and Drosophila MSL3 chromo-barrel domains bind preferentially to peptides representing the mono or dimethyl isoform of lysine 20 on the histone H4 N-terminal tail (H4K20Me1 or H4K20Me2). Mutation of Tyr-31 to Ala in the hMSL3 methyllysine-binding cage resulted in weaker in vitro binding to H4K20Me1. The same mutation in the msl3 gene compromised male survival in Drosophila. Combined mutation of Glu-21 and Pro-22 to Ala in hMSL3 resulted in slightly weaker in vitro binding to H4K20Me1, but the corresponding msl3 mutation had no effect on male survival in Drosophila. We propose MSL3 plays an important role in targeting the male specific lethal complex to chromatin in both humans and flies by binding to H4K20Me1. Binding studies on the related dMRG15 chromo-barrel domain revealed that MRG15 prefers binding to H4K20Me3.

Structure of the Cytoplasmic Domain of the Flagellar Secretion Apparatus Component FlhA from Helicobacter pylori

Using x-ray crystallography we have determined the structure of the cytoplasmic fragment (residues 384–732) of the flagellum secretion system protein FlhA from Helicobacter pylori at 2.4-Å resolution (r = 0.224; Rfree = 0.263). FlhA proteins and their type III secretion homologues contain an N-terminal integral membrane domain (residues 1–350), a linker segment, and a globular C-terminal cytoplasmic region. The tertiary structure of the cytoplasmic fragment contains a thioredoxin-like domain, an RNA recognition motif-like domain inserted within the thioredoxin-fold, a helical domain, and a C-terminal β/α domain. Inter-domain contacts are extensive and the H. pylori FlhA structure appears to be in a closed conformation where the C-terminal domain closes against the RNA recognition motif-fold domain. Highly conserved surface residues in FlhA proteins are concentrated on a narrow surface strip comprising the thioredoxin-like and helical domains, possibly close to the export channel opening. The conformation of the FlhA N-terminal linker segment suggests a likely orientation for the FlhA cytoplasmic fragment relative to the membrane-embedded export pore. Comparison with the recently published structures of the Salmonella FlhA cytoplasmic fragment and its type III secretion counterpart InvA highlight a conformational change where the C-terminal β/α domain in H. pylori FlhA moves 15 Å relative to Salmonella FlhA. The conformational change is complex but primarily involves hinge-like movements of the helical and C-terminal domains. Interpretation of previous mutational screens suggest that the C-terminal domain of FlhAC plays a regulatory role in substrate class switching in flagellum export.

A description of the Mei2-like protein family; structure, phylogenetic distribution and biological context

The Schizosaccharomyces pombe Mei2 gene encodes an RNA recognition motif (RRM) protein that stimulates meiosis upon binding a specific non-coding RNA and subsequent accumulation in a “mei2-dot” in the nucleus. We present here the first systematic characterization of the family of proteins with characteristic Mei2-like amino acid sequences. Mei2-like proteins are an ancient eukaryotic protein family with three identifiable RRMs. The C-terminal RRM (RRM3) is unique to Mei2-like proteins and is the most highly conserved of the three RRMs. RRM3 also contains conserved sequence elements at its C-terminus not found in other RRM domains. Single copy Mei2-like genes are present in some fungi, in alveolates such as Paramecium and in the early branching eukaryote Entamoeba histolytica, while plants contain small families of Mei2-like genes. While the C-terminal RRM is highly conserved between plants and fungi, indicating conservation of molecular mechanisms, plant Mei2-like genes have changed biological context to regulate various aspects of developmental pattern formation.

The 2.2-Å Structure of the HP0958 Protein from Helicobacter pylori Reveals a Kinked Anti-Parallel Coiled-Coil Hairpin Domain and a Highly Conserved Zn-Ribbon Domain

We have determined the 2.2-Å structure of the HP0958 protein from the human gastric pathogen Helicobacter pylori. HP0958 is essential for flagellum formation and motility. It functions as a chaperone for RpoN (σ(54)) and also controls the stability and translation of mRNA for the major flagellin subunit FlaA. The protein is composed of a highly elongated and kinked coiled-coil hairpin domain (residues 1-170), followed by a C(4) Zn-ribbon domain (residues 174-238). The Zn-ribbon domain is rich in aromatic and positively charged amino acid residues. Electrophoretic mobility shift assays identified residues in a positively charged region of the Zn-ribbon domain of HP0958 whose mutation alters the mobility of an HP0958-flaA mRNA complex. Mutation of surface residues in the coiled-coil domain did not result in an observable change in the mobility of the HP0958-flaA transcript complex. The data thus suggest the arrangement of HP0958 into distinct structural and functional domains.

Draft Genome Sequences of Helicobacter pylori Strains 17874 and P79

Helicobacter pylori is a human pathogen that colonizes the human gastric mucosa, causing gastritis, duodenal and gastric ulcers, and gastric carcinoma. Here we announce the draft genomes of H. pylori strain 17874, commonly used for studying motility, and P79, a strain for which plasmid vectors have been developed.

