Lipid Interactions of a Ciliary Membrane TRP Channel: Simulation and Structural Studies of Polycystin-2

Abstract

Polycystin-2 (PC2) is a transient receptor potential (TRP) channel present in ciliary membranes of the kidney. PC2 shares a transmembrane fold with other TRP channels, in addition to an extracellular domain found in TRPP and TRPML channels. Using molecular dynamics (MD) simulations and cryoelectron microscopy we identify and characterize PIP₂ and cholesterol interactions with PC2. PC2 is revealed to have a PIP binding site close to the equivalent vanilloid/lipid binding site in the TRPV1 channel. A 3.0-Å structure reveals a binding site for cholesterol on PC2. Cholesterol interactions with the channel at this site are characterized by MD simulations. The two classes of lipid binding sites are compared with sites observed in other TRPs and in Kv channels. These findings suggest PC2, in common with other ion channels, may be modulated by both PIPs and cholesterol, and position PC2 within an emerging model of the roles of lipids in the regulation and organization of ciliary membranes.

Keywords: TRP channel; cholesterol; cryoelectron microscopy; lipids; molecular dynamics; phosphatidylinositol bisphosphate; polycystin-2.

Graphical abstract

Figure 1

A Phospholipid Interaction Site Identified in Simulations of PC2 in a PC Bilayer (A) The PC2 channel (PDB: 5K47) embedded in a phospholipid bilayer, shown using a snapshot from an atomistic simulation of the protein (color) in a phosphatidylcholine (PC) bilayer (similar results for PDB: 5MKF and 5T4D are shown in Figure S1). The lipid tails are in cyan, phosphates in orange and red, and choline nitrogens in blue. Water molecules are omitted for clarity. (B) The PC2 protein is shown as a gray surface, viewed perpendicular to the central pore axis, with one subunit depicted as a pale purple cartoon. Green isocontour surfaces represent a high probability of occurrence of phospholipid molecules. (C) Zoomed-in view (red box in B) of the S3/S4/S5 pocket and the high phospholipid occurrence density with a PC molecule (taken from a simulation snapshot) shown within the density. See also Figure S1.

Figure 2

Lipid Binding Sites Suggested by cryo-EM Maps of PC2 (A and B) Lipid binding sites suggested by cryo-EM maps of PC2 obtained (A) in the presence of PI(4,5)P₂ (3.0 Å resolution) and (B) in the presence of PI(3,5)P₂ (3.4 Å resolution). Protein density (contoured at 3.2σ) is in gray, detergent density in yellow, lipid density in orange, and cholesterol density in cyan. (C) Expanded view around the proposed lipid binding site between S3, S4, and S5 showing density from the 3.0 Å map (pink; see Figure S9) and from the 3.4 Å map (gray). See also Figures S2, S3, and S5.

Figure 3

Images of the Density and Structure Relating to the Lipid Binding Sites Representative images of the density and structure relating to the lipid binding sites, from the 3.0 Å resolution cryo-EM map. (A and B) The S4 (A) and S5 (B) transmembrane helices, with aromatic and basic side chains labeled. (C) The S4 and S5 helices (yellow structure and blue density) with the density proposed to correspond to lipid (see main text for discussion) in pink. (D) The S4 helix (yellow structure and blue density) with the fitted cholesterol molecule (green structure and darker blue density; see main text and Figure 8). Density is shown filtered to 2.96 Å at 2σ. See also Figures S4 and S5.

Figure 4

Simulations of PC2 in an In Vivo Mimetic Mixed Lipid Bilayer Coarse-grained (CG) simulations of PC2 in an in vivo mimetic mixed lipid bilayer. (A) PC2 (pale purple) in a mixed lipid bilayer, viewed from the intracellular face and showing molecules of PIP₂ (red), cholesterol (cyan), PC (white), PE (orange), and PS (lime) in the inner leaflet of the bilayer. (B) Distance from the binding site as a function of time for four PIP₂ molecules that bind to PC2 during a simulation in a mixed lipid bilayer. The distance is from the center of mass of the head group of each PIP₂ molecule to the center of mass of the S505 and S591 side chains at the site to which that lipid molecule eventually binds. The four colors correspond to the four different PIP₂ molecules that eventually bind. (C) Snapshot from the end of the simulation shown in (A) showing PIP₂ molecules (head group phosphate particles in brown, as shown in the CG representation of PIP₂ on the right) at four sites on the channel tetramer. Side-chain particles of basic residues at the PIP₂ binding site are shown in blue (arginine) and cyan (lysine). See also Figures S6 and S7.

