Structure of Ala20 → Prs/Pro64 → Ala substituted subunit c of Escherichia coli ATP synthase in which the essential proline is switched between transmembrane helices

The structure of the A20P/P64A mutated subunit c of Escherichia coli ATP synthase, in which the essential proline has been switched from residue 64 of the second transmembrane helix (TMH) to residue 20 of the first TMH, has been solved by N-15,H-1 MMR in a monophasic chloroform/methanol/water (4:4:1) solvent mixture. The cA20P/P64A mutant grows as well as wild type, and the F0F1 complex is fully functional in ATPase-coupled H+ pumping. Residues 20 and 64 lie directly opposite to each other in the hairpin-like structure of wild type subunit c, and the prolinyl 64 residue is thought to induce a slight bend in TMH-2 such that it wraps around a more straightened TMH-1. In solution, the A20P/P64A substituted subunit c also forms a hairpin of two alpha -helices, with residues 41-45 forming a connecting loop as in the case of the wild type protein, but, in this case, Pro(20) induces a bend in TMH-1, which then packs against a more straightened TMH-2. The essential prolinyl residue, whether at position 64 or 20, lies close to the aspartyl 61 H+ binding site. The prolinyl residue may introduce structural flexibility in this region of the protein, which may be necessary for the proposed movement of the alpha -helical segments during the course of the H+ pumping catalytic cycle.