Concerted conformational effects of Ca2+ and ATP are required for activation of sequential reactions in the Ca2+ ATPase (SERCA) catalytic cycle

Abstract

We relate solution behavior to the crystal structure of the Ca2+ ATPase (SERCA). We find that nucleotide binding occurs with high affinity through interaction of the adenosine moiety with the N domain, even in the absence of Ca2+ and Mg2+, or to the closed conformation stabilized by thapsigargin (TG). Why then is Ca2+ crucial for ATP utilization? The influence of adenosine 5'-(beta,gamma-methylene) triphosphate (AMPPCP), Ca2+, and Mg2+ on proteolytic digestion patterns, interpreted in the light of known crystal structures, indicates that a Ca2+-dependent conformation of the ATPase headpiece is required for a further transition induced by nucleotide binding. This includes opening of the headpiece, which in turn allows inclination of the "A" domain and bending of the "P" domain. Thereby, the phosphate chain of bound ATP acquires an extended configuration allowing the gamma-phosphate to reach Asp351 to form a complex including Mg2+. We demonstrate by Asp351 mutation that this "productive" conformation of the substrate-enzyme complex is unstable because of electrostatic repulsion at the phosphorylation site. However, this conformation is subsequently stabilized by covalent engagement of the -phosphate yielding the phosphoenzyme intermediate. We also demonstrate that the ADP product remains bound with high affinity to the transition state complex but dissociates with lower affinity as the phosphoenzyme undergoes a further conformational change (i.e., E1-P to E2-P transition). Finally, we measured low-affinity ATP binding to stable phosphoenzyme analogues, demonstrating that the E1-P to E2-P transition and the enzyme turnover are accelerated by ATP binding to the phosphoenzyme in exchange for ADP.

Fig 1

Conformational states of the Ca²⁺-ATPase in the presence and absence of Ca²⁺, substrate and product analogs, based on the coordinates deposited in PDB ID:1SU4 (E1·2Ca²⁺; ref. 8), 1VFP (E1·AMPPCP (10)), 1XP5 (E2·AlF_4⁻(TG) (13)), and 2DQS and 2D88 (E2(TG) ·ATP, (14)). ATP in E1·2Ca²⁺ and ADP in E2·AlF_4⁻(TG) are docked by fitting the N-domain of E1·AMPPCP and E2·MgF₄²⁻(TG) (PDB ID: 1WPG (11)). Color changes gradually from the N-terminus (blue) to the C-terminus (red). The two Ca²⁺ (I and II) bound to the high affinity transmembrane sites are circled when present. Three key residues (E183 in the A domain, D351 and D703 in the P domain) are shown in ball-and-stick. Note the positional changes of headpiece domains in the various conformations, as well as the proteinase K sites (30) in the loops connecting the A domain to the M2 and M3 helices. Note the nucleotide binding to the N domain, and the variable relationship of the nucleotide phosphate chain (and Mg²⁺) with the P and A domains.

Fig 2. ATP binding to the ATPase in the absence of Ca²⁺

A: In the presence (●) and in the absence (○) of Mg²⁺. B: in the presence of Mg²⁺ (●), and following addition of 1 μM TG (○) or 100 μM CPA (▲). ATP was added rapidly to the medium at 2 °C, and the mixture was filtered after 10 seconds incubation as described in METHODS.

Fig 2. ATP binding to the ATPase in the absence of Ca²⁺

A: In the presence (●) and in the absence (○) of Mg²⁺. B: in the presence of Mg²⁺ (●), and following addition of 1 μM TG (○) or 100 μM CPA (▲). ATP was added rapidly to the medium at 2 °C, and the mixture was filtered after 10 seconds incubation as described in METHODS.

Fig 3. ATP utilization for formation of phosphorylated enzyme intermediate

A: Time dependence. B: ATP concentration dependence. The experiments were conducted at 2 °C by adding ATP (10 μM in A, as specified in B), and quenching at the time specified in A, or after 10 seconds incubation in B. Reaction mixture given in METHODS.

Fig 3. ATP utilization for formation of phosphorylated enzyme intermediate

A: Time dependence. B: ATP concentration dependence. The experiments were conducted at 2 °C by adding ATP (10 μM in A, as specified in B), and quenching at the time specified in A, or after 10 seconds incubation in B. Reaction mixture given in METHODS.

Fig 4. Ca²⁺ requirement for enzyme phosphorylation by ATP

The reaction was started by addition of 10 μM ATP to enzyme preincubated with Ca²⁺ (●), addition of 10 μM ATP and 1 mM Ca²⁺ to enzyme preincubated with 1 mM EGTA (○), addition of 1 mM Ca²⁺ to enzyme preincubated with 1 mM EGTA and 10 μM ATP (▲), and addition of 10 μM ATP to enzyme preincubated with 1 mM EGTA in the absence of added Ca²⁺ (□). Reaction conducted as described in METHODS, at 2 °C.

