Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
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Although mutational analysis always opens the possibility of indirect conformational effects, the Ca2+ binding defects produced by simple change of a single acidic residues to the corresponding amides provide strong evidence for direct involvement of those residues in Ca2+ binding. This is the case of Glu309, Glu771, Asp800 and Glu908. Asn796 and Thr799 are also likely to be involved, considering their proximity and the effects produced by their mutations. Asn796 and Thr799 may participate directly in Ca2+ complexation with their oxygens, or through hydrogen bonded water.

It is then apparent that the four transmembrane helices M4, M5, M6 and M8, contribute one or more residues each, to form a tight complex with two calcium ions, corresponding to the E1 state of the enzyme. The four amphiphilic helices are likely to form a cluster, stabilized by the two calcium ions which are bound within a virtual channel formed by the clustered helices. It is apparent from Fig 1B that the transmembrane helices are not actually perpendicular to the plane of the membrane, but rather form a slightly slanted bundle. Of the four helices, M8 appears to be least adherent to the bundle, presenting the greatest inclination with respect to the plane of the membrane (Zhang et al., 1998). It should be recognized that these details cannot be completely clarified until the SERCA structure is determined by high resolution diffraction studies. Nevertheless, the experimental observations described above demonstrate convincingly that the specific, high affinity Ca2+ binding domain resides within the membrane bound region of the SERCA protein.

Vectorial displacement of the bound Ca2+, coupled with enzyme phosphorylation by ATP, is the basic function of active transport. The ATP binding domain, however, resides within the extramembranous domain, at a distance in excess of 40 A from the cytosolic surface of the membrane (Bigelow and Inesi, 1992). It is clear that the catalytic chemistry of ATP utilization, including formation of the phosphorylated enzyme intermediate, occurs within the extramembranous domain, at a relatively large distance from the Ca2+ binding domain. Therefore, the two way signaling of catalytic activation by Ca2+ binding, and vectorial displacement of bound Ca2+ by enzyme phosphorylation, must be operated by a long range intramolecular linkage (Inesi et al., 1992). The peptide sequence intervening between phosphorylation site (Asp351) and Ca2+ binding domain (Glu309) is the S4-M4 segment which retains a very high sequence homology in most cation transport ATPases. Single amino acid mutations within this segment interfere with phosphoenzyme turnover (Zhang et al., 1995). Therefore, it is likely that the S4-M4 segment provides a common device for transmission of signals between phosphorylation and cation binding domains.

With regard to the mechanism of thapsigargin inhibition, interaction of the inhibitor with the S3 segment raises the possibility of a structural perturbation transmitted to the neighboring S4 segment (i.e., the linkage segment), and thereby interference with the long range linkage of catalysis and cation binding. It is noteworthy that while the S4 segment retains a high sequence homology in most cation transport ATPases (consistent with a common transduction mechanism), the S3 segment retains only minimal homology (consistent with specific interaction of SERCA with thapsigargin through this segment).

Demonstration of a long range intramolecular linkage among distant functional domains, places a requirement for the occurrence of protein conformational changes, to explain functional events triggered or inhibited by ligand occupancy of distant domains. Several spectroscopic studies have provided evidence of diverse conformational states of the enzyme in the presence and in the absence of Ca2+, even though major changes of secondary structure could not be observed (review: Bigelow and Inesi, 1992). Most importantly, images derived from recent diffraction studies show significant differences in both the catalytic and transmembrane domains, depending on whether the enzyme is placed in the Ca2+ binding state, or kept in a state of low Ca2+ binding affinity (Ogawa et al., 1998). Thapsigargin plays a role in stabilization of the enzyme in the latter state. It is then apparent that the Ca2+ ATPases, and most likely other cation ATPases, utilize allosteric mechanisms to couple their catalytic and transport functions, and to respond to a specific inhibitor such as thapsigargin.

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