SITE-DIRECTED DISULFIDE MAPPING OF HELICES M4 AND M6 IN THE CA2-TWITCH SKELETAL-MUSCLE SARCOPLASMIC-RETICULUM( BINDING DOMAIN OF SERCA1A, THE CA2+ ATPASE OF FAST)
Wj. Rice et al., SITE-DIRECTED DISULFIDE MAPPING OF HELICES M4 AND M6 IN THE CA2-TWITCH SKELETAL-MUSCLE SARCOPLASMIC-RETICULUM( BINDING DOMAIN OF SERCA1A, THE CA2+ ATPASE OF FAST), The Journal of biological chemistry, 272(50), 1997, pp. 31412-31419
In an attempt to define the spatial relationships among SERCA1a transm
embrane 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 retaine
d 44% of wild type Ca2+ transport activity. Pairs of cysteine residues
were then reintroduced to determine whether their juxtaposition would
result in the formation of disulfide cross-links between transmembran
e helices. In initial studies de signed to map the juxtaposition of Ca
2+ binding residues, Cys was substituted for Glu(309) or Gly(310) in t
ransmembrane sequence M4, in combination with the substitution of Cys
for Glu(771) in M5; for Asn(796), Thr(789), or Asp(800) in M6; or for
Glu(908) in M8. These double mutants all retained the capacity to form
a phosphoenzyme intermediate from P-i (but not from ATP in the presen
ce of Ca2+), and in all but mutants E309C/N796C and G310C/N796C, phosp
hoenzyme formation was insensitive to 100 mu M Ca2+. These results sup
port the view that both Glu(309) and Asn(796) contribute to Ca2+ bindi
ng site II, which is not required for conversion of E-2, the substrate
for P-i phosphorylation, to E-1. Cross linking in mutants E309C/N796C
and G310C/D800C established reference points for the orientation of M
4 and M6 relative to each other and provided the basis for the predict
ion of potential additional cross-links. Strong links were formed with
the pairs T317C/A804C and T317C/L807C near the cytoplasmic ends of th
e two helices and with A305C/L792C and A305C/L793C near the lumenal en
ds. 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 f
or the mutation sensitivity of several pairs of residues in these heli
ces, the proposed association of M4 and M6 supports a new model for th
e orientation of the two Ca2+ binding sites among transmembrane helice
s M4, M5, and M6.