Significance of αThr-349 in the catalytic sites of Escherichia coli ATP synthase.

In a 75 year life span, a typical 70 kg human generates approximately 2.0 million kg of ATP. The cell’s energy currency is generated by converting food into useable energy by oxidation. F1Fo ATP synthase is responsible for the fundamental means of cell energy production in animals, plants, and almost all microorganisms, which occurs by oxidation or photophosphorylation in membranes of bacteria, mitochondria, and chloroplasts. ATP synthase is one of the smallest biological nanomotors and is structurally similar in all species.1−4 In its simplest form, as in Escherichia coli, it contains eight different subunits distributed in the water-soluble F1 sector (subunits α3β3γδe) and the membrane-associated Fo sector (subunits ab2c10). The total molecular size is ∼530 kDa.4 In chloroplasts, there are two isoforms of subunit b. In mitochondria, there are seven to nine additional subunits, depending on the source, but in total, they contribute only a small fraction of additional mass and may have regulatory roles.5−7 The membrane-bound F1Fo ATP synthase enzyme is highly conserved and structurally identical among different species. X-ray structures of bovine enzyme8 established the presence of three catalytic sites at α-subunit−β-subunit interfaces of the α3β3 hexamer. ATP hydrolysis and synthesis occur in the F1 sector, whereas proton transport occurs through the membrane-embedded Fo.8,9 ATP synthesis is a result of proton gradient-driven clockwise rotation of γ (as viewed from the outer membrane), while ATP hydrolysis results in anticlockwise rotation of the γ-subunit. Detailed reviews of ATP synthase structure and function may be found in refs (10−18). A precise knowledge of Pi (inorganic phosphate) binding is not only essential for following the reaction mechanism of ATP synthesis and hydrolysis but also equally important for understanding the relationship between catalytic mechanism and mechanical rotation in this biological nanomotor. For this reason, we have focused our efforts on determining the role of conserved residues in and around catalytic site Pi binding subdomain.19 Knowledge of Pi binding residues and residues surrounding the Pi binding subdomain is imperative for (i) the molecular modulation of the catalytic site(s) for the improved catalytic and motor function of this enzyme, (ii) an explanation of how ATP synthase binds ADP and Pi within its catalytic sites in the face of a relatively high ATP/ADP concentration ratio, and (iii) understanding the relationship between Pi binding and subunit rotation.20−22 Many earlier attempts to measure Pi binding in purified E. coli F1 failed to detect appreciable Pi binding at physiological Pi concentrations,21,23,24 but modification of the assay devised by Perez et al.25 provides a useful measure of Pi binding. In this assay, protection is afforded by Pi against inhibition of ATPase activity induced by covalent reaction with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). X-ray crystallography showed that the covalent interaction of NBD-Cl specifically with β297 occurs in the βE catalytic site26 (see Figure ​Figure1A);1A); thus, protection afforded by Pi indicates that binding of Pi occurs at the βE catalytic site. Modification of the assay described above for E. coli, purified F1 or F1Fo membranes, previously allowed us to investigate the relationship between Pi binding and catalysis for eight residues, namely, βArg-246, βAsn-243, αArg-376, βLys-155, βArg-182, αPhe-291, αSer-347, and αGly-351.a Although all these residues are situated in the proximity of the phosphate analogues AlF3 or SO42– in X-ray structures of catalytic sites,27,28 we found that four residues, namely, βArg-246, αArg-376, βLys-155, and βArg-182, grouped in a triangular fashion, are directly involved in Pi binding while the fifth residue, αSer-347, is indirectly involved in Pi binding through its interaction with βArg-246 (see Figure ​Figure11B).19,29−35 Figure 1 X-ray structures of catalytic sites in mitochondrial ATP synthase showing the spatial relationship of α-subunit VISIT-DG sequence residue αT349. (A) Reacted NBD-O-tyrosyl-297 in the βE site.26 (B) βDP site in the AlF4– ... The mechanism of condensation of Pi with MgADP proposed by Senior et al.9 was strengthened by the X-ray crystallography structure of bovine ATP synthase of Menz et al.28 showing the transition state analogue MgADP·AlF4– trapped in catalytic sites (Figure ​(Figure1B).1B). It is clear from the geometry of this complex that the fluoroaluminate group occupies the position of the ATP γ-phosphate in the predicted transition state. The first transition state-like structure of F1 from rat liver crystallized with the Pi analogue vanadate (Vi), reported by Pedersen’s group, demonstrated that ADP was not essential, suggesting that the MgVi–F1 complex inhibited the catalytic activity to the same extent as that observed for the MgADP–Vi–F1 complex.36 Neither purified F1 nor membrane-bound F1Fo from E. coli is inhibited by MgVi or MgADP–Vi.30 Consequently, we have relied on inhibition of ATPase activity by fluoroaluminate (or fluoroscandium) to assess the potential to stabilize a transition state complex.19,29−32,35 Cingolani and Duncan18 have resolved the first E. coli F1 sector high-resolution crystal structure in an autoinhibited conformation. This structure divulges a wealth of information about the regulatory features of bacterial and chloroplast ATP synthase. Moreover, the E. coli ATP synthase X-ray structure paves the way for the development of new antimicrobial drugs. For E. coli, ATP synthase is naturally a better candidate for antimicrobial drugs in comparison to mitochondrial ATP synthase.4,18 Newly developed anti-tuberculosis drug targeting bacterial ATP synthase corroborates this assertion.37 Because the E. coli high-resolution structure4 does not contain sulfate, phosphate, fluoroaluminate, or fluoroscandium, we have relied on the mitochondrial ATP synthase structure that is very similar to that of E. coli (∼70% homologous sequence) as this study deals with the analogues described above.27,28,38 Fortunately, by mutagenic analysis along with the NBD-Cl protection assay, as well as ATPase inhibition by transition state analogues, we can investigate the direct or indirect role of residues in Pi binding. In this work, we examine the role of the highly conserved α-subunit VISIT-DG sequence residue αThr-349 in the process of Pi binding. Figure ​Figure1B1B shows the position of αThr-349 with respect to other known Pi binding residues. The strategic position of αThr-349 in the Pi binding subdomain leads to the following basic questions: Is αThr-349 involved in Pi binding directly or indirectly? Do the αT349A, αT349D, αT349Q, and αT349R mutations have any effect on transition state formation? Also, can αT349R compensate for βArg-182, a known Pi binding residue?