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Bacterial rotary export ATPases are allosterically regulated by the nucleotide second messenger cyclic-di-GMP.

Trampari E, Stevenson CE, Little RH, Wilhelm T, Lawson DM, Malone JG - J. Biol. Chem. (2015)

Bottom Line: The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro.Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits.Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins.

View Article: PubMed Central - PubMed

Affiliation: From the Molecular Microbiology Department and.

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Clustal alignment of conserved residues between FliI, HrcN, and ClpB2 proteins in this study. Identities between all six residues are marked with asterisks (*), and similarities across all six with periods (.) or colons (:). The mutated FliI Walker A/B residues (see Fig. 6) are marked in red, the capture compound-binding NVLLLMDSLTR peptide is marked in blue, the position of the conserved cdG binding arginine in FlrA (Arg-176) is marked in purple, and the conserved residues of the proposed cdG binding site are marked in green (see Fig. 7).
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Figure 9: Clustal alignment of conserved residues between FliI, HrcN, and ClpB2 proteins in this study. Identities between all six residues are marked with asterisks (*), and similarities across all six with periods (.) or colons (:). The mutated FliI Walker A/B residues (see Fig. 6) are marked in red, the capture compound-binding NVLLLMDSLTR peptide is marked in blue, the position of the conserved cdG binding arginine in FlrA (Arg-176) is marked in purple, and the conserved residues of the proposed cdG binding site are marked in green (see Fig. 7).

Mentions: The ATPase activity of both FliI and HrcN is suppressed by the addition of cdG. In this respect, the relationship between cdG and the export ATPase proteins is reminiscent of the transcriptional motility regulators FleQ (27) and FlrA (71). These proteins both contain AAA+ ATPase domains and bind cdG close to the Walker A motif of the protein. However, there appear to be important differences between the binding characteristics of FleQ/FlrA and the export ATPase proteins described here. In FleQ, cdG interacts with the Walker A site of the protein, leading to competitive inhibition of ATPase activity (27). Similarly, cdG binding to an arginine residue (Arg-176) downstream of the Walker A motif of FlrA inhibits binding to its target promoter sequence (71). In the case of the export ATPase proteins, we were able to uncouple ATPase activity from cdG binding. Our FliI/HrcN K181A mutants bound strongly to cdG but displayed no ATPase activity, whereas the G176A mutant retained substantial ATPase activity but showed severely compromised cdG binding. Furthermore, the FlrA R176 residue is conserved in FleQ but not in FliI/HrcN/ClpB2 (Fig. 9, purple), again suggesting a distinct cdG binding mechanism.


Bacterial rotary export ATPases are allosterically regulated by the nucleotide second messenger cyclic-di-GMP.

Trampari E, Stevenson CE, Little RH, Wilhelm T, Lawson DM, Malone JG - J. Biol. Chem. (2015)

Clustal alignment of conserved residues between FliI, HrcN, and ClpB2 proteins in this study. Identities between all six residues are marked with asterisks (*), and similarities across all six with periods (.) or colons (:). The mutated FliI Walker A/B residues (see Fig. 6) are marked in red, the capture compound-binding NVLLLMDSLTR peptide is marked in blue, the position of the conserved cdG binding arginine in FlrA (Arg-176) is marked in purple, and the conserved residues of the proposed cdG binding site are marked in green (see Fig. 7).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 9: Clustal alignment of conserved residues between FliI, HrcN, and ClpB2 proteins in this study. Identities between all six residues are marked with asterisks (*), and similarities across all six with periods (.) or colons (:). The mutated FliI Walker A/B residues (see Fig. 6) are marked in red, the capture compound-binding NVLLLMDSLTR peptide is marked in blue, the position of the conserved cdG binding arginine in FlrA (Arg-176) is marked in purple, and the conserved residues of the proposed cdG binding site are marked in green (see Fig. 7).
Mentions: The ATPase activity of both FliI and HrcN is suppressed by the addition of cdG. In this respect, the relationship between cdG and the export ATPase proteins is reminiscent of the transcriptional motility regulators FleQ (27) and FlrA (71). These proteins both contain AAA+ ATPase domains and bind cdG close to the Walker A motif of the protein. However, there appear to be important differences between the binding characteristics of FleQ/FlrA and the export ATPase proteins described here. In FleQ, cdG interacts with the Walker A site of the protein, leading to competitive inhibition of ATPase activity (27). Similarly, cdG binding to an arginine residue (Arg-176) downstream of the Walker A motif of FlrA inhibits binding to its target promoter sequence (71). In the case of the export ATPase proteins, we were able to uncouple ATPase activity from cdG binding. Our FliI/HrcN K181A mutants bound strongly to cdG but displayed no ATPase activity, whereas the G176A mutant retained substantial ATPase activity but showed severely compromised cdG binding. Furthermore, the FlrA R176 residue is conserved in FleQ but not in FliI/HrcN/ClpB2 (Fig. 9, purple), again suggesting a distinct cdG binding mechanism.

Bottom Line: The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro.Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits.Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins.

View Article: PubMed Central - PubMed

Affiliation: From the Molecular Microbiology Department and.

Show MeSH