Preexisting domain motions underlie protonation-dependent structural transitions of the P-type Ca²⁺-ATPase

Abstract

We have performed microsecond molecular dynamics (MD) simulations to determine the mechanism for protonation-dependent structural transitions of the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Release of two H+ from the transport sites activates SERCA by inducing a structural transition between low (E2) and high (E1) Ca2+-affinity states (E2-to-E1 transition), but the structural mechanism by which transport site deprotonation facilitates this transition is unknown. We performed microsecond all-atom MD simulations to determine the effects of transport site protonation on the structural dynamics of the E2 state in solution. We found that the protonated E2 state has structural characteristics that are similar to those observed in crystal structures of E2. Upon deprotonation, a single Na+ ion rapidly (<10 ns) binds to the transmembrane transport sites and induces a kink in M5, disrupts the M3–M5 interface, and increases the mobility of M3/A-M3 linker. Principal component analysis showed that counter-rotation of the cytosolic N-A domains about the membrane normal axis, which is the primary motion driving the E2-to-E1 transition, is present in both protonated and deprotonated E2 states; however, protonation-dependent structural changes in the transmembrane domain control the hierarchical organization and amplitude of this motion. We propose that preexisting rigid-body domain motions underlie structural transitions of SERCA, where the functionally important directionality is preserved while transport site protonation controls the dominance and amplitude of motion to shift the equilibrium between the E1 and E2 states. We conclude that ligand-induced modulation of preexisting domain motions is likely a common theme in structural transitions of the P-type ATPase superfamily.

Probing the effects of nonannular lipid binding on the stability of the calcium pump SERCA

“…Furthermore, the small structural rearrangement of the cytosolic M2–M4 interface detected in the absence of the nonannular lipid does not propagate to other transmembrane helices or the large cytosolic headpiece of the pump. These findings suggest that factors other than nonannular lipid binding are important for the structural stability of the E2 state 10,17,40,43 . Indeed, SERCA activity is susceptible to allosteric control by cations, small molecules, lipid composition and endogenous proteins, so it is possible that the nonannular lipid is an allosteric effector of the E2 state.…”

“…SERCA in the E2 state releases 1–2 protons from the transport sites to the cytosol, thus completing a cycle of Ca 2+ and H + countertransport across the ER/SR. Finally, proton-metal ion exchange destabilizes the E2 state and accelerates the structural transitions toward the E1 state required for the next Ca 2+ pumping cycle 5,7–10 . A scheme of the transport cycle of SERCA is shown in Fig.…”

“…The complex mechanism for Ca 2+ transport by SERCA and the transition from one intermediate to another is influenced by metal ions 11–15 , nucleotide binding 16 , protonation/deprotonation of the transport sites 10,17 , posttranslational modifications 18,19 , and endogenous regulatory proteins 20–25 . However, SERCA also operates in an environment made up largely by the lipid bilayer, so lipids and cholesterol play a central role on SERCA activity through their diverse chemical spectrum and the cooperative physical properties of lipid mixtures of variable composition.…”

Horse gluteus is a null-sarcolipin muscle with enhanced sarcoplasmic reticulum calcium transport

“…We used all-atom MD simulation to produce structures of horse SERCA1 embedded in a 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) phospholipid bilayer at simulated physiological conditions: 100 mM KCl, 3 mM MgCl 2 , and pH 7.0 (67). Three independent MD simulations of horse SERCA1 were run using NAMD (165), as previously described for rabbit SERCA1 (76,94,166,167). System set-up consisted of periodic boundary conditions (168), particle mesh Ewald for calculating electrostatic interactions in periodic molecular systems (169,170), and a non-bonded cutoff of 0.9 nm, in order to use the RATTLE algorithm (171) to generate 200 ns trajectories of SERCA with a time-step of 2 fs.…”

Structural basis for relief of the sarcoplasmic reticulum Ca2+-ATPase inhibition by phospholamban at saturating Ca2+conditions

“…In the absence of adequate charge neutralization of the transmembrane transport sites, SERCA denaturalization occurs very rapidly even within the native membrane at physiological pH (49,50). Previous studies have shown that this electric charge can be compensated in the absence of Ca 2+ by transport site protonation (16,17), or by binding of metal ions K + (51), Na + (52)(53)(54) or Mg 2+ (55,56). This suggests that super-physiological concentrations of Ca 2+ used in this study simply satisfy transport site charge neutralization, and that the Ca 2+ -bound state of the SERCA-PLB complex might not represent a functional state in the cell.…”