Fig 5. Partial digestion of ATPase with Proteinase K. Effects of Ca²⁺, Mg²⁺ and AMPPCP

Digestion with Proteinase K and electrophoresis as explained in METHODS, using 0.04 mg ProtK/ml, for 10 minutes. The experiment shows that the ATPase band is optimally protected by AMPPCP when both Ca²⁺ and Mg²⁺ are present (compare lane 5 with 9).

Fig 6. Partial digestion of ATPase with Proteinase K. Protection by AMPPCP and concentration dependence

Digestion with Proteinase K and electrophoresis as explained in METHODS, using 0.015 mg ProtK/ml, for 40 minutes. 20 μM Ca²⁺ and 1 mM Mg²⁺ present in all samples. The experiment shows that protection by AMPPCP requires higher concentration when WT ATPase is used, as compared with the Asp351Ala mutant.

Fig 7. ADP binding to the ATPase and to the fluoroaluminate analog of the phosphorylated enzyme intermediate, in the absence and in the presence of Ca²⁺

(¹⁴C)-ADP was mixed into the reaction mixture and incubated for 30 minutes at 20 °C and then filtered. Control with no fluroaluminate or Ca²⁺ (○). Control with no fluoroaluminate, but with 20 μM Ca²⁺ (●). Fluoroaluminate with no Ca²⁺ (□). Fluoroaluminate with 20 μM Ca²⁺ (■).

Fig 8. ATP binding to the fluoroaluminate analog of the phosphorylated enzyme intermediate

SR vesicles were incubated for 30 minutes with 2 mM KF and 100 μM AlCl₃ at 2 °C, as described in METHODS. ATP was added rapidly to the medium at 2 °C, and the mixture was filtered after 10 seconds incubation. Control with no fluroaluminate or Ca²⁺ (●). Fluoroaluminate with no Ca²⁺ (▲). Fluoroaluminate with 20 μM Ca²⁺ (□).

Fig 9. Decay of radioactive phospoenzyme obtained with ATP or Pi

Decay of phosphoenzyme made with ATP was initiated by a chase with 10 mM EGTA (●) or 1 mM non-radioactive ATP and 10 mM EGTA(■). Decay of phosphoenzyme made with Pi was initiated by a chase with 50 mM Pi (▲) or 50 mM Pi and 1 mM ATP (▼). In both cases the temperature of the chase was 2 °C. The time curve following the EGTA chase (●) includes additional points up to 15 seconds, which were used for exponential fitting, and are not included in the graphics curve to avoid excessive compression of the time scale for the other curves.

Fig 10. Partial digestion of ATPase with Proteinase K. Effect of TG

Digestion with Proteinase K and electrophoresis as explained in METHODS, using 0.04 mg ProtK/ml. The reaction mixture contained 1.0 mM EGTA, 10 μM Ca²⁺ (endogenous), and 1.0 μM TG when indicated. The time of incubation with Proteinase K was for 0 (1), 10 (2), 20 (3), 30 (4), 40 (5), 50 (6), 60 (7) and 90 (8) minutes.

Fig 11. Arrangement of the cytoplasmic domain in the E2-AMPPCP(TG) (2DQS) and E1-AMPPCP-2Ca²⁺ (1VFP) crystal structures

The structures are superimposed by fitting the N-domain, which shows little conformational changes between the two structures. The A-domain movement between the two states is largely approximated by a 110° rotation (indicated by dotted arrows in (a) around an axis (a: thin red rod; b: double circles) nearly perpendicular to the membrane. Views are along the rotation axis (a) and normal to it (b). The residues forming hydrogen bonds between A- and N-domains are clustered (dotted circles) and shown in stick representation. Only E486 is involved in either structure.

FIG 12. Alignment of key residues and substrate in AMPPCP-E1-2Ca²⁺ (1VFP) and ADP-E1-AlFx-2Ca²⁺ (1WPE)

Amino acid residues are shown in atom color, AMPPCP and Mg²⁺ in red for AMPPCP-E1-2Ca²⁺; amino acid residues, ADP, aluminum fluoride and Mg²⁺ are shown in blue for ADP-E1-AlFx-2Ca²⁺. Not withstanding a nearly identical conformation, AMPPCP-E1-2Ca²⁺ is unstable in solution, while ADP-E1-AlFx-2Ca²⁺ is stable (see text).