Transition between conformational states of the TREK-1 K2P channel promoted by interaction with PIP2

Figures

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  Figure 1. Coarse-grained molecular dynamics simulation. (A) Schematic representation of the coarse-grained simulation conducted in this study. Only the headgroup of the lower leaflet lipids (PC: gray, PS: black, and PIP2: orange) are shown. TREK-1 (blue) in the up state is embedded in the phospholipid bilayer. After a 10 μs simulation (right), PIP2 headgroups are distributed more closely at their relative binding site (right) compared with their initial distribution (left). (B and C) The PIP2-binding site on the TREK-1 (B) up state or (C) down state. Binding sites 1 (red) and 2 (yellow) are shown superimposed relative to their crystal structure. Each site is annotated with their residence time within the binding site and their occupancy. To see this figure in color, go online.

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  Figure 2. Calculation of PIP2-binding free energy relative to other lipids. (A) Schematic description of the coarse-grained potential of mean force (CG-PMF) calculations. In this scheme, PIP2 (green) in the binding site was steered away from the protein (blue surface) along the collective variable (arrow). The PMF was calculated using umbrella sampling with the force constraint at 1000 kJ nm–2mol–1 (red). (B) One-dimensional energy landscape calculated from PMF calculation as PIP2 is steered away from the binding site from up (black) or down (red) states to the bulk lipid bilayer (PC). Energy landscape is marked at 0 kJ mol–1 when the PIP2 is >1.2 nm away from the binding site. (C) Schematic description of CG free-energy perturbation. The PIP2 headgroup was alchemically transformed to different lipids (PI4P, PC, PS, PG, PA) in both the bound state (ΔG2) and the bulk state (ΔG3). This transformation allows relative affinities between two lipids species to be calculated. (D) Binding free energy of PIP2, PI4P, PI, PG, PS, and PA relative to PC within the TREK-1 PIP2-binding site in the up (black) or down state (red). Error bars showed 95% confidence interval calculated based on t-statistics…

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  Figure 3. Atomistic simulation of PIP2 and TREK-1. (A and B) Representative interactions between (A) up state and (B) down state of TREK-1. The backbone of the protein is represented in blue. Key contacting residues contacting to the phosphate group on the PIP2 molecule are shown in yellow. (C) PIP2-contact analysis showing the fraction of time that residues are in 4 Å proximity to the PIP2 molecule (contact probability) of the up state (black) and down state (red) of TREK-1. Only residues with an average >50% contact probability are shown. Data are collected from three repeats of 500 ns simulations. Error bars showed 95% confidence interval calculated based on t-statistics (n = 6). Structural models highlighting these binding sites are also available (Supplementary Data S1 and S2). To see this figure in color, go online.

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  Figure 4. PIP2 induces conformational change on TREK-1 C-terminus. (A) The difference between the Cα RMSF in the presence and absence of PIP2 (ΔRMSF) of the up state (black) and down state (red) of TREK-1. The positive ΔRMSF value implies that the backbone is stabilized by PIP2. Shaded region represents the propagation of 95% confidence interval. (B) The conformational change induced by PIP2 in the down state. The initial conformation at 0 ns (orange) is compared with the post-simulation at 500 ns (blue) with PIP2 (dark green) in the binding site. The key conformational change in the C-terminal domain is described with the red arrow. We defined the zipper measurement based on the distance between R207 and W295. Residues coordinated with PIP2 (K304 and R311) are highlighted before (yellow) and after (blue) 500 ns simulation. (C) The magnitude of the conformational change along the vector describing the third principal component from the down state plotted against time in the unbound state (orange) and PIP2-bound state (purple). The darker lines show the running average for each simulation (500 ns) (n = 3). (D) Distribution plot of the zipper distance collected from the final 100 ns of each…