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)()
Wj. Rice et Dh. Maclennan, 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)(), The Journal of biological chemistry, 271(49), 1996, pp. 31412-31419
Scanning mutagenesis was performed on all amino acids in transmembrane
sequences M5, M6, and M8, which, together with M4, make up the Ca2+ b
inding domain of the Ca2+-ATPase of sarcoplasmic reticulum (SERCA1a),
When these transmembrane sequences were displayed on a helical net, ex
amination of the effects of 101 novel point mutations and 95 prior mut
ations carried out on 92 transmembrane amino acids revealed ''patches'
' of sensitivity to mutation in M4, M5, and M6 but not in M8. The patc
hes of mutation-sensitive residues spanned 6 of the 7 tiers of the hel
ical 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 mutati
on-insensitive hydrophobic amino acids was found in M4 and M6 and in t
iers 4 to 7 of M5. A six-residue motif, (E/D)GLPA(T/V) in tiers 3 and
4 of M4 and Mg with Ca2+-binding residues Glu(309) and Asp(800) as the
first residue, was highlighted by mutation sensitivity, Elements of t
he 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 sequence
s were not similar, The motif or its counterpart was found in a region
in M4, M5, and Mg 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 mutat
ions, E(1)P to E(2)P block mutations and E(2)P dephosphorylation mutat
ions, indicating an important role for these central tiers in Ca2+ bin
ding and in the conformational changes that accompany Ca2+ translocati
on. Analysis of M8 revealed only a single mutation-sensitive residue,
the Ca2+-binding amino acid, Glu(908). This residue and a mutation-ins
ensitive residue, Ala(912) ,were the only vestiges of the motif that w
as found in M4 and M6. Additional mutations to Glu(908) provided furth
er evidence for its role in Ca2+ binding, Since mutation of M8 failed
to identify residues involved in blocking conformational changes or al
tering Ca2+ affinity, it is apparent that M8 plays a peripheral role i
n Ca2+ binding and translocation in comparison with M4, M5, and M6.