Characterization of L382 and S383 in the βDELSEED‐motif of ATP synthase

Abstract

BackgroundAntibiotic resistance is rising on an alarming rate. Finding alternative ways to combat microbial infections is a matter of human survival. Selective inhibition of microbial ATP synthase has the potential to eradicate antibiotic resistant microbes. ATP synthase is the chief source of ATP production in almost all organisms. Antimicrobial properties of some venom peptides are connected to the binding and inhibition of ATP synthase. Peptides bind at the bDELSEED‐motif of E. coli ATP synthase and inhibit Escherichia coli ATP synthase to variable degrees. In order to understand the selective binding and inhibition of microbial ATP synthase it is essential to fully elucidate the binding site. Currently our lab is studying the role of Leu‐382 and Ser‐383 of the bDELSEED‐motif in peptide binding.MethodGrowth properties of wild type, null, and bDELSEED‐motif mutant E. coli strains were checked on minimal media with 3% glucose media and succinate media with 1/4th case amino acids. Wild type and mutant membrane bound F1Fo enzymes were isolated by growing on minimal media to late log phase. Cells were harvested in super centrifuge, French Pressed, and finally membrane‐bound ATP synthase isolated by ultracentrifugation. Purity of isolated protein was validated by western blot using anti‐F1‐β antibody. Biochemical inhibition of wild type and mutant ATP synthase was validated by NBD‐Cl. βLeu‐382 and βSer‐383 mutants were inhibited by honey bee venom peptide melittin. Time kinetics for full enzyme inhibition was determined along with reversibility of enzyme inhibition.ResultsOur growth results show that mutant strains grew between wild type and null strain on limiting glucose. Growth on succinate media corroborate the growth on limiting glucose. Melittin inhibited mutant enzymes to variable degrees. Moreover, we found that loss of βLeu‐382 and βSer‐383 residues caused reduced level of peptide binding at βDELSEED‐motif.ConclusionsWe conclude that residue Leu‐382 and Ser‐383 of βDELSEED‐motif are essential for proper orientation and binding of peptides at the peptide‐binding site. Moreover, βLeu‐382 and βSer‐383 play important role in maintenance of the βDELSEED‐motif.Support or Funding InformationThis work was supported by the KCOM Biomedical Sciences Graduate Program Grant Award Fund # 851‐044 to AS and ZA.