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Crystal structure of TNF-?-inducing protein from Helicobacter pylori in active form reveals the intrinsic molecular flexibility for unique DNA-binding.

ABSTRACT: Tip? (TNF-?-inducing protein) from Helicobacter pylori is a carcinogenic effector. Studies on this protein revealed that a homodimer linked by a pair of intermolecular disulfide bridges (Cys25-Cys25 and Cys27-Cys27) was absolutely necessary for its biological functions. The activities of Tip? would be abolished when both disulfide bridges were disrupted. The crystal structures of Tip? reported to date, however, were based on inactive, monomeric mutants with their N-terminal, including residues Cys25 and Cys27, truncated. Here we report the crystal structure of H. pylori Tip? protein, Tip?N(25), at 2.2Å resolution, in which Cys25 and Cys27 form a pair of inter-chain disulfide bridges linking an active dimer. The disulfide bridges exhibit structural flexibility in the present structure. A series of structure-based mutagenesis, biochemical assays and molecular dynamic simulations on DNA-Tip? interactions reveal that Tip? utilizes the dimeric interface as the DNA-binding site and that residues His60, Arg77 and Arg81 located at the interface are crucial for DNA binding. Tip? could bind to one ssDNA, two ssDNA or one dsDNA in experiments, respectively, in the native or mutant states. The unique DNA-binding activities of Tip? indicate that the intrinsic flexible nature of disulfide bridges could endow certain elasticity to the Tip? dimer for its unique bioactivities. The results shed light on the possible structural mechanism for the functional performances of Tip?.

SUBMITTER: Gao M

PROVIDER: S-EPMC3409205 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Crystal structure of TNF-α-inducing protein from Helicobacter pylori in active form reveals the intrinsic molecular flexibility for unique DNA-binding.

Gao Mingming M Li Defeng D Hu Yonglin Y Zhang Ying Y Zou Quanming Q Wang Da-Cheng DC

PloS one 20120731 7

Tipα (TNF-α-inducing protein) from Helicobacter pylori is a carcinogenic effector. Studies on this protein revealed that a homodimer linked by a pair of intermolecular disulfide bridges (Cys25-Cys25 and Cys27-Cys27) was absolutely necessary for its biological functions. The activities of Tipα would be abolished when both disulfide bridges were disrupted. The crystal structures of Tipα reported to date, however, were based on inactive, monomeric mutants with their N-terminal, including residues C ...[more]

PMID: 22860022

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Project description:Many bacterial genomes are highly variable but nonetheless are typically published as a single assembled genome. Experiments tracking bacterial genome evolution have not looked at the variation present at a given point in time. Here, we analyzed the mouse-passaged Helicobacter pylori strain SS1 and its parent PMSS1 to assess intra- and intergenomic variability. Using high sequence coverage depth and experimental validation, we detected extensive genome plasticity within these H. pylori isolates, including movement of the transposable element IS607, large and small inversions, multiple single nucleotide polymorphisms, and variation in cagA copy number. The cagA gene was found as 1 to 4 tandem copies located off the cag island in both SS1 and PMSS1; this copy number variation correlated with protein expression. To gain insight into the changes that occurred during mouse adaptation, we also compared SS1 and PMSS1 and observed 46 differences that were distinct from the within-genome variation. The most substantial was an insertion in cagY, which encodes a protein required for a type IV secretion system function. We detected modifications in genes coding for two proteins known to affect mouse colonization, the HpaA neuraminyllactose-binding protein and the FutB α-1,3 lipopolysaccharide (LPS) fucosyltransferase, as well as genes predicted to modulate diverse properties. In sum, our work suggests that data from consensus genome assemblies from single colonies may be misleading by failing to represent the variability present. Furthermore, we show that high-depth genomic sequencing data of a population can be analyzed to gain insight into the normal variation within bacterial strains.IMPORTANCE Although it is well known that many bacterial genomes are highly variable, it is nonetheless traditional to refer to, analyze, and publish "the genome" of a bacterial strain. Variability is usually reduced ("only sequence from a single colony"), ignored ("just publish the consensus"), or placed in the "too-hard" basket ("analysis of raw read data is more robust"). Now that whole-genome sequences are regularly used to assess virulence and track outbreaks, a better understanding of the baseline genomic variation present within single strains is needed. Here, we describe the variability seen in typical working stocks and colonies of pathogen Helicobacter pylori model strains SS1 and PMSS1 as revealed by use of high-coverage mate pair next-generation sequencing (NGS) and confirmed by traditional laboratory techniques. This work demonstrates that reliance on a consensus assembly as "the genome" of a bacterial strain may be misleading.

Dataset Information

Crystal structure of TNF-?-inducing protein from Helicobacter pylori in active form reveals the intrinsic molecular flexibility for unique DNA-binding.

Publications

Crystal structure of TNF-α-inducing protein from Helicobacter pylori in active form reveals the intrinsic molecular flexibility for unique DNA-binding.

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