(a) The binding sites of the sterols along the hypothesized tunnel in SMO. Sterol binding sites have been identified deep in the TMD (6XBL),44 in the extracellular TMD, at the interface of CRD and TMD (6XBM),44 at the orthosteric binding site in the CRD (5L7D)18 and a dual-bound mode where cholesterol is bound to TMD and CRD (6O3C).45 (b) Example simulation system showing SMO (5L7D, cyan) embedded in a membrane (white/magenta). Water is shown as a surface, while sodium (blue) and chloride (gray) ions are shown as spheres. (c) The two pathways explored in this study for the translocation of cholesterol from membrane to SMO’s TMD. (d) The common pathway followed by cholesterol once cholesterol enters the protein to reach SMO’s CRD. Snapshots in (a) are made from structures in the PDB, while (b-d) are frames taken from MD simulations.

The molecular events as cholesterol enters the core of SMO’s TMD from the outer leaflet of the membrane.

(a) Free energy plot showing the angle of cholesterol with the x-y plane, the plane of the membrane, v/s the z-coordinate of cholesterol for Pathway 1. The pathway followed by cholesterol is α → β → α∗. The experimental structures of SMO are shown as black polygons. (b) Free energy landscape of cholesterol’s y-coordinate plotted v/s cholesterol’s x-coordinate. Cholesterol interacts with residues in TM2-TM3 while entering core TMD of SMO. (c-f) Insets show cholesterol’s interactions with residues at the membrane-protein interface for Pathway 1. (c,e) show cholesterol outside the protein (α), while (d,f) show cholesterol entering the protein (β). All snapshots presented are frames taken from MD simulations.

Effects of mutations along Pathways 1 and 2 on the activation of SMO.

(a) Gli1 mRNA fold change (indicator of SMO activation) plotted for SMO mutants, showing fold change when the mutants are untreated and treated with SHH. Untreated Gli1 levels indicate low SMO activity, while SHH-treated values correspond to the level of SMO activation induced by SHH ligand. t test with a Welch’s correction was used to compute statistical significance. (P values: untreated vs treated: WT: 1.327 × 10−3, G2.57f V: 9.212 × 10−3, IECL2A: 4.2 × 10−5, A2.60f M: 7.1 × 10−5, R5.64f A: 2.062 × 10−3, R5.64f Q: 1.192 × 10−3, F6.36f I: 2.163 × 10−3, L5.62f A: 1.948 × 10−3, treated WT vs treated mutant: G2.57f V: 9.1 × 10−3, IECL2A: 0.02734, A2.60f M: 0.7477, R5.64f A: 0.08858, R5.64f Q: 0.02766, F6.36f I: 1.923 × 10−3, L5.62f A: 2.306 × 10−3 key: Not significant (ns) P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, N=4 for all experiments.) (b) ΔGli1 mRNA fold change (high SHH vs untreated) and Δ PMF (difference of peak PMF) plotted for the mutants in Pathway 1. (c) Example mutant A2.60f M shows that cholesterol is able to enter SMO through Pathway 1 even on a bulky mutation. (d) Same as (b) but for Pathway 2 (e) Example mutant L5.62f A shows that cholesterol is able to enter SMO through Pathway 2 due to lesser steric hinderance. All snapshots presented are frames taken from MD simulations.

Molecular events at the entry of cholesterol from the membrane into the core SMO TMD for Pathway 2.

(a) Free energy plot showing the angle of cholesterol with the x-y plane, the plane of the membrane, v/s the z-coordinate of cholesterol for Pathway 2. The pathway followed by cholesterol is η → θ → α∗. The experimental structures of SMO are shown as black polygons. (b) Free energy landscape of cholesterol’s y-coordinate plotted v/s cholesterol’s x-coordinate for Pathway 2. Cholesterol interacts with residues in TM5-TM6 for Pathway 2 while entering SMO core TMD. (e-h) Insets show cholesterol’s interactions with residues at the membrane-protein interface for Pathway 2. (c,e) show cholesterol outside the protein (η), while (d,f) show cholesterol entering the protein (θ) for Pathway 2. All snapshots presented are frames taken from MD simulations.

Multiple positions of cholesterol as it translocates through the common pathway, including the off-pathway intermediate.

(a) upright (δ) (b) tilted (γ) (c) overtilted (E), the off pathway intermediate and (d) Cholesterol at the CRD binding site (ζ). All snapshots presented are frames taken from MD simulations.

Effects of mutations along the translocation pathway connecting the TMD and CRD binding sites on activation of SMO.

(a) Gli1 mRNA fold change (indicator of SMO activation) plotted for SMO mutants, showing fold change when the mutants are untreated v/s treated with SHH. Untreated Gli1 levels indicate low SMO activity, while SHH-treated values correspond to the level of SMO activation induced by SHH ligand. t test with a Welch’s correction was used to compute statistical significance. (P values: untreated vs treated: WT: 3 × 10−6, YLDA: 2.46 × 10−4, F6.65f A: 1.08 × 10−3, IECL3A: 1.12 × 10−4, treated WT vs treated mutant: F6.65f A: 1.6 × 10−5, IECL3A: 1.6 × 10−5, YLDA: 1.4 × 10−5, key: Not significant (ns) P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, N=4 for all experiments.) (b) ΔGli1 mRNA fold change (high SHH vs untreated) and Δ PMF (difference of peak PMF) plotted for mutants along the TMD-CRD pathway. (c, d) Example mutants YLDA and F6.65f A show that cholesterol is unable to translocate through this pathway because of the loss of crucial hydrophobic contacts provided by Y207 and F484 and along the solvent exposed pathway.

The tunnel profile during cholesterol translocation in SMO.

(a) Free energy plot of the z-coordinate v/s the tunnel diameter when cholesterol is present in the core TMD. The tunnel shows a spike in the radius in the TMD domain, indicating the presence of a cholesterol accommodating cavity. (b) Representative figure for the tunnel when a cholesterol is in the TMD. (c) Same as (a), when cholesterol is at the TMD-CRD interface. (e) same as (b), when cholesterol is at the TMD-CRD interface. (e) same as (a), when cholesterol is at the CRD binding site. (f) same as (b), when cholesterol is at the CRD binding site. Tunnel diameters shown as spheres. All snapshots presented are frames taken from MD simulations.

The timescales associated with the translocation of cholesterol through SMO.

Each major intermediate state has been marked (a-f). Timescales were obtained by calculating the mean first passage time (MFPT) using the markov state model. Errors in timescales are shown as subscripts. The arrows represent the relative flux for the translocation between subsequent steps. The overall process occurs at a timescale of ∼ 1ms.