Biochemical Characterization of Protein Complexes from the Helicobacter pylori Protein Interaction Map

Terradot, Laurent; Durnell, Nathan; Li, Min; Li, Ming; Ory, Jeramia; Labigne, Agnès; Legrain, Pierre; Colland, Frédéric; Waksman, Gabriel

doi:10.1074/mcp.m400048-mcp200

Cited by 45 publications

(5 citation statements)

References 46 publications

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“…Since CagI was detected mostly in the soluble periplasmic fraction and to a limited extent in the membrane fraction we thought it would be practical to look for other CagI interacting proteins, if any, in the periplasmic space. Previously, at least two non- cag -PAI components HP1489 and GyrA have been predicted to interact with CagI by in-silico analysis [23,29]. In this direction, we have selectively labeled periplasmic proteins in wild-type H.…”

Section: Resultsmentioning

confidence: 99%

Cag Type IV Secretion System: CagI Independent Bacterial Surface Localization of CagA

et al. 2013

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Helicobacter pylori Cag type IV secretion system (Cag-T4SS) is a multi-component transporter of oncoprotein CagA across the bacterial membranes into the host epithelial cells. To understand the role of unique Cag-T4SS component CagI in CagA translocation, we have characterized it by biochemical and microscopic approaches. We observed that CagI is a predominantly membrane attached periplasmic protein partially exposed to the bacterial surface especially on the pili. The association of the protein with membrane appeared to be loose as it could be easily recovered in soluble fraction. We documented that the stability of the protein is dependent on several key components of the secretion system and it has multiple interacting partners including a non-cag-PAI protein HP1489. Translocation of CagA across the bacterial membranes to cell surface is CagI-independent process. The observations made herein are expected to assist in providing an insight into the substrate translocation by the Cag-T4SS system and Helicobacter pylori pathogenesis.

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Section: Resultsmentioning

confidence: 99%

Cag Type IV Secretion System: CagI Independent Bacterial Surface Localization of CagA

et al. 2013

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“…E. coli DiaA orthologs are also widely conserved among eubacterial species (15). Notably, the Helicobacter pylori DiaA ortholog HobA is reported to be essential in cell growth and is known to directly bind the cognate DnaA and to stimulate the assembly of the DnaA molecules on the cognate oriC (37)(38)(39). Similarly, it would be possible that dynamics of the initial complexes during replicative helicase loading as well as DiaA-controlled DnaA assembly are conserved among eubacterial species.…”

Section: Discussionmentioning

confidence: 99%

DiaA Dynamics Are Coupled with Changes in Initial Origin Complexes Leading to Helicase Loading

Keyamura¹,

Abe

Higashi³

et al. 2009

Journal of Biological Chemistry

192

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Chromosomal replication initiation requires the regulated formation of dynamic higher order complexes. Escherichia coli ATP-DnaA forms a specific multimer on oriC, resulting in DNA unwinding and DnaB helicase loading. DiaA, a DnaA-binding protein, directly stimulates the formation of ATP-DnaA multimers on oriC and ensures timely replication initiation. In this study, DnaA Phe-46 was identified as the crucial DiaA-binding site required for DiaA-stimulated ATP-DnaA assembly on oriC. Moreover, we show that DiaA stimulation requires only a subgroup of DnaA molecules binding to oriC, that DnaA Phe-46 is also important in the loading of DnaB helicase onto the oriCDnaA complexes, and that this process also requires only a subgroup of DnaA molecules. Despite the use of only a DnaA subgroup, DiaA inhibited DnaB loading on oriC-DnaA complexes, suggesting that DiaA and DnaB bind to a common DnaA subgroup. A cellular factor can relieve the DiaA inhibition, allowing DnaB loading. Consistently, DnaA F46A caused retarded initiations in vivo in a DiaA-independent manner. It is therefore likely that DiaA dynamics are crucial in the regulated sequential progress of DnaA assembly and DnaB loading. We accordingly propose a model for dynamic structural changes of initial oriC complexes loading DiaA or DnaB helicase.In many cellular organisms, multiple proteins form dynamic complexes on the chromosomal origin for the initiation of DNA replication. In Escherichia coli, ATP-DnaA forms a specific multimeric complex on the origin (oriC), resulting in an initiation complex that is competent in the replicational initiation (1-3). ATP-DnaA complexes, but not ADP-DnaA complexes, unwind the DNA duplex within the oriC DNA unwinding element (DUE) 2 with the aid of superhelicity of oriC DNA and heat energy, resulting in the formation of open complexes (4, 5). At the unwound region, the loading of a DnaB replicative helicase is mediated by a DnaC helicase loader, resulting in the formation of the prepriming complex (6, 7). DnaG primase then complexes with DnaB loaded on the single-stranded (ss) region, which leads to primer synthesis and the loading of DNA polymerase III holoenzyme (8). The cellular ATP-DnaA level fluctuates during the replication cycle with a peak around the time of initiation (9). At the post-initiation stage, DnaA-ATP is hydrolyzed in a manner depending on ADP-Hda protein and the DNA-loaded form of the ␤-clamp subunit of the polymerase III holoenzyme, yielding inactive ADP-DnaA (10 -13). This DnaA inactivation system is called RIDA (regulatory inactivation of DnaA). Hda consists of a short N-terminal region bearing a clamp-binding motif and a C-terminal AAA ϩ domain. This protein is activated by ADP binding, which allows interaction with ATP-DnaA in a DNA-loaded ␤-clamp-dependent manner. RIDA decreases the level of cellular ATP-DnaA in a replication-coordinated manner and represses extra initiation events (9 -11).The timing of chromosomal replication initiation is strictly regulated and needs to be linked to the regulation o...

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“…Therefore, they are likely to form a large ATPase complex that energizes substrate transport through the translocation machinery. The architecture of this complex and the contributions of each ATPase to secretion or pilus biogenesis are still unclear.VirB10 interacts directly with VirB4 and VirD4 (REFS 46,48–51). The VirB10–VirD4 interaction involves domains near the N-terminal regions of both proteins that reside within or near the inner membrane 50–52 .…”

Section: The Cytoplasmic Atpasesmentioning

confidence: 99%

The structural biology of type IV secretion systems

2009

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Type IV secretion systems (T4SSs) are versatile secretion systems that are found in both Gram-negative and Gram-positive bacteria and secrete a wide range of substrates, from single proteins to protein–protein and protein–DNA complexes. They usually consist of 12 components that are organized into ATP-powered, double-membrane-spanning complexes. The structures of single soluble components or domains have been solved, but an understanding of how these structures come together has only recently begun to emerge. This Review focuses on the structural advances that have been made over the past 10 years and how the corresponding structural insights have helped to elucidate many of the details of the mechanism of type IV secretion.

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Biochemical Characterization of Protein Complexes from the Helicobacter pylori Protein Interaction Map

Cited by 45 publications

References 46 publications

Cag Type IV Secretion System: CagI Independent Bacterial Surface Localization of CagA

Cag Type IV Secretion System: CagI Independent Bacterial Surface Localization of CagA

DiaA Dynamics Are Coupled with Changes in Initial Origin Complexes Leading to Helicase Loading

The structural biology of type IV secretion systems

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