The Response Regulator OmpR Oligomerizes via β-Sheets to Form Head-to-head Dimers

Maris, A.E.; Walthers, Don; Mattison, Kirsten; Byers, Nicole; Kenney, Linda J.

doi:10.1016/j.jmb.2005.05.057

Cited by 37 publications

(44 citation statements)

References 60 publications

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“…The TcpP dimer bands migrated slightly differently depending on whether C207 or C218 was present, likely due to differences in the location of the disulfide bond relative to the C terminus of the protein. Since proteins in this family tend to dimerize (21,33,(38)(39)(40)(41)(42), and this band was the expected size of a TcpP dimer, we predicted that this band was likely the result of an intermolecular disulfide bond formed between two TcpP molecules. To confirm this, the complex was isolated by immunoprecipitation and analyzed by mass spectrometry (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Formation of an Intramolecular Periplasmic Disulfide Bond in TcpP Protects TcpP and TcpH from Degradation in Vibrio cholerae

et al. 2016

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TcpP and ToxR coordinately regulate transcription of toxT, the master regulator of numerous virulence factors in Vibrio cholerae. TcpP and ToxR are membrane-localized transcription factors, each with a periplasmic domain containing two cysteines. In ToxR, these cysteines form an intramolecular disulfide bond and a cysteine-to-serine substitution affects activity. We determined that the two periplasmic cysteines of TcpP also form an intramolecular disulfide bond. Disruption of this intramolecular disulfide bond by mutation of either cysteine resulted in formation of intermolecular disulfide bonds. Furthermore, disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP. While the decreased stability of TcpP-C207S resulted in a nearly complete loss of toxT activation and cholera toxin (CT) production, the second cysteine mutant, TcpP-C218S, was partially resistant to proteolytic degradation and maintained ϳ50% toxT activation capacity. TcpP-C218S was also TcpH independent, since deletion of tcpH did not affect the stability of TcpP-C218S, whereas wild-type TcpP was degraded in the absence of TcpH. Finally, TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, suggesting that the single periplasmic cysteine in TcpH may assist with disulfide bond formation in TcpP by interacting with the periplasmic cysteines of TcpP. Consistent with this finding, a TcpH-C114S mutant was unable to stabilize TcpP and was itself unstable. Our findings demonstrate a periplasmic disulfide bond in TcpP is critical for TcpP stability and virulence gene expression. IMPORTANCEThe Vibrio cholerae transcription factor TcpP, in conjunction with ToxR, regulates transcription of toxT, the master regulator of numerous virulence factors in Vibrio cholerae. TcpP is a membrane-localized transcription factor with a periplasmic domain containing two cysteines. We determined that the two periplasmic cysteines of TcpP form an intramolecular disulfide bond and disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP and reduced virulence gene expression. Normally TcpH, another membrane-localized periplasmic protein, protects TcpP from degradation. However, we found that TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, indicating that the periplasmic cysteines of TcpP are required for functional interaction with TcpH and that this interaction is required for both TcpP and TcpH stability. C holera is caused by ingestion of the aquatic Gram-negative bacterium Vibrio cholerae. The profuse watery diarrhea characteristic of cholera is induced by cholera toxin (CT), an ADPribosylating toxin that induces cyclic AMP production in intestinal epithelial cells, resulting in massive secretion of water and electrolytes. Microcolony formation and colonization of the intestine require expression of the toxin-coregulated pilus (TCP) (1). Transcription of the genes encoding CT and TCP, as well as several other virulence factors, is regulated by...

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Section: Resultsmentioning

confidence: 99%

Formation of an Intramolecular Periplasmic Disulfide Bond in TcpP Protects TcpP and TcpH from Degradation in Vibrio cholerae

et al. 2016

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“…Therefore, we utilized another high-energy phosphate donor, PA, which has been shown to successfully phosphorylate other RRs in vitro (25). In Fig.…”

Section: Resultsmentioning

confidence: 99%

Oligomerization of the Response Regulator ComE from Streptococcus mutans Is Affected by Phosphorylation

Hung¹,

Downey²,

Kreth³

et al. 2011

Journal of Bacteriology

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We have previously characterized the interactions of the response regulator ComE from Streptococcus mutans and DNA binding sites through DNase I footprinting and electrophoretic mobility shift assay analysis. Since response regulator functions are often affected by their phosphorylation state, we investigated how phosphorylation affects the biochemical function of ComE. Unlike many response regulators, we found that the phosphorylation state of ComE does not likely play a role in DNA binding affinity but rather seems to induce the formation of an oligomeric form of the protein. The role of this oligomerization state for ComE function is discussed.

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“…Joint β-hairpins forming β-sheets are a classical motif of oligomer formation in soluble proteins (Maris et al, 2005;Rimon et al, 2007). Interestingly, the NhaA β-sheet is located parallel to the membrane in the periplasmic space outside the membrane ( Fig.…”

Section: The Nhaa Monomer Is Fully Functional Yet the Dimer Is Cruciamentioning

confidence: 99%

NhaA crystal structure: functional–structural insights

Padan

Kozachkov

Herz

et al. 2009

Journal of Experimental Biology

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). The NhaA crystal structure has provided insights into the pH-regulated mechanism of antiporter action and opened up new in silico and in situ avenues of research. The monomer is the functional unit of NhaA yet the dimer is essential for the stability of the antiporter under extreme stress conditions. Ionizable residues of NhaA that strongly interact electrostatically are organized in a transmembrane fashion in accordance with the functional organization of the cation-binding site, 'pH sensor', the pH transduction pathway and the pH-induced conformational changes. Remarkably, NhaA contains an inverted topology motive of transmembrane segments, which are interrupted by extended mid-membrane chains that have since been found to vary in other ion-transport proteins. This novel structural fold creates a delicately balanced electrostatic environment in the middle of the membrane, which might be essential for ion binding and translocation. Based on the crystal structure of NhaA, a model structure of the human Na + /H + exchanger (NHE1) was constructed, paving the way to a rational drug design.

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The Response Regulator OmpR Oligomerizes via β-Sheets to Form Head-to-head Dimers

Cited by 37 publications

References 60 publications

Formation of an Intramolecular Periplasmic Disulfide Bond in TcpP Protects TcpP and TcpH from Degradation in Vibrio cholerae

Formation of an Intramolecular Periplasmic Disulfide Bond in TcpP Protects TcpP and TcpH from Degradation in Vibrio cholerae

Oligomerization of the Response Regulator ComE from Streptococcus mutans Is Affected by Phosphorylation

NhaA crystal structure: functional–structural insights

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