Major structural changes during SMO Activation. (A) Comparison of the broken D-R-E network and the W-R π-cation lock, and the expanded tunnel, in inactive (magenta, 5L7D32) vs active (green, 6XBL33) SMO (B) Comparison of inactive and active SMO, indicating the outward movement of the TM6 and TM3 and inward movement of TM5 in active SMO.

Molecular metrics integral to SMO Activation. (A) Rearrangement of the WGM motif, a conserved molecular switch across class F GPCRs, undergoes rearrangement on SMO activation. (B) Relative free energies from MSM-weighted simulation data plotted on the TM3-TM6 distance vs TM3-TM5 distance measured at residues W3393.50f, M4496.30f and G4225.65f. (C) Breaking of the D-R-E network on the extracellular end of the TMD. (D) Similar to (B), but for TM3-TM6 distance vs the D-E distance. (E) The π-cation lock breaks by the sidechain rotation of W5357.55f. (F) Same as (B) but for TM3-TM6 distance vs χ2 dihedral measured at W5357.55f.

Overall activation of SMO involves residues at CRD-TMD junction. (A)-(F) Snapshots and probability density plots outlining the salt-bridge rearrangements at the CRD-TMD interface during SMO activation.

(A)Relative free energies from MSM-weighted simulation data of Apo-SMO plotted along tIC1 and tIC2, the 2 slowest components, with the intermediate states I1–3 as shown. The intermediate states I1–3 were defined based on metastable basins and free energy barriers associated with transitioning from an inactive to an active state. A cutoff of 1.8 kcal/mol was used to separate one basin from another. Residues shows as sticks include the π-cation lock, the WGM motif and the salt bridges involved in activation. (B) Overall transition pathway of SMO activation process. The inactive (PDB ID: 5L7D)32 and active (PDB ID: 6XBL)38 structures are separated by the presence of 3 metastable conformations in between, I1–3. Residues shown by sticks correspond to the salt bridges, the WGM motif, the DRE network and the π-cation lock, all residues critical for mediating SMO activation.

Tunnel radius plots for SMO. (A) Free energy plot of the tunnel diameter along the z-coordinate for SANT1-bound SMO. (C) same as (A), but for Apo-SMO. (E) same as (A), but for SAG-bound SMO. SAG-bound SMO clearly shows the expansion of the tunnel as compared to Apo-SMO and SANT1-SMO. (B), (D), (F) - representative figures for SANT-1 SMO, Apo-SMO and SAG-SMO. Tunnel radii were calculated using the HOLE program67

Allosteric pathways between E5187.38f and W3393.50f. (A) Pathway in Apo-Inactive-SMO. Since the tunnel radius is decreased, TM6 outward movement is restricted, and therefore the entire allosteric communications occurs via TM6. (B) In SANT1-SMO, due to slight outward movement of TM6, the pathways switches from TM7 to TM6 to TM3. (C,D) SAG-SMO and Apo-Active SMO show the same allosteric pathway, which spans TM7-TM6-TM5-TM3.