Structure of Ala(20) -> Prs/Pro(64) -> 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 sy nthase, 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 b een 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 th e F0F1 complex is fully functional in ATPase-coupled H+ pumping. Residues 2 0 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. I n 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 ca se of the wild type protein, but, in this case, Pro(20) induces a bend in T MH-1, which then packs against a more straightened TMH-2. The essential pro linyl 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 move ment of the alpha -helical segments during the course of the H+ pumping cat alytic cycle.