Scanning mutagenesis reveals a similar pattern of mutation sensitivity in transmembrane sequences M4, M5, and M6, but not in M8, of the Ca2+-ATPase of sarcoplasmic reticulum (SERCA1a)

Abstract

Scanning mutagenesis was performed on all amino acids in transmembrane sequences M5, M6, and M8, which, together with M4, make up the Ca2+ binding domain of the Ca2+-ATPase of sarcoplasmic reticulum (SERCA1a). When these transmembrane sequences were displayed on a helical net, examination of the effects of 101 novel point mutations and 95 prior mutations carried out on 92 transmembrane amino acids revealed "patches" of sensitivity to mutation in M4, M5, and M6 but not in M8. The patches of mutation-sensitive residues spanned 6 of the 7 tiers of the helical net and covered about 240 degrees at their widest point in tiers 3 or 4 and 140 degrees in tiers 2 and 5. A contiguous column of mutation-insensitive hydrophobic amino acids was found in M4 and M6 and in tiers 4 to 7 of M5. A six-residue motif, (E/D)GLPA(T/V) in tiers 3 and 4 of M4 and M6 with Ca2+-binding residues Glu309 and Asp800 as the first residue, was highlighted by mutation sensitivity. Elements of the motif could also be discerned in M5, but reading in the C-terminal to N-terminal direction. Mutation sensitivity in tier 5 of M4 mirrored mutation sensitivity of tier 5 in M6, although the amino acid sequences were not similar. The motif or its counterpart was found in a region in M4, M5, and M6 that is made up of tiny or small amino acids but is bounded by tiers with a larger percentage of bulky amino acids. Tiers 3, 4, and 5 of M4, M5, and M6 contain Ca2+ binding and affinity mutations, E1P to E2P block mutations and E2P dephosphorylation mutations, indicating an important role for these central tiers in Ca2+ binding and in the conformational changes that accompany Ca2+ translocation. Analysis of M8 revealed only a single mutation-sensitive residue, the Ca2+-binding amino acid, Glu908. This residue and a mutation-insensitive residue, Ala912, were the only vestiges of the motif that was found in M4 and M6. Additional mutations to Glu908 provided further evidence for its role in Ca2+ binding. Since mutation of M8 failed to identify residues involved in blocking conformational changes or altering Ca2+ affinity, it is apparent that M8 plays a peripheral role in Ca2+ binding and translocation in comparison with M4, M5, and M6.