Glycine 105 as Pivot for a Critical Knee-like Joint between Cytoplasmic and Transmembrane Segments of the Second Transmembrane Helix in Ca2+-ATPase

Abstract

The cytoplasmic actuator domain of the sarco(endo)plasmic reticulum Ca²⁺-ATPase undergoes large rotational movements that influence the distant transmembrane transport sites, and a long second transmembrane helix (M2) connected with this domain plays critical roles in transmitting motions between the cytoplasmic catalytic domains and transport sites. Here we explore possible structural roles of Gly¹⁰⁵ between the cytoplasmic (M2c) and transmembrane (M2m) segments of M2 by introducing mutations that limit/increase conformational freedom. Alanine substitution G105A markedly retards isomerization of the phosphoenzyme intermediate (E1PCa₂ → E2PCa₂ → E2P + 2Ca²⁺), and disrupts Ca²⁺ occlusion in E1PCa₂ and E2PCa₂ at the transport sites uncoupling ATP hydrolysis and Ca²⁺ transport. In contrast, this substitution accelerates the ATPase activation (E2 → E1Ca₂). Introducing a glycine by substituting another residue on M2 in the G105A mutant (i.e. "G-shift substitution") identifies the glycine positions required for proper Ca²⁺ handling and kinetics in each step. All wild-type kinetic properties, including coupled transport, are fully restored in the G-shift substitution at position 112 (G105A/A112G) located on the same side of the M2c helix as Gly¹⁰⁵ facing M4/phosphorylation domain. Results demonstrate that Gly¹⁰⁵ functions as a flexible knee-like joint during the Ca²⁺ transport cycle, so that cytoplasmic domain motions can bend and strain M2 in the correct direction or straighten the helix for proper gating and coupling of Ca²⁺ transport and ATP hydrolysis.

Keywords: calcium ATPase; domain motion; enzyme kinetics; enzyme mechanism; enzyme mutation; enzyme structure; glycine; phosphoenzyme intermediate; sarco(endo)plasmic reticulum; transmembrane helix.

FIGURE 1.

Reaction cycle and structural changes of Ca²⁺-ATPase. A, the structural changes are modeled on the crystal structures E1Ca₂·AlF₄⁻·ADP as the E1∼P·ADP·Ca₂^‡ analog (PDB entry 1T5T (10)), E2·BeF₃⁻ as the E2P ground state analog (7) (PDB 2ZBE (11)), E2·AlF₄⁻(TG) as the transition state (E2∼P^‡) analog for E2P hydrolysis (7) (PDB 2ZBG (11)), E2(TG) as the E2 state fixed with thapsigargin (PDB 1IWO (6)), E1Mg²⁺ (PDB 3W5A (14)), and E1Ca₂ (PDB 1SU4 (4)). The structures are aligned with the static M8–M10 helices. The nucleotide binding (N), phosphorylation (P), and actuator (A) domains and M1–M6 helices are colored as indicated. The approximate position of membrane is shown by light green lines. The binding sites for two Ca²⁺ (purple spheres) consist of residues on M4, M5, M6, and M8. The yellow, pink, and red arrows indicate the approximate motions of the A and P domains and M2 (depicted in red), respectively, to the next structural state during the EP processing (isomerization and hydrolysis) and the E2 → E1 transition. The light blue arrow indicates the unwound M2 part. B, the α-carbon of residues on M2 mutated in this study is indicated in the crystal structure E1Ca₂·AlF₄⁻·ADP. Ca²⁺ ligand Glu³⁰⁹, which is occluding Ca²⁺ as a closed cytoplasmic gate, and Leu⁶⁵ on M1, which is fixing the Glu³⁰⁹ side chain configuration for the Ca²⁺ occlusion, are also depicted. The position of Gly¹⁰⁵ is indicated by a pink triangle.

FIGURE 2.

Total amount of EP (EP_total) at steady state and E2P fraction. Microsomes expressing wild type or mutants were phosphorylated with [γ-³²P]ATP at 0 °C for 30 s in 50 μl of a mixture containing 1–5 μg of microsomal protein, 10 μm [γ-³²P]ATP, 1 μm A23187, 0.1 m KCl, 7 mm MgCl₂, 10 μm CaCl₂, and 50 mm MOPS/Tris (pH 7.0). The EP_total formed (open bars) was determined by acid quenching. For determination of ADP-insensitive EP (E2P, closed bars), an equal volume of a mixture containing 2 mm ADP, 1 μm A23187, 0.1 m KCl, 7 mm MgCl₂, 10 mm EGTA, and 50 mm MOPS/Tris (pH 7.0) was added to the above phosphorylation mixture, and the reaction was quenched at 1 s after the ADP addition. ADP-sensitive EP (E1P) disappeared entirely within 1 s after the ADP addition. Error bars, S.D.

