Lipid-protein interactions mapped by Markov state modeling and cryo-electron microscopy.

(A) Overview of the MD simulation system with GLIC (gray surface) embedded in a lipid bilayer (light blue carbon atoms) with interacting lipids highlighted (yellow carbon atoms). Lipid oxygens are shown in red; phosphorus, orange; nitrogen, dark blue. Water molecules and ions have been omitted for clarity. (B) Markov state models were used to cluster simulations conducted under resting (R) or activating (A) conditions into five states, including closed (left of the light or dark orange lines) and open (right of the light or dark blue lines). Black lines mark edges of other state clusters derived from MSM eigenvectors. Experimental structures are highlighted as white circles. (C) To characterize the stability of lipid interactions, duration times were measured in individual, unclustered, trajectories. (D) To identify lipid binding sites, lipid-occupied densities were obtained from state-clustered frames for closed (orange) and open (blue) states. (E) Further simulations were performed to test the role of residues involved in extensive lipid interactions through single residue mutations around lipid binding sites. (F) View of a manually built cryo-EM model of closed GLIC (gray) with computationally derived lipid occupancies in orange (semi-transparent). (G) Cryo-EM reconstruction of closed GLIC (gray), with partly defined non-protein densities (orange). (H) Equivalent view of a manually built cryo-EM model as in F, with newly built lipids shown as sticks (yellow, heteroatom; phosphorus, orange; oxygen, red; nitrogen, blue).

Cryo-EM data collection and model refinement statistics

Cryo-EM and simulations reveal lipid interaction sites.

(A) Cryo-EM reconstruction of closed GLIC with distance to the interacting lipids colored according to the bar below. (B) Mean duration time of lipid-residue interactions, scaled (white–red) according to the color bar below, projected onto the closed-state GLIC structure (PDB ID: 4NPQ). For both panels, a single subunit is displayed in cartoon. (C) Secondary structure schematic for GLIC (top), mean duration time of each lipid-residue interaction (middle), and number of lipids interacting with each residue during simulations (bottom). Light gray points represent numbers from individual trajectories and black lines ensemble averages (500 trajectories).

Protein and lipid determinants of state-independent inner-leaflet binding.

(A) Computationally derived densities from the open (blue) and closed (orange) states generally agree. (B) Modeled lower leaflet lipids (yellow, stick, heteroatom coloring) from cryo-EM densities (brown mesh). (C) Zoom-in view of the lower leaflet buried lipid interaction site at the M1-M3 subunit interface. Lipids particularly interact with residues T274 (purple) and W217 (magenta). (D) The 100 simulation frames that best fit the computational densities clustered into two distinct binding poses at the T274 binding site. When the tail with a double bond (yellow sphere) is situated in the pocket lipid heads are directed out from the channel (left panel), while a larger variety of poses are sampled when the lipid tail without a double bond occupies the site, allowing the head to enter into a crevice on the bottom of the channel, approaching the pore (right panel). The M4 helix is not shown for clarity in the top panels. (E) Number of contacts made by specific POPC lipid atoms with residue T274 in simulations at both resting and activating conditions (colors span 0 contacts, white, to 137015 contacts, dark red). For the tail with an unsaturation, interactions are concentrated around the double-bond region while the saturated tail displays interactions interspersed along the tail. (F) Mutation of residue W217, lining this pocket, reveals shortened interactions at the T274 binding site (magenta).

State-dependent protein-lipid interactions in the upper leaflet.

(A) Zoom view of the outer transmembrane domain of the closed GLIC cryo-EM structure (left, gray), showing non-protein cryo-EM densities (brown mesh) overlaid with built lipids (yellow). For comparison, the same region is shown of an open state x-ray structure (PDB ID: 6HZW) (Hu et al., 2018) with built lipid and detergent in equivalent positions (right). Conformational changes of the M2-M3 loop, including residue P250, in channel opening are highlighted. Lipids are colored by heteroatom (phosphorus, orange; oxygen, red; nitrogen, blue). (B) Same region as in (A) showing densities occupied by lipids (>40%) in simulation frames clustered as closed (left, orange) or open (right, blue) states (top). Open-state differences include an enhanced density on the right-hand side of the intersubunit cleft, and a more deeply penetrating density in the intrasubunit cleft (Figure 4—??). Overlaid densities for each state represent simulations conducted under resting (dark shades) or activating (light shades) conditions, which were largely superimposable within each state. (C) Radial distribution of the lipid atoms closest to the upper pore, showing closer association of lipids in the open (light blue, dark blue) versus closed (orange, red) states. (D) Same region and cryo-EM densities (mesh) as in (A) overlaid with lipids from a few simulation frames where the lipids had the highest correlation with the closed-state computational occupancies in (B). (E) Snapshots from simulation frames that had the highest correlation with the occupancies (activating conditions) in (B). The open states are characterized by an additional lipid at the intersubunit site, interacting with the oxygen of P250. (F) The minimum distance between P250 and the closest lipid projected onto the free energy landscapes obtained from Markov state modeling. P250-lipid interactions are possible in the open state, but not in the closed state.

Lipid sites identified by cryo-EM and simulations.

(A) In the closed state, five lipid interaction sites were independently identified through both cryo-EM and simulations. In the outer leaflet, two intrasubunit sites are situated at the principal (P) and complementary (C) subunits, with the lipid at the principal subunit site interacting with S196 of the pre-M1 loop. Particularly, no lipid site could be found at the subunit interface. In the inner leaflet, a buried lipid pose was found at the subunit interface, determined by interactions with T274 and W217 of the M3 and M1 helices, respectively. (B) In the open state, six lipid interaction sites were identified from simulations, out of which three sites were known from previous structures and one presumably occupied by a detergent molecule (Hu et al., 2018) (thin blue stripes). In the outer leaflet, an additional lipid site could be found at the subunit interface in the open state, interacting with P250 on the M2-M3 loop. Additionally, the site at the principal intrasubunit site is slightly closer to the M4 helix compared to the closed state, and the lipid site at the principal intrasubunit site has an increased likelihood of tail penetration into an allosteric pocket compared to the closed state. Lipid sites in the inner leaflet are at positions equivalent to those in the closed state.