The critical roles of residues P235 and F236 of subunit A of the motor protein A-ATP synthase in P-loop formation and nucleotide binding.

The mutants P235A and F236A have been generated and their crystal structure was determined to resolutions of 2.38 and 2.35 A, respectively, in order to understand the residues involved in the formation of the novel arched P-loop of subunit A of the A-ATP synthase from Pyrococcus horikoshii OT3. Both the structures show unique, altered conformations for the P-loop. Comparison with the previously solved wild type and P-loop mutant S238A structures of subunit A showed that the P-loop conformation for these two novel mutants occupy intermediate positions, with the wild type fully arched and the well-relaxed S238A mutant structures taking the extreme positions. Even though the deviation is similar for both mutants, the curvature of the P-loop faces the opposite direction. Deviations in the GER-loop, lying above the P-loop, are similar for both mutants, but in F236A, it moves towards the P-loop by around 2 A. The curvature of the loop region V392-V410, located directly behind the P-loop, moves close by 3.6 A towards the P-loop in the F236A structure and away by 2.5 A in the P235A structure. Two major deviations were observed in the P235A mutant, which are not identified in any of the subunit A structures analyzed so far, one being a wide movement of the N-terminal loop region (R90-P110) making a rotation of 80 degrees and the other being rigid-body rotation of the C-terminal helices from Q520-A588 by around 4 degrees upwards. Taken together, the data presented demonstrate the concerted effects of the critical residues P235A, F236, and S238 in the unique P-loop conformation of the A-ATP synthases.