Glutamate 90 at the Luminal Ion Gate of Sarcoplasmic Reticulum Ca2+-ATPase Is Critical for Ca2+ Binding on Both Sides of the Membrane

Abstract

(4 -7), an increasingly detailed picture of the structural changes relating to Ca 2ϩ transport by the Ca 2ϩ pump is steadily emerging. The Ca 2ϩ -ATPase consists of 10 membrane-spanning helices (M1 through M10) connecting three major cytoplasmic domains named A (actuator), P (phosphorylation), and N (nucleotide binding) and some smaller luminal loops. Transmembrane helices M4-M6 and M8 contain the residues that coordinate the two Ca 2ϩ ions bound side-by-side in a binding pocket in the Ca 2 E1 and Ca 2 E1P states. A key element in the transport cycle is the action of the A domain, which is directly linked to M1-M3 via flexible linkers (see Fig. 1). Thus, occlusion of the Ca 2ϩ ions at the high affinity sites of Ca 2 E1 is achieved through an ATPinduced ϳ30°tilting of the A domain and accompanying partial unfolding and bending of the N-terminal part of M1 (3). After transfer of the ␥-phosphate of ATP to Asp 351 , a further ϳ90°r otation of the A domain during the Ca 2 E1P 3 Ca 2 E2P transition propagates to the transmembrane domain, disrupting the high affinity Ca 2ϩ sites and exposing the Ca 2ϩ ions to the lumen. The subsequent release of the Ca 2ϩ ions to the lumen is succeeded by dephosphorylation of the aspartyl phosphate in E2P, catalyzed by the 181 TGES phosphatase motif of the A domain (8) that has been brought into position in the catalytic site in E2P (5, 6). The cycle is completed by reversal of the A domain rotation and restoration of the cytoplasmically facing high affinity Ca 2ϩ sites (E2 3 E1 in Scheme 1). The molecular nature and properties of the Ca 2ϩ binding pocket in the membrane-spanning region of the Ca 2 E1 and Ca 2 E1P states are known in great detail. Little, however, is known about the exact entry and exit pathways to and from the Ca 2ϩ binding pocket and the structural elements involved in extrusion of the Ca 2ϩ ions to the endoplasmic reticulum lumen.

Modulatory ATP Binding Affinity in Intermediate States of E2P Dephosphorylation of Sarcoplasmic Reticulum Ca2+-ATPase

“…The cDNA encoding the mutant Ca 2ϩ -ATPases studied in the present work was the same as that applied in our previous studies (16,17,(33)(34)(35). The cDNA was inserted into the expression vector pMT2 (36).…”

“…To form the Ca 2 E2P state of mutant 4Gi-46/47 (31,35), phosphorylation was carried out for 10 min at 25°C in 25 mM MOPS/tetramethyl ammonium hydroxide (pH 7.0), 10 mM MgCl 2 , 15% DMSO, 38 M calcium ionophore A23187, 1 mM EGTA, and 0.5 mM P i , followed by cooling on ice. Immediately prior to photolabeling, 2 l of the phosphorylated microsomes were supplemented with 2 l of ice-cold 42 mM CaCl 2 , to give a final free Ca 2ϩ concentration of 20.5 mM (on both the lumenal and the cytoplasmic sides of the membrane, because of the presence of the calcium ionophore).…”

Structure and mechanism of Zn2+-transporting P-type ATPases

“…3b), this hints at an additional role of Lys693 for electrostatic repulsion against re-entry of Zn 2+ , possibly further stimulated by Glu202 guiding Zn 2+ to the extracellular environment. The equivalent residues of Glu202 in SERCA and CopA (Glu90 and Glu189, respectively) have been proposed to serve a similar purpose 11,20 and supporting this notion, Glu202 is critical for function (Fig. 2a) 21 .…”

Structural identification of cation binding pockets in the plasma membrane proton pump

“…Recently, a low affinity Ca 2þ binding site was suggested in the luminal Ca 2þ exit pathway of the SR Ca 2þ -ATPase (42). This site involves Glu90 of the SR Ca 2þ -ATPase, which is homologous to Asp95 in the PM H þ -ATPase, part of the Ho 3þ cation binding site identified in this work.…”