Significance of the highly conserved α VISITDG sequence in the catalytic sites of ATP synthase
Authors: Raheem Samah , Ahmad Zulfiqar 
Published: June 2021

BackgroundATP synthase is the main source of cellular energy ATP. ATP is generated and hydrolyzed in the catalytic sites of ATP synthase. The highly conserved αVISITDG sequence is located in close proximity to the Pi binding subdomain in the catalytic site of ATP synthase. Previously all αVISITDG sequence residues except αV345 and αD350 have been shown to contribute in Pi binding or in the maintenance of pi binding sub‐domain. The non‐polar aliphatic αVal‐345 and negatively charged acidic αAsp‐350 residues are uniquely positioned in close proximity to two known Pi binding residues αR376 and βR182. The functional role of αV345 and αD350 are still unknown. NBD‐Cl interaction with βY297 caused variable degree of inhibition of wild type and mutant enzymes and was reversed by DTT. AlFx and ScCl3 inhibition of wild type and mutants resulted in various transition states end products. Molecular modulation of αVISITDG sequence residues provided insight into their role in motor and catalytic function. Moreover, Pi protection assay results against NBD‐Cl inhibition suggest that αD350 may be involved Pi binding through its interaction with neighboring Pi binding residues. In contract, αV345, although strongly conserved and important for function, appears not to participate on Pi binding.MethodsGrowth properties of wild type and mutant strains were observed on fermentable and non‐fermentable carbon sources. The functional properties of αVal‐345 and αAsp‐350 were studied using a variety of mutants. Molecular modulation of neighboring residues was performed by addition of positive and negative charges. For Pi protection against NBD‐Cl inhibition assay wild type and mutant enzymes were pre‐incubated with varying concentrations of 0 mM, 2.5 mM, 5.0 mM, or 10.0 mM MgPi before treating with 100 uM NBD‐Cl. Level of protection against NBD‐Cl inhibition in presence of Pi were assessed by OD700.ResultsNBD‐Cl completely inhibited the wild‐type E. coli enzyme while caused variable degree of inhibition of mutant enzymes. About 50–90 % inhibition was observed for the mutant enzymes. 4 mM DTT fully revered the NBD‐Cl caused inhibition of all enzymes confirming NBD‐Cl interaction and binding to βY297. MgPi protected wild type and αVal‐345 mutant enzyme against NBD‐Cl inhibition while αD350 mutant were resisted the protection.ConclusionsThe highly conserved αVISITDG residues play an important role either in Pi binding or maintenance Pi binding sub‐domain. Catalytic activity and motor function of ATP synthase can be enhanced by molecular modulations of Pi binding sub‐domain residues. Both αV345 and αD350 are important for the function of ATP synthase. While αD350 is required for Pi binding, αV345 is not.Support or Funding InformationThis work was supported by A.T Still University Kirksville College of Osteopathic Medicine Biomedical Sciences Graduate Program Grant Award number 851‐036 to SR and ZA and A.T Still University Warner/Fermanturo Research Grant Award number 501‐605 to ZA.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.