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The structure of CrgA from Neisseria meningitidis reveals a new octameric assembly state for LysR transcriptional regulators.

Sainsbury S, Lane LA, Ren J, Gilbert RJ, Saunders NJ, Robinson CV, Stuart DI, Owens RJ - Nucleic Acids Res. (2009)

Bottom Line: LysR-type transcriptional regulators (LTTRs) form the largest family of bacterial regulators acting as both auto-repressors and activators of target promoters, controlling operons involved in a wide variety of cellular processes.The LTTR, CrgA, from the human pathogen Neisseria meningitidis, is upregulated during bacterial-host cell contact.Here, we report the crystal structures of both regulatory domain and full-length CrgA, the first of a novel subclass of LTTRs that form octameric rings.

View Article: PubMed Central - PubMed

Affiliation: The Oxford Protein Production Facility and Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.

ABSTRACT
LysR-type transcriptional regulators (LTTRs) form the largest family of bacterial regulators acting as both auto-repressors and activators of target promoters, controlling operons involved in a wide variety of cellular processes. The LTTR, CrgA, from the human pathogen Neisseria meningitidis, is upregulated during bacterial-host cell contact. Here, we report the crystal structures of both regulatory domain and full-length CrgA, the first of a novel subclass of LTTRs that form octameric rings. Non-denaturing mass spectrometry analysis and analytical ultracentrifugation established that the octameric form of CrgA is the predominant species in solution in both the presence and absence of an oligonucleotide encompassing the CrgA-binding sequence. Furthermore, analysis of the isolated CrgA-DNA complex by mass spectrometry showed stabilization of a double octamer species upon DNA binding. Based on the observed structure and the mass spectrometry findings, a model is proposed in which a hexadecameric array of two CrgA oligomers binds to its DNA target site.

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Model of a hexadecameric array of two CrgA oligomers binding to the DNA target site. (A) Two CrgA octamers bound to the 63-bp CrgA DNA footprint (48). The first octamer (blue) binds to CrgA site 1 and the second octamer (gray) to site 2. The LTTR motifs (boxed in green in the sequence) are bound between the two recognition helices of a DBD pair. One strand of the DNA is coloured to represent the sequence elements depicted in the schematic. The LTTR motifs are indicated by green colouring of the DNA. The three base pairs between CrgA site 1 and 2 are shown in yellow. The bases overexposed to DMS methylation (+) or hypersensitive to DNase I (*) are indicated. The predicted transcription and translation start points are represented with bent and broad arrows, respectively. The predicted –10 promoter element for crgA is underlined. The location of the –35 promoter (underlined) element of mdaB is indicated with a dashed orange line. (B) EMSA of purified CrgA binding to its DNA target site. The amount of DNA was kept constant at 2.5 pmol per reaction and the molarity of CrgA was varied as indicated. (C) Asymmetric unit of Crystal form B of CrgA.
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Figure 7: Model of a hexadecameric array of two CrgA oligomers binding to the DNA target site. (A) Two CrgA octamers bound to the 63-bp CrgA DNA footprint (48). The first octamer (blue) binds to CrgA site 1 and the second octamer (gray) to site 2. The LTTR motifs (boxed in green in the sequence) are bound between the two recognition helices of a DBD pair. One strand of the DNA is coloured to represent the sequence elements depicted in the schematic. The LTTR motifs are indicated by green colouring of the DNA. The three base pairs between CrgA site 1 and 2 are shown in yellow. The bases overexposed to DMS methylation (+) or hypersensitive to DNase I (*) are indicated. The predicted transcription and translation start points are represented with bent and broad arrows, respectively. The predicted –10 promoter element for crgA is underlined. The location of the –35 promoter (underlined) element of mdaB is indicated with a dashed orange line. (B) EMSA of purified CrgA binding to its DNA target site. The amount of DNA was kept constant at 2.5 pmol per reaction and the molarity of CrgA was varied as indicated. (C) Asymmetric unit of Crystal form B of CrgA.

Mentions: As mentioned above, the published DNA footprinting data for CrgA show that the protein interacts with two adjacent LTTR-binding motifs in the CrgA/MdaB promoter region (48). We have shown that CrgA assembles as an octamer and have argued from an examination of the crystal structure that geometric constraints preclude the simultaneous binding of both sites by the same CrgA octamer. The MS of CrgA–DNA complexes showed the binding of single octamers to DNA and sedimentation velocity experiments confirmed that single octamers are the predominant species observed in the presence of DNA. We conclude that in this case, only one of the two LTTR-binding motifs in the DNA are involved in CrgA binding. Consistent with this, EMSA assays showed that in addition to binding to the 63-bp CrgA DNA footprint, CrgA binds to the individual CrgA site 1 and site 2 sequences (Figure 7B). The mobilities of these 30-bp DNA fragments were clearly shifted by CrgA. No shift was observed for a randomized control oligonucleotide of similar GC base content, indicating that the interaction with the CrgA sites was sequence specific (Figure 7B). In contrast to the EMSA with the 63-bp sequence, no defined band corresponding to a CrgA–DNA complex for these shorter DNA fragments was observed. This possibly indicates that the presence and occupancy of both binding sites is required for the formation of a stable complex in the EMSA. In these experiments, we did observe some material that did not enter the gel, which may correspond to high-molecular-weight complexes. However, given the propensity of the protein to aggregate it could also include non-specifically aggregated material.Figure 7.


