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

Abstract

Refs. 1 and 2). The membrane-buried region of the Ca 2ϩ -ATPase is made up of 10 membrane spanning helices and is connected to a large cytoplasmic headpiece, which is further separated into three distinct domains, denoted A ("actuator"), P ("phosphorylation"), and N ("nucleotide binding"). Ca 2ϩ transport is achieved by means of a reaction cycle (Scheme 1) involving the formation and decay of a phosphorylated intermediate and extensive protein conformational changes between four major states, E1, E1P, E2P, and E2. The catalytic function in E1 (autokinase activity) and E2P (autophosphatase activity) as well as the movement of Ca 2ϩ ions across the membrane can be understood on the basis of the sequential gathering and displacement of certain conserved amino acid motifs of the N-and A-domains relative to the catalytic site in the P-domain and the coupling of these events to rearrangements of the transmembrane helices containing the Ca 2ϩ sites. In the E1 and E1P states, the highly conserved 181 TGES loop of the A-domain is distant from the catalytic center containing nucleotide binding residues and the phosphorylated Asp 351 of the P-domain, which in this condition is able to react with ATP/ADP. However, during the Ca 2 E1P 3 E2P transition the A domain rotates ϳ90°around an axis nearly perpendicular to the membrane, thereby moving the 181 TGES loop into close contact with the catalytic site, such that Glu 183 can catalyze dephosphorylation of E2P by hydrolysis (3-5). During the dephosphorylation, Glu 183 likely coordinates the water molecule attacking the aspartyl phosphoryl bond and withdraws a hydrogen.ATP in addition to being the substrate in the phosphorylation of the Ca 2 E1 state also functions in a non-phosphorylating mode (boxed ATP in Scheme 1), enhancing the rates of the steps involved in phosphoenzyme turnover (Ca 2 E1P 3 E2P and E2P 3 E2) as well as the E2 3 Ca 2 E1 transition of the dephosphoenzyme (6 -17). The mechanisms underlying these modulatory effects of ATP remain largely unresolved. A debated issue is whether the modulatory ATP molecule binds at the same site as the phosphorylating ATP or at a distinct, "allosteric" site (14, 18 -23). During dephosphorylation the 181 TGES loop of the A-domain occupies the position in close contact with the P-domain taken up by part of the ATP and ADP in Ca 2 E1 and Ca 2 E1P, however, ATP may still bind to residues of the N-and

Critical Roles of Interdomain Interactions for Modulatory ATP Binding to Sarcoplasmic Reticulum Ca2+-ATPase

“…The concentration of expressed Ca 2ϩ -ATPase was determined by an enzyme-linked immunosorbent assay (37) and by determination of the maximum capacity for phosphorylation with ATP or P i ("active site concentration" (38) The enzyme-inhibitor complexes were formed immediately prior to the initiation of the photolabeling experiments and were kept on ice throughout (Ͻ1 h). The high stability of the E2⅐AlF, E2⅐BeF, and E2⅐orthovanadate complexes during the course of the photolabeling experiments has been documented previously (17).…”

“…Table 3. The binding site for TNP-8N 3 -ATP overlaps sufficiently with the binding site for modulatory ATP in the E2 states to allow competition binding assays in which the affinity for modulatory ATP is obtained from the ATP concentration dependence of inhibition of TNP-8N 3 -ATP photolabeling (17). Results of such competition experiments are shown in Fig.…”

“…The cDNA encoding mutants E439A and R678A was the same as used previously (17,28). To express wild type and mutant cDNA, COS-1 cells were transfected using the calcium phosphate precipitation method (35).…”

“…Like the wild type enzyme, the mutants formed stable complexes of the E2 state with the metal fluorides, as proved by measuring the phosphorylation from [␥- Because of the mutually exclusive binding of these metal fluoride phosphoryl analogs and the ␥-phosphate of ATP at the phosphorylation site, and the low rate of dissociation of the metal fluorides, the 32 P incorporation determined under these conditions was inhibited. Hence, as demonstrated below, we were able to determine the affinity of these complexes for the modulatory ATP by use of our previously validated TNP-8N 3 -ATP photolabeling assay (13,17). Table 3.…”

“…Some of the residues that contribute to ATP binding in the phosphorylating mode in E1 are also involved in ATP binding in the modulatory mode, in particular N-domain residues. When the A-domain approaches the P-domain in E2 and E2P-like states, the N-domain is partially displaced, leading to a less compact structure of the ATP site and affinities for modulatory ATP 10 -100-fold lower than the affinity of E1 for phosphorylating ATP (17). The apparent affinity for ATP binding to E2P is further lowered in the presence of Mg 2ϩ , because only free ATP and not MgATP can bind to this state, which already contains Mg 2ϩ bound with the phosphoryl group (26,27).…”