Abstract
The amino acid sequence (L67) intervening between the M6 and M7 transmembrane segments of the Ca(2+) transport ATPase was subjected to mutational analysis. Mutation of Pro(820) to Ala interferes with protein expression even though transcription occurs at normal levels. Single mutations of Lys(819) or Arg(822) to Ala, Phe, or Glu allow good expression, but produce strong inhibition of ATPase activity. The main defect produced by these mutations is strong interference with enzyme phosphorylation by ATP in the presence of Ca(2+), and also by P(i) in the absence of Ca(2+). The Lys(819) and Arg(822) mutants undergo slight and moderate reduction of Ca(2+) binding affinity, respectively. Reduction of overall steady state ATPase velocity is then due to inhibition of phosphorylated intermediate formation. On the other hand, a cluster of conservative mutations of Asp(813), Asp(815), and Asp(818) to Asn interferes strongly with enzyme activation by Ca(2+) binding and formation of phosphorylated enzyme intermediate by utilization of ATP. Enzyme phosphorylation by Pi in the absence of Ca(2+) undergoes slight or no inhibition by the triple aspartate mutation. Therefore, the triple mutation interferes mainly with the calcium-dependent activation of the ATPase. The effect of the triple mutation can be to a large extent reproduced by single mutation of Asp(813) (but not of Asp(815) or Asp(818)) to Asn. Functional and structural analysis of the experimental data demonstrates that the L67 loop plays an important role in protein folding and function. This role is sustained by linking the cytosolic catalytic domain and the transmembrane Ca(2+) binding domain through a network of hydrogen bonds.
Highlights
The sarcoplasmic reticulum (SR)1 ATPase is a membranebound enzyme that plays a crucial role in sequestration of cytosolic Ca2ϩ in muscle fibers [1,2,3]
The loops between the remaining transmembrane segments are relatively small, attention has been brought to L67 (Phe809–Gly831) by Falson et al [10] and Menguy et al [11], who found that cluster mutations of aspartic residues to alanine raise the Ca2ϩ concentration required for ATPase activation
It is of interest that Menguy et al [11] obtained expression of a totally inactive Pro820 3 Ala mutant. This suggests that mutation of Pro820 interferes profoundly with protein folding, resulting in degradation of product by the COS-1 cells used for expression in our experiments, and production of inactive protein by the yeast cells used by Menguy et al [11]
Summary
The sarcoplasmic reticulum (SR) ATPase is a membranebound enzyme that plays a crucial role in sequestration of cytosolic Ca2ϩ in muscle fibers [1,2,3]. Mutational [7] and structural studies [8] have shown that the Ca2ϩ binding domain resides within the membrane-bound region of the ATPase, at a 50-Å distance from the phosphorylation domain in the cytosolic region of the enzyme. The cytosolic region of the ATPase includes a short N-terminal segment (Met1–Glu58) and the loop (Trp107-Ser261) between the M2 and M3 transmembrane segments, folded in the A domain. The cytosolic region includes the large loop between the M4 and M5 transmembrane segments, folded in separate P (phosphorylation) and N (nucleotide binding) domains [8]. The loops between the remaining transmembrane segments are relatively small, attention has been brought to L67 (Phe809–Gly831) by Falson et al [10] and Menguy et al [11], who found that cluster mutations of aspartic residues to alanine raise the Ca2ϩ concentration required for ATPase activation.
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