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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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DNA binding and recognition in CrgA. (A) Sequence alignment of the DBD of CrgA with two further LTTR family members [E. coli, CysB and OxyR) and the three HTH proteins (DtxR from Corynebacterium diphtheriae (56), OhrR from Bacillus subtilis (55) and NarL from E. coli (54)] used for modeling. Residues boxed in yellow, contact the DNA in the co-structures of DtxR (PDB code 1F5T), OhrR (PDB code 1Z9C) and NarL (PDB code 1ZG5) or are predicted to contact DNA in CrgA. Residues important for DNA binding identified by mutational analysis in CysB, OxyR and CrgA are shown in red (11,57). Residues underlined form the activating region identified in CysB (64). The residue conservation scores of CrgA obtained with Consurf, based on 70 LTTRs from the Swissprot database, are shown above the alignment. Only residues of the structurally equivalent α2 and α3 are shown for DtxR, OhrR and NarL. (B) EMSA on CrgA wild type (2.5 pmol) and CrgA R55Q mutant (1.5 pmol) mixed in a 1:1 molar ratio with the reported 63-bp CrgA DNA footprint (18,48). (C) One DBD of CrgA with modeled DNA showing selected residues discussed in this report. (D) Structural comparison of a DBD and LH pair from CrgA and CbnR (Cα RMSD 1.1 Å2 for 86 equivalent residues). R55 of CrgA is labeled for orientation purposes in panel E. (E) Electrostatic surface potential of the CrgA octamer showing one DNA-binding pair with modeled DNA (DxtR, PDB code 1F5T).
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Figure 6: DNA binding and recognition in CrgA. (A) Sequence alignment of the DBD of CrgA with two further LTTR family members [E. coli, CysB and OxyR) and the three HTH proteins (DtxR from Corynebacterium diphtheriae (56), OhrR from Bacillus subtilis (55) and NarL from E. coli (54)] used for modeling. Residues boxed in yellow, contact the DNA in the co-structures of DtxR (PDB code 1F5T), OhrR (PDB code 1Z9C) and NarL (PDB code 1ZG5) or are predicted to contact DNA in CrgA. Residues important for DNA binding identified by mutational analysis in CysB, OxyR and CrgA are shown in red (11,57). Residues underlined form the activating region identified in CysB (64). The residue conservation scores of CrgA obtained with Consurf, based on 70 LTTRs from the Swissprot database, are shown above the alignment. Only residues of the structurally equivalent α2 and α3 are shown for DtxR, OhrR and NarL. (B) EMSA on CrgA wild type (2.5 pmol) and CrgA R55Q mutant (1.5 pmol) mixed in a 1:1 molar ratio with the reported 63-bp CrgA DNA footprint (18,48). (C) One DBD of CrgA with modeled DNA showing selected residues discussed in this report. (D) Structural comparison of a DBD and LH pair from CrgA and CbnR (Cα RMSD 1.1 Å2 for 86 equivalent residues). R55 of CrgA is labeled for orientation purposes in panel E. (E) Electrostatic surface potential of the CrgA octamer showing one DNA-binding pair with modeled DNA (DxtR, PDB code 1F5T).

Mentions: Although the fold of each CrgA subunit is similar to that observed for CbnR, the overall assembly of the complex is strikingly different. CbnR is tetrameric whereas CrgA assembles into square-like hollow octameric rings with dimensions of 125 × 125 × 67 Å. The regulatory domains are sandwiched between the DBDs that are located in pairs (DBD pairs) at the four corners of the disc. Viewed from above, the hollow of the octamer has a cross-like shape with a distance of ∼95 Å between opposing LHs and ∼50 Å between opposing regulatory domains (Figure 2). On first inspection the octamer, which occupies one asymmetric unit, appears to have 4-fold symmetry. However, there are significant differences in the orientation of the LHs and DBDs within the eight subunits, which break this apparent symmetry. Due to the nature of the assembly, each CrgA subunit contacts just two of the other subunits. Each subunit forms an interface with one adjacent subunit, principally through their equivalent N-terminal domains, (residues 1–89, N-term interface) and to a second subunit, through their regulatory domains (RD interface) (Figure 2). The sizes of these two interfaces are similar, each burying an average surface area of ∼1300 Å2. The RD interface contains two sets of equivalent interactions, between residues of the RD-I and RD-II on the adjacent chain while the N-terminal interface is primarily formed by dimerization of the LHs through intermolecular anti-parallel coiled coil interactions (Figures 4A and 6D).Figure 2.