FIGURE 3.

Ca²⁺-ATPase activity and oxalate-dependent Ca²⁺ transport activity. A, the activities of the expressed SERCA1a mutants were determined as described under “Experimental Procedures” and shown as the values relative to the respective wild-type activities (ATP hydrolysis, 47.8 ± 1.8 nmol P_i/min/mg microsomal protein (n = 5); oxalate-dependent Ca²⁺ transport, 12.7 ± 0.5 nmol Ca²⁺/min/mg microsomal protein (n = 5)). Typical time courses of P_i liberation and Ca²⁺ accumulation in the wild type and mutant G105A are shown in the inset. B, the ratio, Ca²⁺ transport activity per Ca²⁺-ATPase activity (Ca²⁺/ATP) is shown as the percentage of the wild-type ratio. The wild type (light gray), G105A and G-substitution mutant (open), G-shift substitution mutant (closed), and GG-substitution mutant (lattice) are shown as indicated. C and D, M2 is viewed from a direction parallel to the membrane plane (C) or from the position indicated by the red arrow in C (D) in the crystal structure as indicated (E1Ca₂·AlF₄⁻·ADP). The effect of G-shift substitution of residues on Ca²⁺/ATP in B is visualized with α-carbon coloring as follows. Green, wild type (Gly¹⁰⁵)-like coupled transport; red, <36% of wild type (severely uncoupled). Error bars, S.D.

FIGURE 4.

EP isomerization. A, microsomes expressing wild type or mutant were first phosphorylated with [γ-³²P]ATP at 0 °C for 30 s as in Fig. 2. The phosphorylation reaction was terminated at zero time by Ca²⁺ removal by the addition of an equal volume of a buffer containing 10 mm EGTA, 0.1 m KCl, 7 mm MgCl₂, and 50 mm MOPS/Tris (pH 7.0) at 0 °C, and the amounts of EP were determined at the indicated times. Note that the wild type and all of the mutants accumulate mostly E1P at steady state (Fig. 2); therefore, the rate-limiting E1P to E2P isomerization rate was determined by EP decay kinetics. The inset shows typical examples; solid lines show the least squares fit to a single exponential, and the E1P decay rates thus obtained are shown in the main panel. Bars are colored as in Fig. 3. B and C, the effects of G-shift substitution of residues in A are visualized with α-carbon coloring on M2. Green, rate >0.060 s⁻¹, which is comparable with or higher than the wild-type (Gly¹⁰⁵) rate, 0.078 ± 0.015 (n = 4) s⁻¹); red, rate <0.025 s⁻¹ (i.e. marked retardation). M2 is viewed as in Fig. 3, C and D. Error bars, S.D.

FIGURE 5.

E2P hydrolysis. A, microsomes expressing wild type or mutant were phosphorylated with ³²P_i at 25 °C for 10 min in 5 μl of a mixture containing 1–5 μg of microsomal protein, 0.1 mm ³²P_i, 1 μm A23187, 0.2 mm EGTA, 7 mm MgCl₂, 50 mm MOPS/Tris (pH 7.0), and 30% (v/v) Me₂SO. The mixture was then cooled and diluted at 0 °C by the addition of 95 μl of a mixture containing 2.1 mm non-radioactive P_i, 105 mm KCl, 7 mm MgCl₂, 50 mm MOPS/Tris (pH 7.0), and 3 mm EGTA, and E2P hydrolysis was followed. Typical time courses of E2P hydrolysis are shown with the wild type and some mutants in the inset. The amounts of E2P formed with ³²P_i at zero time are normalized to 100%. Solid lines show the least squares fit to a single exponential, and the rates thus obtained are in the main panel. Bars are colored as in Fig. 3. B and C, the effects of G-shift substitution of residues in A are visualized with α-carbon coloring on M2 in E2·AlF₄⁻(TG) structure. Green, rate >0.25 s⁻¹, which is comparable with or higher than the wild type (Gly¹⁰⁵) rate, 0.59 ± 0.08 (n = 6) s⁻¹; yellow, 0.25–0.15 s⁻¹ (i.e. moderate retardation); red, <0.15 s⁻¹ (i.e. marked retardation). Error bars, S.D.

FIGURE 6.

