High sensitivity to site directed mutagenesis of the peptide segment connecting phosphorylation and Ca2+ binding domains in the Ca2+ transport ATPase.

Nine residues (Leu321, Lys329, Asn330, Val333, Arg334, Leu336, Pro337, Val339 and Glu340), within the peptide segment intervening between the catalytic domain and the Ca2+ binding domain of the sarcoplasmic reticulum (SERCA 1) ATPase, were individually mutated to Ala. The mutated proteins were recovered in the microsomal fraction of COS-1 cells following transient expression, and exhibited inhibition of Ca2+ uptake and ATPase hydrolytic activity, while forming discernable levels of phosphorylated intermediate. Mutation of Glu340 to Gln (rather than to Ala) was much less effective, suggesting that the functional consequence of the mutation is related to structural perturbation, rather than loss of the acidic side chain. The high sensitivity of this peptide segment to single mutations suggests that its structural integrity is required for functional linkage of the phosphorylation and Ca2+ binding domains.