Multiscale Simulation Reveals Passive Proton Transport Through SERCA on the Microsecond Timescale

Abstract

The sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) transports two Ca²⁺ ions from the cytoplasm to the reticulum lumen at the expense of ATP hydrolysis. In addition to transporting Ca²⁺, SERCA facilitates bidirectional proton transport across the sarcoplasmic reticulum to maintain the charge balance of the transport sites and to balance the charge deficit generated by the exchange of Ca²⁺. Previous studies have shown the existence of a transient water-filled pore in SERCA that connects the Ca²⁺ binding sites with the lumen, but the capacity of this pathway to sustain passive proton transport has remained unknown. In this study, we used the multiscale reactive molecular dynamics method and free energy sampling to quantify the free energy profile and timescale of the proton transport across this pathway while also explicitly accounting for the dynamically coupled hydration changes of the pore. We find that proton transport from the central binding site to the lumen has a microsecond timescale, revealing a novel passive cytoplasm-to-lumen proton flow beside the well-known inverse proton countertransport occurring in active Ca²⁺ transport. We propose that this proton transport mechanism is operational and serves as a functional conduit for passive proton transport across the sarcoplasmic reticulum.

Figure 1

Location of the luminal water pore of SERCA. (A) Structure of SERCA bound to PLB embedded in a lipid bilayer. The proteins are shown as ribbons, and the phosphate groups of the lipids are shown as gold spheres. For clarity, the TM helices are colored as follows: TM6, blue; TM8, green; TM9, purple; TM10, orange. (B) Luminal water pore of SERCA located between transmembrane helices TM6, TM8, TM9, and TM10; the pore is shown as a yellow surface. To see this figure in color, go online.

Figure 2

(A) Two-dimensional potential of mean force for proton transport from E908 to H944. The errors in the PMF are within 1.5 kcal/mol and the error plot can be found in Supporting Material. (B) Free energy along the minimum free energy path (MFEP) with error bars are shown. Errors indicate the standard deviations from block analysis. To see this figure in color, go online.

Figure 3

Representative configurations along the minimum free energy path, with labels showing the positions on the 2D PMF (Fig. 2A). The position of the excess proton defect CEC is rendered as a purple sphere. The hydrogen atoms of S767, T799, V798, V905, S902, and W794 are not shown for clarity. (A) The protonated E908 forms a hydrogen bond with S767. (B) The channel becomes hydrated and the E908-S767 hydrogen bond breaks. (T) The transition state of the PT reaction in which the excess proton is solvated in the water close to the hydrophobic V798 and V905 residues. (C) The excess proton shuttles to H944 via water wires. To see this figure in color, go online.

Figure 4

(A) 2D PMF for PT from H944 to the lumen. The errors in the PMF are within 1 kcal/mol, and the detailed error plot can be found in the Supporting Material. (B) Free energy along the MFEP with error bars are shown. Note that the vertical scale in this figure is less about half that of Fig. 2B. (C) Representative configuration at the transition state. Errors indicate the standard deviations from block analysis. To see this figure in color, go online.