Three-dimensional cryo-electron microscopy of the calcium ion pump in the sarcoplasmic reticulum membrane

Abstract

The ATP-driven calcium pump (Ca(2+)-ATPase) is an integral membrane protein (M(r) 110K) which relaxes striated muscle by pumping calcium out of the cytoplasm into the sarcoplasmic reticulum against a large concentration gradient. Recent efforts have attempted to relate the sequence of Ca(2+)-ATPase to its structure and function. In particular, site-directed mutagenesis has identified critical amino-acid residues, and its predicted secondary structure, which includes ten transmembrane helices, has gained experimental support. But direct visualization of the molecule has so far been limited to the cytoplasmic domains at low resolution. We present here the three-dimensional structure of Ca(2+)-ATPase in the native sarcoplasmic reticulum membrane at 14 A resolution, determined by cryo-electron microscopy and helical image analysis. The structure shows an unexpected transmembrane organization, consisting of three distinct segments, one of which is highly inclined. These features can be related to earlier predictions of secondary structure.

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

“…(15), and dashed line is a fit using an alternate model that is identical to the proposed model except SERCA is assumed to sequentially bind three protons instead of four. Calculated dependence of various SERCA species concentration on free calcium concentration [Ca 2+ ] at fixed values of pH (6,7,8) and free Mg 2+ (0, 0.1 and 10 mM) in the solution; the calculated curves are obtained by using Eqs. (1)- (11) and parameter values listed in Table 2.…”

“…The functional unit is a protein monomer comprising 994 amino acid residues. The sequence is folded into a cluster of 10 segments forming a transmembrane region, and three relatively large domains ("N", "P" and "A") protruding from the cytosolic surface of the membrane [5,6]. The ATPase cycle begins with high affinity binding of Ca 2+ derived from the cytosolic medium ("outside"), followed by ATP utilization to form a phosphorylated enzyme intermediate.…”

Topology of sarcoplasmic reticulum Ca2+‐ATPase: An infrared study of thermal denaturation and limited proteolysis

“…The structural basis for the study is the 14 A resolution picture obtained by cryoelectron microscopy (Toyoshima et al, 1993) and the structural model deduced from the sequences of several P-type ATPases together with other available data (Stokes et al, 1994). The transmembrane domain has 10 helical segments, whereas the cytoplasmic domain possesses a-helix and @-sheet structure.…”

“…The structure of sarcoplasmic reticulum Ca2+-ATPase has been predicted from the amino acid sequence (MacLennan et al, 1985) and from electron microscopy observations (Toyoshima et al, 1993). The protein appears to consist of an extensive beak-shaped cytoplasmic domain containing interconnected a-helical and P-strand segments, a stalk connecting the beak with the membrane, and the ten transmembrane segments characteristic of P-type ion pumps (Stokes et al, 1994).…”

“…Sarcoplasmic reticulum Ca2+-ATPase is an integral membrane protein that pumps calcium out of the cytoplasm during striated muscle relaxation (Martonosi, 1996). This ATPase is part of a family of P-type ion pumps that includes several cation-activated ATPases having in common ten predicted transmembrane helical segments (Stokes et al, 1994).The structure of sarcoplasmic reticulum Ca2+-ATPase has been predicted from the amino acid sequence (MacLennan et al, 1985) and from electron microscopy observations (Toyoshima et al, 1993). The protein appears to consist of an extensive beak-shaped cytoplasmic domain containing interconnected a-helical and P-strand segments, a stalk connecting the beak with the membrane, and the ten transmembrane segments characteristic of P-type ion pumps (Stokes et al, 1994).…”