Overlapping Effects of S3 Stalk Segment Mutations on the Affinity of Ca²⁺-ATPase (SERCA) for Thapsigargin and Cyclopiazonic Acid

Abstract

Chimeric exchanges and mutations were produced in the Ca(2+)-ATPase (SERCA) to match (in the majority of cases) corresponding sequences of the Na(+),K(+)-ATPase. The effects of these mutations on the concentration dependence of the specific Ca(2+)-ATPase inhibition by thapsigargin (TG) and cyclopiazonic acid (CPA) were then determined. Extensive chimeric mutations on the large cytosolic loop, on the S4 stalk segment, and on the M3 transmembrane segments produced little or no modification of the Ca(2+)-ATPase sensitivity to either inhibitor. On the other hand, the presence of a six amino acid Na(+), K(+)-ATPase sequence within the S3 stalk segment of the Ca(2+)-ATPase raised 60-fold the apparent K(i) for TG and 250-fold the apparent K(i) for CPA. More limited mutations within the same S3 segment, however, affected differently the concentration dependence of the Ca(2+)-ATPase inhibition by TG or CPA. Specifically, single mutation of Phe256 to Val increased 20-fold the apparent K(i) for TG, while having very little effect on the apparent K(i) for CPA. These findings indicate significant overlap of the TG and CPA binding domains within the S3 stalk segment of the Ca(2+)-ATPase, where the contribution of each protein residue is dependent on the structures of the two inhibitors. Saturating concentrations of either or both TG and CPA produce an identical reduction of the affinity of the ATPase for ATP, suggesting that only one inhibitor can bind at any time due to significant overlap of their binding domains. It is suggested that perturbations produced by binding of either inhibitor within the stalk segment interfere with the long-range functional linkage between ATP utilization in the ATPase cytosolic region and Ca(2+) binding in the membrane-bound region.

Pharmacological Modulation of Sarcoplasmic Reticulum Function in Smooth Muscle

“…Interestingly, in this respect, the fluorescent analog 8-O-(4-aminocinnamoyl)-8-O-debutanoylthapsigargin is almost as potent as thapsigargin in inhibiting 45 Ca 2ϩ uptake by rabbit SERCA1-expressing skeletal muscle microsomes (IC 50 ϭ 168 versus 123 nM, respectively), and its fluorescence is sensitive to the E 1 -E 2 conformational equilibrium, thus making it a conformational probe (Procida et al, 1998). This mechanism of action is also consistent with the localization of the thapsigargin-specific binding site on the SERCA within the S3 stalk segment, as shown in chimerical studies (Ma et al, 1999). The CPA-specific PHARMACOLOGICAL MODULATION OF SMOOTH MUSCLE SR binding site was also shown to significantly overlap it, making CPA a competitor of thapsigargin at the molecular level.…”

Interdomain communication in calcium pump as revealed in the crystal structures with transmembrane inhibitors

“…It binds to the ATPase in the E2 state and affects the enzymatic activity of Ca 2ϩ -ATPase in a way very similar to TG (21,22). Because of some structural similarity to TG, which binds to the space surrounded by the M3, M5, and M7 transmembrane helices (4), it has been supposed that they share the binding sites (21).…”

“…It binds to the ATPase in the E2 state and affects the enzymatic activity of Ca 2ϩ -ATPase in a way very similar to TG (21,22). Because of some structural similarity to TG, which binds to the space surrounded by the M3, M5, and M7 transmembrane helices (4), it has been supposed that they share the binding sites (21). Site-directed mutagenesis and chimera results have been interpreted to support this idea, although the most influential residue on TG binding, that is, Phe-256 on the M3 helix, affected the CPA binding only slightly (21,23).…”

“…Because of some structural similarity to TG, which binds to the space surrounded by the M3, M5, and M7 transmembrane helices (4), it has been supposed that they share the binding sites (21). Site-directed mutagenesis and chimera results have been interpreted to support this idea, although the most influential residue on TG binding, that is, Phe-256 on the M3 helix, affected the CPA binding only slightly (21,23). It has also been shown that a malarial Ca 2ϩ -ATPase (PfATP4) is sensitive to CPA but insensitive to TG (24).…”

Unravelling the Interaction of Thapsigargin with the Conformational States of Ca2+-ATPase from Skeletal Sarcoplasmic Reticulum

“…Location at the membrane interface and interaction with the S3 stalk segment between Asp 254 and Leu 260 have been suggested (14). The S3 segment has also been related with the binding domain of the high affinity inhibitor cyclopiazonic acid (15). In this sense, the region around Phe 256 , where the structural elements M3, M4, and L67 gather, seems to be critical for Ca 2ϩ binding (16).…”