EP formation from E2 and E1Ca₂ states. A, microsomes expressing wild type or mutant were preincubated for 20 min at 25 °C in 50 μl of a mixture containing 1–5 μg of microsomal protein, 1 μm A23187, 0.1 m KCl, 7 mm MgCl₂, 50 mm MOPS/Tris (pH 7.0), and 1 mm EGTA with and without 1.2 mm CaCl₂ to form the Ca²⁺-bound (open bar, E1Ca₂) and unbound (gray bar, E2) states, respectively. After cooling, an equal volume of a phosphorylation mixture containing 10 μm [γ-³²P]ATP and 1 mm EGTA with 1.2 or 2.4 mm CaCl₂ (to give 0.2 mm Ca²⁺ for both Ca²⁺-bound and Ca²⁺-unbound states in the above preincubation), otherwise as above, was added at 0 °C, and the EP formation time course was followed. Typical examples are shown with the wild type and the mutant G105A in the inset. Solid lines, least squares fit to a single exponential; the rates thus determined are shown in the main panel. The Ca²⁺-unbound state was denoted as “E2” for simplicity, and the ratio of the two rates is shown in B. Bars are colored as in Fig. 3. C and D, residues of which G-substitution severely retarded the E2 → E1 transition rate are highlighted (*). The effects of G-shift substitution of residues on the ratio (i.e. on the rate-limiting E2 → E1 transition in B) are visualized with α-carbon coloring on M2 in the E1Mg²⁺ structure. Green, ratio 28–37%, comparable with the wild type 31.4 ± 2.4% (n = 4); red, 66–93% (i.e. marked acceleration); blue, <3% (i.e. marked retardation). Error bars, S.D.

FIGURE 7.

Ca²⁺ occlusion in E1PCa₂ state. A, phosphorylation was performed with ATP in 10 μm ⁴⁵Ca²⁺ or with [γ-³²P]ATP in 10 μm Ca²⁺, otherwise as in Fig. 2, in which EP accumulated is mostly E1P. The amounts of occluded Ca²⁺ and EP_total were determined as described under “Experimental Procedures,” and the amount of Ca²⁺ occluded in EP is shown as the relative value (mean ± S.D. (n = 10–20)). Bars are colored as in Fig. 3. B and C, the effect of G-shift substitution of residues on the Ca²⁺ occluded/EP_total in A are visualized with α-carbon coloring on M2 in the E1Ca₂·AlF₄⁻·ADP structure. Green, 1.7–1.9 (i.e. occlusion as in the wild type (Gly¹⁰⁵)); red, <0.9 (i.e. severe disruption of the occlusion). Error bars, S.D.

FIGURE 8.

Ca²⁺ occlusion in transient E2PCa₂ state. A, to trap the transient E2PCa₂ state, the A/M1′-linker was elongated by four-glycine insertion between Gly⁴⁶ and Lys⁴⁷ (4Gi-46/47) in the indicated M2 mutants explored in this study. The microsomes expressing the wild type, these M2 mutants with the elongated A/M1′-linker (+4Gi-46/47 as indicated), or the A/M1′-linker elongated mutant without M2 mutation (4Gi-46/47) were phosphorylated with [γ-³²P]ATP, and the amounts of EP_total (open bars) and E2P (closed bars) were determined as described in the legend to Fig. 2. B, microsomes expressing wild type or mutants were first phosphorylated with [γ-³²P]ATP at 0 °C for 30 s as in A. Then the EP decay rate was determined and shown as in Fig. 4. Note that the A/M1′-linker elongation causes almost exclusive E2P accumulation (A) and almost completely blocks its decay (B). C, the amount of Ca²⁺ occluded in EP_total was determined and shown as the mean ± S.D. (n = 10–20), otherwise as in Fig. 7. D and E, the effect of G-shift substitution of residues on the Ca²⁺-occluded/EP_total in C are visualized with α-carbon coloring on M2 in the E2·BeF₃⁻ structure. Green, 1.6–1.8 (i.e. occlusion as in the wild type (Gly¹⁰⁵)); red, <0.5 (i.e. severe disruption of the occlusion). Error bars, S.D.

FIGURE 9.