The structure of CrgA from Neisseria meningitidis reveals a new octameric assembly state for LysR transcriptional regulators.

Sainsbury S, Lane LA, Ren J, Gilbert RJ, Saunders NJ, Robinson CV, Stuart DI, Owens RJ - Nucleic Acids Res. (2009)

Model of a hexadecameric array of two CrgA oligomers binding to the DNA target site. (A) Two CrgA octamers bound to the 63-bp CrgA DNA footprint (48). The first octamer (blue) binds to CrgA site 1 and the second octamer (gray) to site 2. The LTTR motifs (boxed in green in the sequence) are bound between the two recognition helices of a DBD pair. One strand of the DNA is coloured to represent the sequence elements depicted in the schematic. The LTTR motifs are indicated by green colouring of the DNA. The three base pairs between CrgA site 1 and 2 are shown in yellow. The bases overexposed to DMS methylation (+) or hypersensitive to DNase I (*) are indicated. The predicted transcription and translation start points are represented with bent and broad arrows, respectively. The predicted –10 promoter element for crgA is underlined. The location of the –35 promoter (underlined) element of mdaB is indicated with a dashed orange line. (B) EMSA of purified CrgA binding to its DNA target site. The amount of DNA was kept constant at 2.5 pmol per reaction and the molarity of CrgA was varied as indicated. (C) Asymmetric unit of Crystal form B of CrgA.
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Figure 7: Model of a hexadecameric array of two CrgA oligomers binding to the DNA target site. (A) Two CrgA octamers bound to the 63-bp CrgA DNA footprint (48). The first octamer (blue) binds to CrgA site 1 and the second octamer (gray) to site 2. The LTTR motifs (boxed in green in the sequence) are bound between the two recognition helices of a DBD pair. One strand of the DNA is coloured to represent the sequence elements depicted in the schematic. The LTTR motifs are indicated by green colouring of the DNA. The three base pairs between CrgA site 1 and 2 are shown in yellow. The bases overexposed to DMS methylation (+) or hypersensitive to DNase I (*) are indicated. The predicted transcription and translation start points are represented with bent and broad arrows, respectively. The predicted –10 promoter element for crgA is underlined. The location of the –35 promoter (underlined) element of mdaB is indicated with a dashed orange line. (B) EMSA of purified CrgA binding to its DNA target site. The amount of DNA was kept constant at 2.5 pmol per reaction and the molarity of CrgA was varied as indicated. (C) Asymmetric unit of Crystal form B of CrgA.
Mentions: As mentioned above, the published DNA footprinting data for CrgA show that the protein interacts with two adjacent LTTR-binding motifs in the CrgA/MdaB promoter region (48). We have shown that CrgA assembles as an octamer and have argued from an examination of the crystal structure that geometric constraints preclude the simultaneous binding of both sites by the same CrgA octamer. The MS of CrgA–DNA complexes showed the binding of single octamers to DNA and sedimentation velocity experiments confirmed that single octamers are the predominant species observed in the presence of DNA. We conclude that in this case, only one of the two LTTR-binding motifs in the DNA are involved in CrgA binding. Consistent with this, EMSA assays showed that in addition to binding to the 63-bp CrgA DNA footprint, CrgA binds to the individual CrgA site 1 and site 2 sequences (Figure 7B). The mobilities of these 30-bp DNA fragments were clearly shifted by CrgA. No shift was observed for a randomized control oligonucleotide of similar GC base content, indicating that the interaction with the CrgA sites was sequence specific (Figure 7B). In contrast to the EMSA with the 63-bp sequence, no defined band corresponding to a CrgA–DNA complex for these shorter DNA fragments was observed. This possibly indicates that the presence and occupancy of both binding sites is required for the formation of a stable complex in the EMSA. In these experiments, we did observe some material that did not enter the gel, which may correspond to high-molecular-weight complexes. However, given the propensity of the protein to aggregate it could also include non-specifically aggregated material.Figure 7.

Bottom Line: LysR-type transcriptional regulators (LTTRs) form the largest family of bacterial regulators acting as both auto-repressors and activators of target promoters, controlling operons involved in a wide variety of cellular processes.The LTTR, CrgA, from the human pathogen Neisseria meningitidis, is upregulated during bacterial-host cell contact.Here, we report the crystal structures of both regulatory domain and full-length CrgA, the first of a novel subclass of LTTRs that form octameric rings.

View Article: PubMed Central - PubMed

Affiliation: The Oxford Protein Production Facility and Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.

ABSTRACT
LysR-type transcriptional regulators (LTTRs) form the largest family of bacterial regulators acting as both auto-repressors and activators of target promoters, controlling operons involved in a wide variety of cellular processes. The LTTR, CrgA, from the human pathogen Neisseria meningitidis, is upregulated during bacterial-host cell contact. Here, we report the crystal structures of both regulatory domain and full-length CrgA, the first of a novel subclass of LTTRs that form octameric rings. Non-denaturing mass spectrometry analysis and analytical ultracentrifugation established that the octameric form of CrgA is the predominant species in solution in both the presence and absence of an oligonucleotide encompassing the CrgA-binding sequence. Furthermore, analysis of the isolated CrgA-DNA complex by mass spectrometry showed stabilization of a double octamer species upon DNA binding. Based on the observed structure and the mass spectrometry findings, a model is proposed in which a hexadecameric array of two CrgA oligomers binds to its DNA target site.

Show MeSH
Related in: MedlinePlus