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

Abstract

Roles of hydrogen bonding interaction between Ser 186 of the actuator (A) domain and Glu 439 of nucleotide binding (N) domain seen in the structures of ADP-insensitive phosphorylated intermediate (E2P) of sarco(endo)plasmic reticulum Ca2؉ -ATPase were explored by their double alanine substitution S186A/E439A, swap substitution S186E/E439S, and each of these single substitutions. All the mutants except the swap mutant S186E/E439S showed markedly reduced Ca 2؉ -ATPase activity, and S186E/E439S restored completely the wild-type activity. In all the mutants except S186E/E439S, the isomerization of ADP-sensitive phosphorylated intermediate (E1P) to E2P was markedly retarded, and the E2P hydrolysis was largely accelerated, whereas S186E/E439S restored almost the wildtype rates. Results showed that the Ser 186 -Glu 439 hydrogen bond stabilizes the E2P ground state structure. The modulatory ATP binding at sub-mMϳmM range largely accelerated the EP isomerization in all the alanine mutants and E439S. In S186E, this acceleration as well as the acceleration of the ATPase activity was almost completely abolished, whereas the swap mutation S186E/E439S restored the modulatory ATP acceleration with a much higher ATP affinity than the wild type. Results indicated that Ser 186 and Glu 439 are closely located to the modulatory ATP binding site for the EP isomerization, and that their hydrogen bond fixes their side chain configurations thereby adjusts properly the modulatory ATP affinity to respond to the cellular ATP level.Sarcoplasmic reticulum Ca 2ϩ -ATPase (SERCA1a) 2 is a representative member of P-type ion-transporting ATPases and catalyzes Ca 2ϩ transport coupled with ATP hydrolysis (Fig. 1) (1-9). In the catalytic cycle, the enzyme is activated by binding of two Ca 2ϩ ions at the transport sites (E2 to E1Ca 2 , steps 1-2) and then autophosphorylated at Asp 351 with MgATP to form ADP-sensitive phosphoenzyme (E1P, step 3), which can react with ADP to regenerate ATP. Upon formation of this EP, the bound Ca 2ϩ ions are occluded in the transport sites (E1PCa 2 ). The subsequent isomeric transition to ADP-insensitive form (E2P) results in a change in the orientation of the Ca 2ϩ binding sites and reduction of their affinity, and thus Ca 2ϩ release into lumen (steps 4 and 5). Finally, the hydrolysis takes place and returns the enzyme into an unphosphorylated and Ca 2ϩ -unbound form (E2, step 6). E2P can also be formed from P i in the presence of Mg 2ϩ and the absence of Ca 2ϩ by reversal of its hydrolysis.The cytoplasmic three domains N, A, and P largely move and change their organization states during the Ca 2ϩ transport cycle (10 -22). These changes are linked with the rearrangements in the transmembrane helices. In the EP isomerization (loss of ADP sensitivity) and Ca 2ϩ release, the A domain largely rotates (by ϳ110°parallel to membrane plane), intrudes into the space between the N and P domains, and the P domain largely inclines toward the A domain. Thus in E2P, these domains produce the most compactly organized state (see Fi...

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

“…6, 8, and 9), the Gln 202 side chain amide is within hydrogen bonding distance (ϳ3 Å) of the backbone carbonyl of Gly 626 , which is a critical residue at the catalytic site (15,44 186 and Glu 439 substantially increased the basal E2P dephosphorylation rate determined in the absence of ATP, and the swap mutation S186E/E439S almost restored the rate to a wild type-like level. These findings are in accordance with those of Liu et al (30), who from analysis of the E2⅐BeF crystal structures (9,45) pointed to a role for the hydrogen bond between Ser 186 and Glu 439 in stabilization of the interaction between domains A, N, and P in the E2P ground state. The pK a of the introduced glutamate in S186E was estimated to be extremely high, consistent with protonation and hydrogen bond formation between this glutamate and Glu 439 , thereby explaining the minimal effect of the mutation S186E (30).…”

“…These findings are in accordance with those of Liu et al (30), who from analysis of the E2⅐BeF crystal structures (9,45) pointed to a role for the hydrogen bond between Ser 186 and Glu 439 in stabilization of the interaction between domains A, N, and P in the E2P ground state. The pK a of the introduced glutamate in S186E was estimated to be extremely high, consistent with protonation and hydrogen bond formation between this glutamate and Glu 439 , thereby explaining the minimal effect of the mutation S186E (30). Structural modeling suggested that in the swap mutant, serine and glutamate may still be able to interact by hydrogen bonding in the E2⅐BeF state, thus explaining the gain of function seen for S186E/E439S with respect to attaining a wild type-like basal E2P dephosphorylation rate.…”

“…The present study, furthermore, encompasses mutant S186P, which was found to display the highest basal E2P dephosphorylation rate of the Ser 186 mutants, likely a consequence of the influence of the unique properties of proline on the backbone conformation of the 181 TGES loop. Also in accordance with Liu et al (30) we found that S186A, S186E, and E439S, as well as the S186E/E439S swap mutation, abolished the stimulatory effect of ATP on E2P dephosphorylation ( Fig. 3 and Table 2).…”

Nucleotide Binding in an Engineered Recombinant Ca2+-ATPase N-Domain

“…Thereafter, the N-domain internal dynamics would serve to position and attain the linear conformation of ATP required for γ-phosphate transfer to the P-domain . Interestingly, molecular interaction of ATP in binding mode I is similar to that observed in Ca 2+ -ATPase in complex with the inhibitor thapsigargin and Mg 2+ in the absence of Ca 2+ (PDB entry 3AR4), except that the polyphosphate tail of ATP in a complex with Mg 2+ interacts with Glu439 on the opposite site. − With regard to Ca 2+ -ATPase function, it has been reported that mutations Thr441Ala, Glu442Ala, Lys515Ala, Arg560Leu, Arg560Val, Arg560Glu, Cys561Ala, and Leu562Phe significantly decrease ATPase activity, the rate of phosphorylation, and the rate of Ca 2+ transport . Interestingly, Cys561Ala and Cys561Trp mutations decrease ATP binding affinity and generate only a slight decrease (∼20%) in the rate of Ca 2+ transport. , Cys561 also seems to be involved in protein stability as the recombinant N-domain Cys561Ala mutant shows a tendency to quickly precipitate …”

Conformational Transitions and Alternating-Access Mechanism in the Sarcoplasmic Reticulum Calcium Pump

“…The most detailed study of inter-domain interaction was conducted on the Glu439-Ser186 contact; mutations of one or both residues were shown to affect the E1P to E2P transition [59, 60]. But the swap mutation (E439S/S186E) was found to retain normal behavior, clearly indicating the importance of this interaction.…”