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)

DNA binding and recognition in CrgA. (A) Sequence alignment of the DBD of CrgA with two further LTTR family members [E. coli, CysB and OxyR) and the three HTH proteins (DtxR from Corynebacterium diphtheriae (56), OhrR from Bacillus subtilis (55) and NarL from E. coli (54)] used for modeling. Residues boxed in yellow, contact the DNA in the co-structures of DtxR (PDB code 1F5T), OhrR (PDB code 1Z9C) and NarL (PDB code 1ZG5) or are predicted to contact DNA in CrgA. Residues important for DNA binding identified by mutational analysis in CysB, OxyR and CrgA are shown in red (11,57). Residues underlined form the activating region identified in CysB (64). The residue conservation scores of CrgA obtained with Consurf, based on 70 LTTRs from the Swissprot database, are shown above the alignment. Only residues of the structurally equivalent α2 and α3 are shown for DtxR, OhrR and NarL. (B) EMSA on CrgA wild type (2.5 pmol) and CrgA R55Q mutant (1.5 pmol) mixed in a 1:1 molar ratio with the reported 63-bp CrgA DNA footprint (18,48). (C) One DBD of CrgA with modeled DNA showing selected residues discussed in this report. (D) Structural comparison of a DBD and LH pair from CrgA and CbnR (Cα RMSD 1.1 Å2 for 86 equivalent residues). R55 of CrgA is labeled for orientation purposes in panel E. (E) Electrostatic surface potential of the CrgA octamer showing one DNA-binding pair with modeled DNA (DxtR, PDB code 1F5T).
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Related In: Results  -  Collection

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Figure 6: DNA binding and recognition in CrgA. (A) Sequence alignment of the DBD of CrgA with two further LTTR family members [E. coli, CysB and OxyR) and the three HTH proteins (DtxR from Corynebacterium diphtheriae (56), OhrR from Bacillus subtilis (55) and NarL from E. coli (54)] used for modeling. Residues boxed in yellow, contact the DNA in the co-structures of DtxR (PDB code 1F5T), OhrR (PDB code 1Z9C) and NarL (PDB code 1ZG5) or are predicted to contact DNA in CrgA. Residues important for DNA binding identified by mutational analysis in CysB, OxyR and CrgA are shown in red (11,57). Residues underlined form the activating region identified in CysB (64). The residue conservation scores of CrgA obtained with Consurf, based on 70 LTTRs from the Swissprot database, are shown above the alignment. Only residues of the structurally equivalent α2 and α3 are shown for DtxR, OhrR and NarL. (B) EMSA on CrgA wild type (2.5 pmol) and CrgA R55Q mutant (1.5 pmol) mixed in a 1:1 molar ratio with the reported 63-bp CrgA DNA footprint (18,48). (C) One DBD of CrgA with modeled DNA showing selected residues discussed in this report. (D) Structural comparison of a DBD and LH pair from CrgA and CbnR (Cα RMSD 1.1 Å2 for 86 equivalent residues). R55 of CrgA is labeled for orientation purposes in panel E. (E) Electrostatic surface potential of the CrgA octamer showing one DNA-binding pair with modeled DNA (DxtR, PDB code 1F5T).
Mentions: Although the fold of each CrgA subunit is similar to that observed for CbnR, the overall assembly of the complex is strikingly different. CbnR is tetrameric whereas CrgA assembles into square-like hollow octameric rings with dimensions of 125 × 125 × 67 Å. The regulatory domains are sandwiched between the DBDs that are located in pairs (DBD pairs) at the four corners of the disc. Viewed from above, the hollow of the octamer has a cross-like shape with a distance of ∼95 Å between opposing LHs and ∼50 Å between opposing regulatory domains (Figure 2). On first inspection the octamer, which occupies one asymmetric unit, appears to have 4-fold symmetry. However, there are significant differences in the orientation of the LHs and DBDs within the eight subunits, which break this apparent symmetry. Due to the nature of the assembly, each CrgA subunit contacts just two of the other subunits. Each subunit forms an interface with one adjacent subunit, principally through their equivalent N-terminal domains, (residues 1–89, N-term interface) and to a second subunit, through their regulatory domains (RD interface) (Figure 2). The sizes of these two interfaces are similar, each burying an average surface area of ∼1300 Å2. The RD interface contains two sets of equivalent interactions, between residues of the RD-I and RD-II on the adjacent chain while the N-terminal interface is primarily formed by dimerization of the LHs through intermolecular anti-parallel coiled coil interactions (Figures 4A and 6D).Figure 2.

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