Two Duplicated Genes DDI2 and DDI3 in Budding Yeast Encode a Cyanamide Hydratase and Are Induced by Cyanamide

Background: DDI2 and DDI3 are two uncharacterized identical genes found in budding yeast. Results: They encode novel cyanamide hydratases and are massively induced by cyanamide. Their deletion causes cellular sensitivity to cyanamide. Conclusion: The two genes function in cyanamide detoxification and are tightly regulated. Significance: This is the first attempt to understand the duplicated gene cluster in budding yeast. Two DNA damage-inducible genes in Saccharomyces cerevisiae, DDI2 and DDI3, are identical and encode putative HD domain-containing proteins, whose functions are currently unknown. Because Ddi2/3 also shows limited homology to a fungal cyanamide hydratase that converts cyanamide to urea, we tested the enzymatic activity of recombinant Ddi2. To this end, we developed a novel enzymatic assay and determined that the Km value of the recombinant Ddi2/3 for cyanamide is 17.3 ± 0.05 mm, and its activity requires conserved residues in the HD domain. Unlike most other DNA damage-inducible genes, DDI2/3 is only induced by a specific set of alkylating agents and surprisingly is strongly induced by cyanamide. To characterize the biological function of DDI2/3, we sequentially deleted both DDI genes and found that the double mutant was unable to metabolize cyanamide and became much more sensitive to growth inhibition by cyanamide, suggesting that the DDI2/3 genes protect host cells from cyanamide toxicity. Despite the physiological relevance of the cyanamide induction, DDI2/3 is not involved in its own transcriptional regulation. The significance of cyanamide hydratase activity and its induced expression is discussed.

Statistical characterization of the GxxxG glycine repeats in the flagellar biosynthesis protein FliH and its Type III secretion homologue YscL

BackgroundFliH is a protein involved in the export of components of the bacterial flagellum and we herein describe the presence of glycine-rich repeats in FliH of the form AxxxG(xxxG)mxxxA, where the value of m varies considerably in FliH proteins from different bacteria. While GxxxG and AxxxA patterns have previously been described, the long glycine repeat segments in FliH proteins have yet to be characterized. The Type III secretion system homologue to FliH (YscL, AscL, PscL, etc.) also contains a similar GxxxG repeat, and hence the presence of the repeat is evolutionarily conserved in these proteins, suggesting an important structural role or biological function.ResultsA set of FliH and YscL protein sequences was downloaded from GenBank, and then filtered to reduce redundancy, to ensure the soundness of the sequences, and to eliminate, as much as possible, confounding phylogenetic signal between individual sequences by implementing a pairwise 25% sequence identity cut-off. The general features of the glycine-rich repeats in these proteins were examined, and it was found that the length of these repeat segments varied substantially among FliH proteins but was fairly consistent for the Type III (YscL) homologue sequences, with values of m ranging from 0 to 12 for FliH and 0 to 2 for YscL. The amino acid sequence distribution of each of the three positions in the GxxxG repeats was found to differ significantly from the overall amino acid composition of the FliH/YscL proteins. The high frequency of Glu, Gln, Lys and Ala residues in the repeat positions, which is not likely indicative of any contaminating phylogenetic signal, suggests an α-helical structure for this motif. In addition, we sought to determine whether certain pairs of amino acids, in certain pairs of positions, were found together significantly more often than would be predicted by chance. Several statistically significant correlations were uncovered, which may be important for maintaining helical stability or for forming helix-helix interactions. These correlations are likely not of a phylogenetic origin as the originating sequences for the pair correlations are derived from a low similarity set and the individual incidences of the pair correlations do not cluster in any obvious phylogenetic sense, nor is there much evidence of strict sequence conservation outside the positions of the glycine residues. Finally, the α-helices from a non-redundant set of proteins from the Protein Data Bank were searched for GxxxG repeats similar in length to those found in FliH, however there were no helices containing more than three contiguous glycine repeat segments; thus, long glycine repeats similar to those found in FliH are presumably quite rare in nature.ConclusionThe glycine repeats in YscL and particularly FliH represent an intriguing amino acid sequence motif that is very rare in nature. Although we do not attempt to offer a mechanism whereby these repeats may have evolved, we do place the existence of the motif and some residue pairings within a rational structural context. While crystal structures of these proteins are necessary to fully elucidate the structural and functional significance of these repeats, the characterization reported here represents a first step in understanding this unique sequence feature.

The HP0256 gene product is involved in motility and cell envelope architecture of Helicobacter pylori

BackgroundHelicobacter pylori is the causative agent for gastritis, and peptic and duodenal ulcers. The bacterium displays 5-6 polar sheathed flagella that are essential for colonisation and persistence in the gastric mucosa. The biochemistry and genetics of flagellar biogenesis in H. pylori has not been fully elucidated. Bioinformatics analysis suggested that the gene HP0256, annotated as hypothetical, was a FliJ homologue. In Salmonella, FliJ is a chaperone escort protein for FlgN and FliT, two proteins that themselves display chaperone activity for components of the hook, the rod and the filament.ResultsAblation of the HP0256 gene in H. pylori significantly reduced motility. However, flagellin and hook protein synthesis was not affected in the HP0256 mutant. Transmission electron transmission microscopy revealed that the HP0256 mutant cells displayed a normal flagellum configuration, suggesting that HP0256 was not essential for assembly and polar localisation of the flagella in the cell. Interestingly, whole genome microarrays of an HP0256 mutant revealed transcriptional changes in a number of genes associated with the flagellar regulon and the cell envelope, such as outer membrane proteins and adhesins. Consistent with the array data, lack of the HP0256 gene significantly reduced adhesion and the inflammatory response in host cells.ConclusionsWe conclude that HP0256 is not a functional counterpart of FliJ in H. pylori. However, it is required for full motility and it is involved, possibly indirectly, in expression of outer membrane proteins and adhesins involved in pathogenesis and adhesion.

Correction for Clancy et al., Draft Genome Sequences of Helicobacter pylori Strains 17874 and P79

Volume 194, no. 9, p. [2402][1], 2012. It was brought to our attention that one of two Helicobacter pylori genome sequences we reported may be derived from an incorrectly identified strain. The genome announcement described the genomes of H. pylori strains 17874 and P79. According to analyses shared