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 sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA), one of the most prominent members of the large P-type ATPase superfamily that transports ions across biological membranes. The 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 the 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.

Figure 1

Effects of transport site protonation on the structural dynamics of E2. (A) Crystal and MD structures at the end of the trajectories of the protonated and deprotonated E2 states. SERCA is colored according to its four functional domains: N (green), P (blue), A (red), and TM (gray). RMSD evolution of SERCA domains in the (B) protonated and (C) deprotonated states of the pump. The RMSD of the TM domain of SERCA was calculated using backbone alignment for TM helices of the pump. The RMSD of N, P, and A domains was calculated by aligning the backbone of the cytosolic headpiece with the structure at t=0 μs of each trajectory.

Figure 2

Structural arrangement of the transport sites. (A) Structure of the transport sites at the end of the MD trajectories of protonated and deprotonated E2 states. The TM helices are shown in gray, and transport site residues are shown as sticks. The Na⁺ ion bound to the transport sites of the deprotonated E2 state is shown as a yellow sphere. (B) Time-dependent evolution of the interaction between transport site residue pairs Val304^O-Glu309^Cγ and Glu771^Cγ-Asn796^Cγ.

Figure 3

Na⁺ ion interactions in the transport sites of the deprotonated E2 state. (A) Location of the three different positions occupied by Na⁺ (yellow spheres) in the transport sites of the deprotonated E2 state. The dashed circles show the approximate location of the Ca²⁺-binding site in the transport site I. (B) Time-dependent changes in the location of Na⁺ in the transport site I. The plot shows the changes in the RMSD of Na⁺ relative to the configuration of the bound ion at the beginning of the MD trajectory (Position 1). The shaded areas correspond to the Na⁺ configurations shown in panel (A).

Figure 4

Protonation-dependent structural changes in the TM helices of SERCA. (A) Average changes in the RMSD of each TM helix in the trajectories of protonated and deprotonated E2 state. Data are presented as the mean ± SD. The location of each TM helix is shown in the right panel as a cartoon representation. (B) Evolution of the secondary structure of the TM helix M5 in the 3.6 μs trajectory of protonated and deprotonated E2 SERCA. Secondary structure is colored as a-helix (pink), turn (cyan), and coil (white).

Figure 5

Kink formation in TM helix M5 induced by deprotonation of the transport sites. (A) Structure of M5 at the end of the trajectories of protonated and deprotonated E2 SERCA. The TM helices are shown as gray cartoons; M5 is shown in orange. Residues Ile765 through Asn768 are shown in magenta. (B) Time-dependent changes in the kink angle of M5.

Figure 6

Na⁺ binding to site I induces kink of M5. (A) Representation of the structural arrangement of M5 between residues Ser767 and Glu771; transport site protonation stabilizes a backbone hydrogen bond between Ser767-Glu771, and H⁺-Na⁺ exchange in this site destabilizes it. M5 is shown as orange cartoon, Ser767 and Glu771 as sticks, and Na⁺ as a yellow sphere. (B) Time-dependent changes in the distance between atoms Ser767^O and Glu771^N. The shadowed area corresponds to the formation of M5 kink described in Figure 5.

Figure 7

Effects of transport site protonation on the M3–M5 interface. (A) Structure of the M3–M5 interface at the end of the trajectories of protonated and deprotonated E2 SERCA. TM helices M3 and M5 are shown as purple and orange cartoons, respectively. The ovals show the approximate location of the binding site for the inhibitor thapsigargin. Residues Phe256 and Ile765, which play a role in binding and stabilization of thapsigargin, are shown as spheres. (B) Evolution of the native contacts between M3 and M5 in the trajectories of protonated and deprotonated E2 SERCA.

Figure 8

Effects of transport site protonation on the mobility of M3 and the A-M3 linker. (A) Location of the M3 and A-M3 linker. For clarity, the luminal and cytosolic regions of M3 are shown as pink and blue cartoons, respectively. The A-M3 linker and the cytosolic A domain are shown in lime and red, respectively. (B) Mean square displacement of M3 and the A-M3 linker calculated in the MD trajectories of protonated and deprotonated E2 SERCA. The shaded areas indicate the location of cytosolic/luminal segments of M3, and the A-M3 linker.

Figure 9

Time-dependent structural transitions of the cytosolic headpiece. (A) MD trajectories of protonated and deprotonated E2 state were used to compute the time-dependent Cα-Cα distances between residues Thr171 and Lys515 (N and A domains, residues Lys515 and Glu680 (N and P domains) and residues Thr171 and Glu680 (A and P domains). For comparison, discrete distances for the same pairs of residues were calculated from crystal structures of E2. (B) Location of the residues used to calculate interdomain distances (cyan spheres). N, A and P domains are colored in green, red and blue, respectively.

Figure 10

Probability distributions for the first 10 PCs extracted in the trajectories of (A) protonated and (B) deprotonated E2 state. These histograms show examples of a non-Gaussian distribution (e.g., PC 1), a distribution that maintains some non-Gaussian features (e.g., PCs 2–4) and purely Gaussian distributions (e.g., PC 10). The r² values shown inside each plot corresponds to the coefficient of determination obtained after fitting each histogram to a one-Gaussian distribution by least squares analysis.

Figure 11

Preexisting domain motions of the cytoplasmic headpiece detected by cPCA. (A) Counter-rotation motion of the N and A domains along the E2-to-E1 transition of SERCA detected by cPCA of ensembles of x-ray structures of E1 and E2 states. The counter-rotation motion of the N-A domains is present in both (B) protonated and (C) deprotonated E2 states. The contribution of the PC corresponding to this motion is shown in parentheses. (D) Cα RMSF for PC1 and PC4 of deprotonated and protonated E2, respectively. The shaded areas indicate the location of A and N domains in the sequence of the protein.