ATP-binding Modes and Functionally Important Interdomain Bonds of Sarcoplasmic Reticulum Ca2+-ATPase Revealed by Mutation of Glycine 438, Glutamate 439, and Arginine 678

Abstract

ATP binds to sarcoplasmic reticulum Ca 2؉ -ATPase both in a phosphorylating (catalytic) mode and in a nonphosphorylating (modulatory) mode, the latter leading to acceleration of phosphoenzyme turnover (Ca 2 E 1 P 3 E 2 P and E 2 P 3 E 2 reactions) and Ca 2؉ binding (E 2 3 Ca 2 E 1 ). P, as well as ATP/MgATP binding in modulatory modes to E 2 P and E 2 , whereas the effects on ATP/ MgATP acceleration of the Ca 2 E 1 P 3 E 2 P transition were small, suggesting that the nucleotide that accelerates Ca 2 E 1 P 3 E 2 P binds differently from that modulating the E 2 P 3 E 2 and E 2 3 Ca 2 E 1 reactions. Mutation of Glu 439 hardly affected nucleotide binding to E 1 , Ca 2 E 1 P, and E 2 , but it led to disruption of the modulatory effect of ATP on E 2 P 3 E 2 and acceleration of the latter reaction, indicating that ATP normally modulates E 2 P 3 E 2 by interfering with the interaction between Glu 439 and Ser 186 . Gly 438 seems to be important for this interaction as well as for nucleotide binding, probably because of its role in formation of the helix containing Glu 439 and Thr 441 .The sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (1) is a membrane-bound energy transducer ("nanomotor") that couples ATP hydrolysis with Ca 2ϩ translocation against a concentration gradient by means of a reaction cycle (Scheme 1) in which the ATPase enzyme is transiently phosphorylated at a conserved aspartic acid residue and undergoes major conformational transitions between Ca 2 E 1 /Ca 2 E 1 P and E 2 /E 2 P forms (2, 3). In recent years, several high resolution crystal structures of the Ca 2ϩ -ATPase, each thought to represent a particular intermediate state in the pump cycle, have been determined (4 -11). The Ca 2ϩ -ATPase consists of a membrane-spanning domain of 10 mostly helical segments and a large cytoplasmic headpiece, comprising three distinct domains, named N (nucleotide binding), P (phosphorylation), and A (actuator) (4). In the Ca 2 E 1 and Ca 2 E 1 P conformations, the catalytic ATPbinding site is made up by residues in the N-and P-domains, and during the Ca 2 E 1 P 3 E 2 P transition the departing ADP molecule is replaced by the TGES loop of the A-domain, which subsequently assists in catalysis of E 2 P dephosphorylation (8,9,12). In addition to being the phosphorylating substrate in the E 1 state, ATP exerts modulatory effects on various steps of the Ca 2ϩ -ATPase cycle (boxed ATP in Scheme 1). Hence, the Ca 2 E 1 P 3 E 2 P, E 2 P 3 E 2 , and E 2 3 Ca 2 E 1 transitions are all accelerated by the binding of ATP or MgATP in a nonphosphorylating mode (13-23). The apparent affinity for the nucleotide is generally lower when it binds in the nonphosphorylating modulatory mode as compared with the phosphorylating mode, but it varies depending on the step being modulated. A subject of much controversy is the question whether the phosphorylating and modulatory ATP molecules are at the same locus, exhibiting variable affinity during the transport cycle depending on conformational state, or whether a separate low affinity allosteric s...

Roles of Interaction between Actuator and Nucleotide Binding Domains of Sarco(endo)plasmic Reticulum Ca2+-ATPase as Revealed by Single and Swap Mutational Analyses of Serine 186 and Glutamate 439

“…The mutations S186A, S186E, E439A, E439S, and S186A/E439A caused significant reduction of the activity, whereas the swap mutation S186E/E439S completely restored the wild-type activity. The result for E439A agrees with the previous finding by Clausen et al (29).…”

“…3). The result on E439A is consistent with the previous observation by Clausen et al (29). The activity was increased only slightly in E439S with MgATP and not increased at all in S186E.…”

“…As compared with the wild type, the rate was markedly increased in the alanine mutants S186A, E439A, S186A/E439A, and also in E439S at a lower extent. The result of E439A is consistent with the previous observation by Clausen et al (29). The rate was slightly decreased in S186E, in contrast to the marked increase in S186A and the Darier disease mutant S186F of SERCA2b (30).…”

“…The E1PCa 2 decay time courses of the wild type and mutants were apparently fitted to a single exponential kinetics but not strictly (data not shown). This complicated kinetics may be because Mg 2ϩ bound at the catalytic site was likely removed by the added EDTA in some E1PCa 2 fraction as previously also noted (29). Nevertheless, for simplicity, the single exponential rates estimated were plotted in Fig.…”

“…2 (27,28). The importance of the interdomain interaction between Arg 678 (P) and Asp 203 (A) in stabilizing the E2P and E2 intermediates and its influence on modulatory ATP activation were pointed out by the mutation R678A (29). Regarding the N domain, the importance of Glu 439 in the EP isomerization and E2P hydrolysis was previously noted by its alanine substitution, and possible importance of its interaction with Ser 186 on the A domain has been suggested since Glu 439 forms a hydrogen bond with Ser 186 in the E2P analog structures (29) (see Fig.…”