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 Ca-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 Gly 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 (E1PCa → E2PCa → E2P + 2Ca), and disrupts Ca occlusion in E1PCa and E2PCa at the transport sites uncoupling ATP hydrolysis and Ca transport. In contrast, this substitution accelerates the ATPase activation (E2 → E1Ca). 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 Ca 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 Gly facing M4/phosphorylation domain. Results demonstrate that Gly functions as a flexible knee-like joint during the Ca 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 Ca transport and ATP hydrolysis.

Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging

“…4 f). Considering that State 2 includes transformation of E 2PCa 2 to E 2P and E 2P hydrolysis, this rate constant is consistent with the rate constant of E 2P hydrolysis of 0.6–0.3 s −1 in the wild type 29 , 36 , 37 . The observed angular change between the two dwell states of ψ S1S2 = 59.5° ± 24.3° (Fig.…”

“…Phosphorylation of SERCA1a in microsomes with [γ- 32 P]ATP was performed essentially as described previously 37 . Microsomes expressing wild type or TCi-196/197 mutant SERCA1a were phosphorylated with [γ- 32 P]ATP at 0 °C for 30 s in 50 μl of a mixture containing 1.2 μg of microsomal protein, 10 μM [γ- 32 P]ATP, 1 μM A23187, 0.1 M KCl, 7 mM MgCl 2 , 10 μM CaCl 2 , and 50 mM MOPS/Tris (pH 7.0).…”

Myosin and Other Energy-Transducing ATPases: Structural Dynamics Studied by Electron Paramagnetic Resonance

“…How is the ion transport coupled to the ATPase reaction and structural change from E1 to E2? Recent studies have reached the conclusion that the structural transition from E1-P.Ca 2+ to E2-P.Ca 2+ precedes Ca 2+ release [161,162]. The FRET from the fluorescently labeled cytoplasmic domains A and P of the Listeria Ca pump directly shows that the structural transition (thermal large-scale domain movement) from E1 to E2 is faster than Ca 2+ release [162].…”