Calcium binding and allosteric signaling mechanisms for the sarcoplasmic reticulum Ca²+ ATPase

Abstract

The sarcoplasmic reticulum Ca²⁺ ATPase (SERCA) is a membrane-bound pump that utilizes ATP to drive calcium ions from the myocyte cytosol against the higher calcium concentration in the sarcoplasmic reticulum. Conformational transitions associated with Ca²⁺-binding are important to its catalytic function. We have identified collective motions that partition SERCA crystallographic structures into multiple catalytically-distinct states using principal component analysis. Using Brownian dynamics simulations, we demonstrate the important contribution of surface-exposed, polar residues in the diffusional encounter of Ca²⁺. Molecular dynamics simulations indicate the role of Glu309 gating in binding Ca²⁺, as well as subsequent changes in the dynamics of SERCA's cytosolic domains. Together these data provide structural and dynamical insights into a multistep process involving Ca²⁺ binding and catalytic transitions.

Figure 1

(a) Holo SERCA (PDB ID 1su4) showing the N, A, and P domains as well as the transmembrane helices (teal). (b) Transmembrane region of SERCA with important TM helices labeled. (c) Holo SERCA (PDB ID 1su4) with both Ca²⁺ ions bound (gray) surrounded by several coordinating residues (licorice representation).

Figure 2

(a) APBS-produced electrostatic map of apo SERCA (red = −3.0 kT/e, blue= +3.0 kT/e). Brownian dynamics simulations were used to investigate cation binding to the L3 region of SERCA. (b) Umbrella sampling simulations were used to model the cation moving from the protein/solvent/lipid boundary to approximately 10 Å deep within the TM region where Ca²⁺ binding-site II is located. (c) Upon Ca²⁺ binding, a gradual motion of the N and A cytosolic domains was observed in MD simulations.

Figure 3

Top view of SERCA trans-membrane domain with L1 and L3 binding pathways and sites I and II Ca²⁺ coordination sites. Tyr 763 is represented with space-filling spheres.

Figure 4

Potential of mean force corresponding to translation of Ca²⁺ from bulk (ψ(x) = 18Å) to the bound conformation with D800 (ψ(x) = 2Å). Figure showing gating of E309 with Ca²⁺ in purple space-filling spheres.

Figure 5

Plot of distance between Ca²⁺ and D800 of the bound configuration (red) versus E309 open/closed state. E309 is considered open when its C_α/C_β angle is approximately −50 degrees.

Figure 6

Left: PC1 linking E2 and E1 states. Right: PC2 linking E1 and E1P structures.

Figure 7

PCA projections of 75 ns conventional MD run 1 (top), 45 ns conventional MD (middle), and 225 ns aMD (bottom) simulations of (a,c) apo and (b,d) holo states. PC2 (y axis) and PC1(x axis) separate crystallographic data into distinct regions: E1(green), E1P (blue), E2P (black) and E2 (red).

Figure 8

Overall RMSD for domains A,N, and P (a,c,e) apo and (b,d,f) holo configurations. Rows correspond to cMD run 1 (a,b), cMD run 2 (c,d) and aMD run (e,f). Plots are colored according to cytosolic domains A (red), N (green), P (blue), and bundle helices (purple).

Figure 9

RMSF distribution for (a) apo and (b) holo based on cMD run1 1 simulation. Residues are colored according to cytosolic domains A (red), N (green), P (blue), cytosolic loops (gray/yellow), bundle helices (purple), and bundle loops (black).