Site-directed disulfide mapping of helices M4 and M6 in the Ca2+ binding domain of SERCA1a, the Ca2+ ATPase of fat twitch skeletal muscle sarcoplasmic reticulum

Abstract

In an attempt to define the spatial relationships among SERCA1a transmembrane helices, M4, M5, M6, and M8, involved in Ca2+ binding, all six cysteine residues were removed from predicted transmembrane sequences by substitution with Ser or Ala. The cysteine-depleted protein retained 44% of wild type Ca2+ transport activity. Pairs of cysteine residues where then reintroduced to determine whether their juxtaposition would result in the formation of disulfide cross-links between transmembrane helices. In initial studies designed to map the juxtaposition of Ca2+ binding residues, Cys was substituted for Glu309 or Gly310 in transmembrane sequence M4, in combination with the substitution of Cys for Glu771 in M5; for Asn796, Thr799, or Asp800 in M6; or for Glu908 in M8. These double mutants all retained the capacity to form a phosphoenzyme intermediate from P(i) (but not from ATP in the presence of Ca2+), and in all but mutants E309C/N796C and G310C/N796C, phosphoenzyme formation was insensitive to 100 μM Ca2+. These results support the view that both Glu309 and Asn796 contribute Ca2+ binding site II, which is not required for conversion of E2, the substrate for P1 phosphorylation, to E1. Cross-linking in mutants E309C/N796C and G310C/D8006 established reference points for the orientation of M4 and M6 relative to each other and provided the basis for the prediction of potential additional cross-links. Strong links were formed with the pairs T317C/A804C and T317C/L807C near the cytoplasmic ends of the two helices and with A305C/L792C and A305C/L793C near the lumenal ends. These combined results support the conclusion that M4 and M6 form a right-handed coiled- coil structure that forms part of the pathway of Ca2+ translocation. In addition to providing a possible explanation for the mutation sensitivity of several pairs of residues in these helices, the proposed association of M4 and M6 supports a new model for the orientation of the two Ca2+ binding sites among transmembrane helices M4, M5, and M6.