Structural analysis of E2PCa₂ state by limited proteolysis. Microsomes expressing wild type or the mutants shown in Fig. 8 were phosphorylated at 25 °C for 10 s in 6 μl of a mixture containing 0.12 mg/ml microsomal protein, 0.5 mm ATP, 0.1 m KCl, 7 mm MgCl₂, 5 mm CaCl₂, 1 μm A23187, and 50 mm MOPS/Tris (pH 7.0), and then 0.72 mg/ml trypsin (top panels) or prtK (bottom panels) was added in a small volume and incubated for the indicated time periods. In the wild type, EP accumulated was exclusively E1PCa₂, and its decay was extremely slowed during the proteolysis periods due to the feedback inhibition by the high concentration of Ca²⁺. In the mutant, EP accumulated was exclusively E2P, and its decay was extremely slow (see Fig. 8, A and B). The proteolysis was terminated by 2.5% (v/v) trichloroacetic acid, and the digests were subjected to Laemmli SDS-PAGE. The ATPase chain and its fragments separated on the gel were blotted onto a polyvinylidene difluoride membrane and visualized by immunodetection with a monoclonal antibody that recognizes the Ala¹⁹⁹–Arg⁵⁰⁵ peptide (tryptic fragment A1) of SERCA1a, as described under “Experimental Procedures.” The tryptic fragments were as follows; A, Met¹–Arg⁵⁰⁵; A1, Ala¹⁹⁹–Arg⁵⁰⁵. The fragments formed by prtK were p95 (Lys¹²⁰–Gly⁹⁹⁴), p81 (Met¹–Met⁷³³), and p83 (Glu²⁴³–Gly⁹⁹⁴) (48, 49). The positions of the Ca²⁺-ATPase chain and its fragments and those of the molecular mass markers are indicated on the left and right, respectively.

FIGURE 10.

Schematic for structural change in EP processing (A) with M2-M4C contact in an E2P model (B) and sequence alignment around M2 for P-type ATPases (C). A, proposed model of Gly¹⁰⁵ functions as a flexible joint of M2c-M2m segments for tilting, bending, and loosening and extending of M2 helix during EP formation E1Ca₂ → E1PCa₂, EP isomerization E1PCa₂ → E2PCa₂, and subsequent Ca²⁺ release E2PCa₂ → E2P + 2Ca²⁺. The A and P domain and selected transmembrane helices are shown; the N domain, M3, and M7–M10 are not depicted for simplicity. The positions of Gly¹⁰⁵, Ala¹¹², and Leu¹¹⁹ are indicated. Open red arrows, movements of M2c and M2m. The yellow arrow on E1Ca₂ and E1PCa₂ and the red arrow on E2PCa₂ indicate the motions (tilting and rotation) of the A domain and the tilting of associated A-P domains, respectively, for the subsequent step. In E1Ca₂ → E1PCa₂, the A domain slightly tilts due to the P domain's conformational change upon the Mg²⁺ ligation and phosphorylation, thereby pulling up M1 and M2 (broken purple arrows), and the M1/M2m is produced and fixes the Glu³⁰⁹ cytoplasmic gate; thus, the Ca²⁺ occlusion is accomplished. In E1PCa₂ → E2PCa₂, the A domain largely rotates and docks on the P domain; thereby, M2 connected with the A domain is pulled and moved, M2c is detached from the cytoplasmic part of M4 (M4C) and strained, and the top part of M2c (M2top) is unwound (as found with Leu¹¹⁹ exposed to prtK in E2PCa₂ (see Fig. 9)). In the latter, E2PCa₂ → E2P + 2Ca²⁺, the associated A-P domains are inclined due to the strain imposed on the A/M1′-linker in E2PCa₂ (23); thereby, the M2m/M1 rigid V-shaped body pushes M4L to open the luminal path (gate) to release Ca²⁺ (9). B, as a consequence, in E2P (depicted with its model E2·BeF₃⁻ (PDB 2ZBE (11)), M2 straightens a steric collision of the Gly¹⁰⁵ region with M4C that inclines toward Gly¹⁰⁵-M2c by the P domain inclination, and the structure is stabilized by interactions in the Tyr¹²²-hydrophobic cluster (Leu¹¹⁹/Tyr¹²² on M2c/M2top with the A and P domains), the M1′-M2c (Val¹⁰⁶–Arg¹¹⁰) interaction, and the M2m/M1 V-shaped body (M2m). Gly¹⁰⁵ (or Gly¹¹² of G-shift G105A/A112G mutant) with its conformational freedom is critical for rapid processing of these large motions while keeping the cytoplasmic gate closed. C, the protein sequence of rabbit SERCA1a Ca²⁺-ATPase (UniProt P04191) is aligned using ClustalW version 2.1 with pig H⁺,K⁺-ATPase (UniProt P19156), pig Na⁺,K⁺-ATPase (UniProt P05024), and human flippase ATP8A2 (UniProt Q9NTI2). Letter colors denote fully conserved (green) or highly conserved residues (blue). The color bars above the sequence alignment indicate the M2m, M2c, and M2top regions of Ca²⁺-ATPase. The glycine residue at the M2m-M2c connecting region is shown in red.