Role of Phosphate Binding Residues in the Catalytic Sites of Escherichia coli ATP synthase

Abstract

Background ATP synthase is the smallest known biological nanomotor in the living organisms. ATP synthase generates ATP from ADP and Pi through oxidative phosphorylation in membranes of bacteria, mitochondria, and chloroplasts. ATP is the main cellular energy currency and it is generated catalytic sites of ATP synthase. Designated Pi binding residues play critical role in the interaction of ADP + Pi to form ATP. Our lab is involved in elucidating the role of phosphate binding residues in the catalytic sites of ATP synthase. So far, we have investigated twelve residues, bAsn-243, bArg-246, bLys-155, bArg-182, aPhe-291, αIle-346, aSer-347, αIle-348, αThr-349, aAsp-350, aGly-351, and aArg-376 based their proximity to the phosphate analogs AlF3 or SO42- in x-ray structures of catalytic sites. Many other catalytic site residues remain to be characterized including highly conserved αVISIT-DG sequence residue αVal-345 which seems to have important functional role in the catalytic sites. Methods Mutagenic analysis of αVal-345 was performed to generate single and double mutants of αV345A, αV345D, αV345Q, and αV345R. Comparative growth was done on limiting glucose and succinate plates. MgPi protection assay against NBD-Cl induced inhibition was performed to reveal the direct or indirect role of αVal-345 in Pi binding. Results Recently, we found that aAsp-350 of the VISIT-DG sequence is the seventh important residue involved in phosphate binding other than bLys-155, bArg-182, Arg-376, bArg-246, and αSer-347, and aThr-347 while αIle-346, αIle-348, bAsn-243, aPhe-291, and aGly-351 are required for the maintenance of catalytic site function