Bacterial rotary export ATPases are allosterically regulated by the nucleotide second messenger cyclic-di-GMP.
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.
Affiliation: From the Molecular Microbiology Department and.Show MeSH
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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.