Roles of Conserved P Domain Residues and Mg2+ in ATP Binding in the Ground and Ca2+-activated States of Sarcoplasmic Reticulum Ca2+-ATPase

Abstract

Residues in conserved motifs 625 The sarcoplasmic reticulum Ca 2ϩ -ATPase 1 from rabbit muscle couples the hydrolysis of ATP to the transport of Ca 2ϩ to the reticular lumen, and the lowering of the sarcoplasmic Ca 2ϩ concentration leads to muscle relaxation. This ion pump is a well studied example of P-type ATPases, the catalytic cycle of which includes a phosphorylated aspartyl intermediate and progression through E1 and E2 states. ATP-dependent phosphorylation takes place upon Ca 2ϩ binding in the E1 form, and hydrolysis of the aspartyl phosphoryl bond occurs in the E2P phosphoenzyme conformation following Ca 2ϩ translocation across the membrane. Two atomic structures of the protein have been elucidated by x-ray crystallography, one in the presence of Ca 2ϩ (designated E1(Ca 2 )) and another in the absence of Ca 2ϩ and presence of the specific inhibitor thapsigargin (E2(TG)) (1, 2). They reveal 10 membrane-spanning helices, connected through a stalk section to the phosphorylation (P) domain in association with nucleotide (N) and actuator (A) domains. The three head domains are loosely attached in E1(Ca 2 ) and closer together, forming a more compact entity, in E2(TG). For ATP-dependent phosphorylation of Asp 351 to occur in the Ca 2ϩ bound E1 state, domain N must close over the phosphorylation site, such that the nucleotide site is brought close to Asp 351 . ATP likely straddles both domains, the nucleoside portion anchored in domain N and the phosphates stretching into domain P (cf. Fig. 1A). Domain P contains the highly conserved segments 625 TGD, 676 FARXXPXXK, and 701 TGDGVND that surround the phosphorylation loop 351 DKTGTLT. Many of these residues have been implicated in ATP binding and catalysis on the basis of chemical labeling and cross-linking experiments, and mutagenesis of Ca 2ϩ -ATPase and related pumps (for review, see Ref.3). Most of them recur at the active site of a large class of soluble phosphohydrolases and phosphotransferases believed to have a similar reaction mechanism (4).

Molecular mechanisms in therapy of acid-related diseases

“…This lysine substitutes for a serine present in the Na,K ATPase isoforms. Site-directed mutagenesis of residues of the Na,K ATPases [36,37], the SR Ca ATPase [38] and the H,K ATPase [27,35,39] showed that the ion-binding domain and other features of the gastric ATPase were similar to the Na,K and SERCA Ca ATPases.…”

Structural and functional analysis of the phosphoryl transfer reaction mediated by the human small C‐terminal domain phosphatase, Scp1

“…For example, it was reported that mutation at this residue in P-type ATPase (Asp707) totally abolishes its activity. 35 In summary, our current study provided definitive evidence for the existence of the phosphorylaspartate intermediate in the reaction catalyzed by Fcp/Scp phosphatase family. We identified Asp206 as a key residue for catalysis and metal binding, whose mutation can result in product trapping.…”

Biochemical basis of regulation of human copper-transporting ATPases

“…Therefore, the loss of transport activity in this mutant appears to be due to uncoupling between the catalytic and transport activity of ATP7A. Alternatively, or in addition, the marked decrease in copper transport for this mutant may be caused by a significant shift in conformation of ATP7A towards E1P form as was earlier observed for the D>A or D>N substitutions of the equivalent aspartate residue in some other P-type ATPases (90). Overall, the position of DxxK in a flexible linker connecting the N-and P-domain and the effect of D1230 mutation on ATP binding and transport activity suggest an important role for this Asp residue in regulating movement of the domains upon ATP binding.…”