Structural and energetic analysis of metastable intermediate states in the E1P–E2P transition of Ca ²⁺ -ATPase

Figures

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  Fig. 1.. Structures of SR Ca2+-ATPase embedded in a DOPC membrane in E1P⋅ADP⋅2Ca2+ (PDB ID: 2ZBD) (A) and E2P (PDB ID: 2ZBE) (B). The three cytoplasmic domains (A, N, and P) are colored in red, purple, and green, respectively. (C) A structural transition pathway predicted using the mean-force string method is projected onto a two-dimensional map along with the two distance RMSDs (dRMS) from the representative structures in MD simulations of E1P and E2P_dp. In the simulation, Glu908 at the Ca2+-binding sites is protonated to mimic the same protonation states of E1P. Only the atomic coordinates that are involved in the collective variables (CVs) are used for dRMS calculations. The five substates (SSs) are defined along the pathway via the fixed radius clustering method (1 to 17: red; 18 to 27: yellow; 28 to 35: green; 36 to 51: cyan; and 52 to 64: blue). Among the 64 images, 5 images (10, 23, 31, 44, and 57) are selected as five representative SSs.

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  Fig. 2.. Conformational changes of the Ca2+-ATPase in the E1P–E2P transition. (A) MT for centroid images of SSs on the pathway from E1P to E2P_dp. From Left to Right, four representative SS transitions between images 10 to 23, 23 to 31, 31 to 44, and 44 to 57. Rigid domains derived by MT are shown with different colors at each SS transition. In other words, parts with the same color are considered as a single rigid domain. The domains are further divided (as shown in white, blue, orange, yellow, and green parts), and the patterns of division become more complex at latter transitions (31–44 and 44–57). Selected residues are shown in stick representation with labeling on the SS transition 10 to 23 (Left). (B) Movements of the cytoplasmic domains between central images of two adjacent SSs. Colored and gray structures represent later and earlier SS structures, respectively. A-, P-, N-domains are shown in red, green, and magenta. Yellow and blue arrows indicate the rotation axes of the A-, and N-domains, respectively, with respect to the P-domain. (C) Changes of contacts between the cytoplasmic domains. Left, Middle, and Right correspond to the SS transitions between images 10 to 23, 23 to 31,…

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  Fig. 3.. Interactions between the TM helices and lipid molecules in the cytoplasmic (A, C, and E) and lumen (B, D, and F) sides. (Left) Snapshot of M1–M6 helices and surrounding lipid molecules. (Right) Distribution of phosphorus atoms in lipid molecules around the protein. Dotted boxes mark the areas of the snapshots shown on the Left, and pink arrows indicate the direction of view. C atoms of the lipid molecules bound to the ATPase are shown in yellow, and C atoms of the other lipid molecules are shown in gray. The protein’s backbone is shown in rainbow spectrum with the N terminal in blue. C, N, and O atoms in the sidechain are shown in green, blue, and red, respectively.

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  Fig. 4.. Energetics of the E1P–E2P transitions. The structures near the Ca2+-binding sites in the first and last images in the string-method simulations (A) from E1P to E2P_dp, (B) from E2P_dp to image 50 (image 16r), and (C) from E2P to image 50 (image 16r). The protonated Glu are highlighted with dashed circles. Ca2+, K+, and water are shown in blue, yellow, and red spheres. Only sidechains of the Ca2+-binding residues are shown with stick representations. C, N, and O atoms in the stick representations are shown in green, blue, and red, respectively. The protein’s backbone is shown in rainbow spectrum with the N terminal in blue. (D) The structures of M4 and M6 in images 64 from E1P to E2P_dp (orange), 1r from E2P_dp to image 50 (blue), and 1r from E2P to image 50 (pink). (E) Free energy profiles obtained in the string-method calculations using E1P (orange), E2P_dp (blue), and E2P (pink). In E1P and E2P_dp, only Glu908 is protonated, while Glu309, Glu771, and Glu908 are protonated in E2P.

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  Fig. 5.. A schematic view of the transition from E1P⋅ADP⋅2Ca2+ to E2P. Structural features of the two intermediates (E1P⋅2Ca2+ and E2P⋅2Ca2+) are predicted in the current simulations. HP contact indicates the transient hydrophobic interactions between A- and P-domains.