Figure 5

Free Energy Landscape for Lipid Interactions Free energy landscape for lipid interactions at the binding site on PC2. (A) Potentials of mean force of the interactions of PIP₂ (orange), PIP₃ (green), PI(4)P (yellow), PS (purple), and PC (blue) with the lipid binding site on PC2. The inset shows a schematic representation of the reaction coordinate corresponding to the distance between centers of mass of the head group of a lipid molecule and of the two serines (S505 and S591) within the lipid binding sites. (B) PIP strip data for interactions of different phospholipid species with PC2. Note the null results for the anionic PA, PS, and S1P lipids, and for the zwitterionic PE and PC controls. See also Figure S8.

Figure 6

Protein-Lipid Interactions of the Head Group of PIP₂ Protein-lipid interactions showing the head group of PIP₂ coordinated by five basic residues (R504, K572, K575, R592, and K595) at the binding site. (A) Snapshot structure corresponding to the CG energy minimum was converted into an atomistic representation and used as the basis of 30 ns distance restrained simulations (see text for details) followed by 250 ns of unrestrained simulations (with snapshots at 100 and 250 ns). (B) H-bonding interactions between key basic side chains and the PIP₂ head group during three replicates (run 1, 0–250 ns; run 2, 250–500 ns; run 3, 500–700 ns) of the unrestrained atomistic simulations.

Figure 7

Comparison of PI Lipids Bound to PC2 and to TRPV1 Comparison of PI lipids bound to PC2 and to TRPV1 with cryo-EM density. (A–C) (A) PIP₂ bound to PC2 (as revealed by the current simulation study); (B) lipid-like density in the cryo-EM maps of PC2 obtained in the presence of PI(3,5)P₂ (3.4 Å resolution; see Figure 5); (C) PI bound to TRPV1 (as revealed by cryoelectron microscopy, PDB ID 5IRZ). In each case the lipid molecule or density is located between the S3, S4, and S5 helices of the VSLD. (D) A sequence alignment colored on contacts with PIP₂ in the mixed lipid simulations. Residues of the region around the binding pocket between the S3, S4, and S5 helices are colored (on a white to red scale) based on the mean duration of the interactions of PIP₂ head groups with each residue. The five basic residues of PC2 which form interactions with the head group of PIP₂ at the binding site (i.e., R504, K572, K575, R592, and K595) are boxed. See also Figure S5.

Figure 8

Cholesterol Interactions with PC2 (A) Cryo-EM density (from the 3.0 Å map, contoured at 2.2σ; see Figure 2A) corresponding to a binding site for cholesterol located between the S3 and S4 helices and helix S6 of the adjacent subunit. (B) Cholesterol observed to bind to the same site in CG simulations of PC2 in an in vivo mimetic mixed lipid bilayer (see Figure 3). (C) Distance from the binding site as a function of time for four cholesterol molecules which bind to PC2 during a simulation in a mixed lipid bilayer. (D) Potential of mean force for the interaction of cholesterol with the binding site on PC2. See also Figures S5 and S10.

Figure 9

Simulations of Cholesterol-Bound PC2 Atomistic simulations of cholesterol-bound PC2, with the cholesterol molecule initially built into the cryo-EM density. (A) Root-mean-square deviation (RMSD) versus time for the four cholesterol molecules bound to PC2 during a 250-ns atomistic MD simulation. The large fluctuations in RMSD for two molecules (purple and green curves) demonstrate the relatively loose binding of cholesterol at this site. (B) Snapshot structures of cholesterol (stick representation; taken every 1 ns) at the binding site on PC2. Each of these structures is colored according to the corresponding simulation time on the red, white, and blue color scale shown; thus structures at the start of the simulation are colored red, and those at the end in blue. (C) Final simulation snapshot (t = 250 ns) showing the arrangement of key binding site residues. See also Figure S10.

Figure 10

Comparison of Lipid Binding Sites of TRP and Kv Channels (A) Anionic lipid (blue acyl tails and red phosphate oxygens) binding sites close to the S4 linker: TRPV1 and PI (PDB: 5IRZ), TRPV5 and PIP₂ (PDB: 6DMU), Kv1.2/Kv2.1 chimera and PG (PDB: 2R9R), and PC2 and PIP₂ (this study). (B) Cholesterol (green carbons and red oxygens) binding sites between the VSD/VSLD and the central pore domains of: TRPM2 (PDB: 6CO7), TRPML3 (PDB: 5W3S), Kv10.1 (PDB: 5K7L), and PC2 (this study). In each case the transmembrane region of the channel structure is shown, with the location of the lipid bilayer shown via the broken gray lines.