Effect of Ca2+ binding on the profile structure of the sarcoplasmic reticulum membrane using time-resolved x-ray diffraction

Abstract

A number of studies have indicated that Ca(2+)-ATPase, the integral membrane protein of the sarcoplasmic reticulum (SR) membrane, undergoes some structural change upon Ca2+ binding to its high affinity binding sites (i.e., upon conversion of the E1 to the CaxE1 form of the enzyme). We have used x-ray diffraction to study the changes in the electron density profile of the SR membrane upon high-affinity Ca2+ binding to the enzyme in the absence of enzyme phosphorylation. The photolabile Ca2+ chelator DM-nitrophen was used to rapidly release Ca2+ into the extravesicular spaces throughout an oriented SR membrane multilayer and thereby synchronously in the vicinity of the high affinity binding sites of each enzyme molecule in the multilayer. A critical control was developed to exclude possible artifacts arising from heating and non-Ca2+ photolysis products in the membrane multilayer specimens upon photolysis of the DM-nitrophen. Upon photolysis, changes in the membrane electron density profile arising from high-affinity Ca2+ binding to the enzyme are found to be localized to three different regions within the profile. These changes can be attributed to the added electron density of the Ca2+ bound at three discrete sites centered at 5, approximately 30, and approximately 67 A in the membrane profile, but they also require decreased electron density within the cylindrically averaged profile structure of the Ca(2+)-ATPase immediately adjacent (< 15 A) to these sites. The locations of these three Ca2+ binding sites in the SR membrane profile span most of the membrane profile in the absence of enzyme phosphorylation,in agreement with the locations of lanthanide (Tb3+ and La3+) binding sites in the membrane profile determined independently by using resonance x-ray diffraction.

Changes in the relative occupancy of metal-binding sites in the profile structure of the sarcoplasmic reticulum membrane induced by phosphorylation of the Ca2+ATPase enzyme in the presence of terbium: a time-resolved, resonance x-ray diffraction study

“…Independent evidence supporting our conclusion that the metal-binding/transport sites identified by our terbium resonance x-ray diffraction study are involved in active calcium transport is provided by the results from a separate, timeresolved x-ray diffraction study of calcium binding to the SR membrane (DeLong and Blasie, 1993). In that work, a calcium cage molecule (DM-nitrophen; Ellis-Davies and Kaplan, 1988) was used to synchronously release calcium in otherwise calcium-free, oriented SR membrane multilayer specimens, thereby allowing the study of the profile structure of the membrane for the transition from the calcium-free, unphosphorylated conformation of the enzyme to the calcium-bound, unphosphorylated conformation (i.e., E-> CaxE).…”

“…75* 100. 125. results from independent, time-resolved x-ray diffraction Z (A) studies of calcium binding to the ATPase (DeLong and URE 3 GFSDM-calculated resonant metal atom electron density pro- Blasie, 1993).…”

The structure of the Ca2+-ATPase of sarcoplasmic reticulum.

“…The absence of lanthanide binding in the membrane domain (Toyoshima et al, 2000) is consistent with earlier observations that most of the lanthanides are bound to Ca 2+ -ATPase in the stalk region and near the phospholipid head groups, outside the membrane domain (Asturias & Blasie, 1991;DeLong & Blasie, 1993;Asturias et al, 1994a).…”

“…These structural differences between Ca 2+free and Ca 2+ -ligated enzyme forms are consistent with the large structural changes seen in earlier X-ray diffraction studies on lamellar arrays of SR vesicles (DeLong et al, 1993).…”

“…Photolysis of the calcium chelator DM nitrophen can be used to rapidly release Ca 2+ in SR multilayers (DeLong & Blasie, 1993). Ca 2+ -binding to the SR in the absence of ATP caused increased electron density in three regions of the membrane profile, corresponding to the intravesicular membrane surface, the center of the bilayer and the junction of the stalk and headpiece regions of the cytoplasmic domain of the Ca 2+ -ATPase (DeLong & Blasie, 1993). The Ca 2+ -binding site in the center of the bilayer corresponds to the location identified by X-ray analysis of multilamellar Ca 2+ -ATPase crystals (Toyoshima et al, 2000).…”