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
Mentions: To confirm the importance of these residues to cdG binding, specific amino acid substitutions were produced (R170H, E208Q, and R337H) in FliIΔ1–18. The solubility of the resulting FliIΔ1–18 alleles was confirmed by gel filtration (data not shown), and then cdG binding and ATPase activity were tested. All three substitutions showed seriously compromised cdG binding, with KD values that were too high to be accurately determined (Fig. 8A and Table 3) and a complete abolition of ATPase activity (Fig. 8B). Finally, we detected no cdG binding for the hexameric ATPase protein NtrC from Azotobacter vinelandii, which shares a tertiary structure fold with the export ATPases but does not have the residues of the proposed binding site (Fig. 8C). Together, these results strongly indicate that the binding indeed occurs at the proposed site shown in Fig. 7D.