Mutations in a Helix-1 Motif of the ATP Synthase c-Subunit of Bacillus pseudofirmus OF4 Cause Functional Deficits and Changes in the c-Ring Stability and Mobility on Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis

Abstract

The ATP synthase of the alkaliphile Bacillus pseudofirmus OF4 has a tridecameric c-subunit rotor ring. Each c-subunit has an AxAxAxA motif near the center of the inner helix, where neutralophilic bacteria generally have GxGxGxG. Here, we studied the impact of four single and six multiple Ala-to-Gly chromosomal mutations in the A16xAxAxA22 motif on the capacity for non-fermentative growth and, for most of the mutants, on ATP synthesis by ADP + Pi-loaded membrane vesicles at pH 7.5 and 10.5. SDS-PAGE analyses of the holo-ATP synthases were used to probe stability of the mutant c-rotors and mobility properties of the c-rotors as well as the monomeric c-subunits that are released from them by trichloroacetic acid treatment. Mutants containing an Ala16-to-Gly mutation exhibited the most severe functional defects. On SDS-PAGE, most of the mutant c-monomers exhibited increased mobility relative to the WT c-subunit, but among the intact c-rings, only Ala16-to-Gly containing mutants exhibited significantly increased mobility relative to the WT c-ring. The hypothesis that these c-rings have a decreased c-subunit stoichiometry is still untested but the functional impact of an Ala16-to-Gly mutation clearly depended upon additional Ala-to-Gly mutation(s) and their positions. The double A16/20G mutant exhibited a larger functional deficit than both the A16G and A16/18G mutants. Most of the mutant c-rings showed in vitro instability relative to wild-type (WT) c-ring. However, the functional deficits of mutants did not correlate well with the extent of c-ring stability loss, so this property is unlikely to be a major factor in vivo.

The c-ring stoichiometry of ATP synthase is adapted to cell physiological requirements of alkaliphilic Bacillus pseudofirmus OF4

“…The A16/20G mutants exhibited the most pronounced phenotype of the alanine mutants. The double mutant defect, described previously, was more severe in a screening assay involving a single 14-h A 600 measurement than in a growth curve experiment conducted under different conditions (24). Interestingly, the extA16/ 20G double mutant grew somewhat better compared with its nonextended homolog on malate at both pH values.…”

“…In a previous site-directed mutagenesis study, we showed that selected alanine-to-glycine mutations, all of which included an A16G change, shifted the c-ring migration profiles on SDS gels (24,26). To uncover structural evidence that these mutant B. pseudofirmus OF4 ATP synthases have altered c-ring stoichiometries, we analyzed their c-rings on the single protein complex level by AFM and at the c/c-subunit contacting interface by X-ray crystallography.…”

“…1A). Mutants within this motif exhibit altered c-ring migration profiles, suggesting reduced c-subunit stoichiometry (24). Concurrent with this observation, it was recently structurally demonstrated that replacement of glycine with alanine can augment the c-ring stoichiometry (25).…”

“…We examined four different B. pseudofirmus OF4 c-rings: WT (control), extended WT (extWT; see the next paragraph), and two mutants, extA16G and extA16/20G (24). For this, the WT B. pseudofirmus OF4 c 13 ring was purified and densely reconstituted in monogalactosyldiacylglycerol (MGDG) vesicles.…”

“…Thus, we performed a structural analysis using atomic force microscopy (AFM) and X-ray crystallography with purified ATP synthase c-rings to image individual mutant protein complexes with potentially altered stoichiometries (24). We also performed concomitant growth studies, the results of which underscore the importance of c-ring stoichiometry for the cell's capacity for growth at high pH under ATP synthase-dependent conditions.…”