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 further investigate the site of cdG binding on FliI, we constructed a homology model for SBW25 FliI based on the crystal structure of its Salmonella homolog (54) (Fig. 7). The location of ATP and the conserved residues across the six cdG-binding ATPases in this study were then mapped onto the model, and a predicted FliI hexameric complex was produced (Fig. 7A). Next, purified FliIHis was incubated and UV cross-linked to the cdG capture compound. Following tryptic digestion and mass spectrometry, mass-shifted peptides were identified using MS-PSA, a recently developed analysis method for the identification of unexpected/unknown peptide modifications (70) (supplemental Fig. S1). Two MS-PSA analyses were performed, treated (i.e. cross-linked to cdG) versus untreated FliIHis (S1A) and treated FliIHis alone (S1B). In the treated sample, we expected to identify both pure FliIHis and FliIHis with bound cdG. Accordingly, many spectra relations corresponding to modified and unmodified peptides were identified (supplemental Fig. S1). Importantly, the most densely modified peptide following cdG capture compound cross-linking comprised residues 259–269 (NVLLLMDSLTR; supplemental Fig. S1). We identified 36 spectra relations where the lighter peptide was Mascot annotated NVLLLMDSLTR, and the heavier not-annotated partner carried a modification of >150 Da. By only comparing spectra between the treated and untreated sample, we identified 52 corresponding NVLLLMDSLTR spectra relations. The NVLLLMDSLTR peptide represents the central strand of a β-sheet at the core of the SBW25 FliI homology model, plus short loops at either end (Fig. 7B, green). The C terminus of the β-strand also contains the conserved aspartate (Asp-265) of the Walker B motif (Fig. 7B, pink). Interestingly, the end of the capture compound cross-linked peptide emerges close to a cluster of highly conserved residues that could form a pocket at the interface between two FliI subunits in our model (Fig. 7, C and D, red). As well as several glycine and proline residues, this conserved pocket contains two arginines (Arg-170 and Arg-337) from one subunit and a glutamate (Glu-208) from the second. Both arginine and glutamate are highly important for dinucleotide binding in all previously characterized cdG binding proteins (40, 42).