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

Abstract

Time-resolved, terbium resonance x-ray diffraction experiments have provided the locations of three different high-affinity metal-binding/transport sites on the Ca2+ATPase enzyme in the profile structure of the sarcoplasmic reticulum (SR) membrane. By considering these results in conjunction with the known, moderate-resolution profile structure of the SR membrane (derived from nonresonance x-ray and neutron diffraction studies), it was determined that the three metal-binding sites are located at the "headpiece/stalk" junction in the Ca2+ATPase profile structure, in the "transbilayer" portion of the enzyme profile near the center of the membrane phospholipid bilayer, and at the intravesicular surface of the membrane profile. All three metal-binding sites so identified are simultaneously occupied in the unphosphorylated enzyme conformation. Phosphorylation of the ATPase causes a redistribution of metal density among the sites, resulting in a net movement of metal density toward the intravesicular side of the membrane, i.e., in the direction of calcium active transport. We propose that this redistribution of metal density is caused by changes in the relative binding affinities of the three sites, mediated by local structural changes at the sites resulting from the large-scale (i.e., long-range) changes in the profile structure of the Ca2+ATPase induced by phosphorylation, as reported in an accompanying paper. The implications of these results for the mechanism of calcium active transport by the SR Ca2+ATPase are discussed briefly.

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).…”

“…This site accounts for about 80% of the bound lanthanides. The remainder is bound with lower affinity near the phospholipid head groups on the cytoplasmic and lumenal membrane surface (Asturias & Blasie, 1991;Asturias et al, 1994a;Blasie et al, 1992). Tb 3+ , in contrast to La 3+ , also binds to the site in the center of the bilayer presumably in competition with Ca 2+ (Asturias et al, 1994a;Blasie et al, 1992).…”

“…The remainder is bound with lower affinity near the phospholipid head groups on the cytoplasmic and lumenal membrane surface (Asturias & Blasie, 1991;Asturias et al, 1994a;Blasie et al, 1992). Tb 3+ , in contrast to La 3+ , also binds to the site in the center of the bilayer presumably in competition with Ca 2+ (Asturias et al, 1994a;Blasie et al, 1992). Phosphorylation of Ca 2+ -ATPase induced by Tb 3+ causes the movement of metal density toward the intravesicular surface presumably due to changes in the affinity of the various metal binding sites (Asturias et al, 1994a).…”

“…Tb 3+ , in contrast to La 3+ , also binds to the site in the center of the bilayer presumably in competition with Ca 2+ (Asturias et al, 1994a;Blasie et al, 1992). Phosphorylation of Ca 2+ -ATPase induced by Tb 3+ causes the movement of metal density toward the intravesicular surface presumably due to changes in the affinity of the various metal binding sites (Asturias et al, 1994a).…”

Preparation and analysis of large, flat crystals of Ca(2+)-ATPase for electron crystallography

“…For example, a large number of spectroscopic studies have measured distances between probes on various chemically defined sites on the Ca2+-ATPase molecule (Bigelow and Inesi, 1992). Ongoing x-ray diffraction studies of partially oriented pellets of SR have determined the mass distribution across the membrane , have documented changes in this distribution upon phosphorylation , and most recently have located sites for lanthanides before and after phosphorylation (Asturias et al, 1994). In addition, three different crystal forms have been discovered (Dux and Martonosi, 1983;Dux et al, 1985Dux et al, , 1987 and have lead to several three-dimensional reconstructions by electron microscopy.…”