MD simulations of Human Sigma1 Receptor Trimer Uncovers Cholesterol Dependent Stabilization and Ligand Specific Dynamics

Key results

1. ShownThe S1R trimer stayed structurally stable in a cholesterol-containing, MAM-mimicking bilayer. In pure POPC, Cα RMSD was higher and fluctuated more. Bilayer thickening followed membrane composition, not ligand identity.

   Thickness was 41.10 ± 0.04 Å (Hal) and 41.19 ± 0.03 Å (PnT) in POPC/POPE/CHOL vs 38.29 ± 0.02 Å and 38.21 ± 0.02 Å in pure POPC. RMSD differences are described only qualitatively, from three 1 µs replicates per system. · Ligand-bound Sigma1 receptor trimers modulate…; Supporting Information

2. SuggestedS1R pulls the bilayer into a local bowl-shaped pit above its core. This comes from the lipid imbalance between leaflets and from strong tilting of lipids packed against the apolar α4/α5 protein platform.

   Mean and Gaussian curvature were both positive (H>0, K>0) near S1R. Lipids within 6 Å of the protein tilted up to 90°, while bulk lipids sat mostly near 20°. The inner leaflet started with 400 lipids vs 450 in the outer leaflet. Crystal structures show ordered lipid density near α1. · Ligand-bound Sigma1 receptor trimers modulate…; Supporting Information; Figure legends

3. ShownCholesterol gathered around S1R on its own, with no restraints applied. The longest-lasting contacts were at the transmembrane CARC-like motif and the α1/α4/α5 junction. Contacts with α5 were most frequent; contacts with α1 were rarer but longest-lived. The pattern was similar with either ligand.

   Persistence was measured as the longest unbroken cholesterol contact within 6 Å, averaged over monomers and three replicas. It peaked at CARC-like residues 7 to 14 and at the α1-α4-α5 junction, a site occupied by ordered lipids in the crystal structures. Hal and PnT complexes gave matching patterns. · Cholesterol-protein contacts; Cholesterol binds S1R in specific hotspots; Figure legends

4. ShownThe two ligands contacted mostly the same residues. Haloperidol, whose more elongated structure penetrates deeper into the cavity, engaged the β6 strand through its chlorophenyl ring more persistently. That strand carries W136, which sits at the interface with the neighbouring protomer.

   Contacts within 3.5 Å differed mainly at residues 83-87 and 132-137. In the crystal structures, both ligands contact W89, Y103 and F107 and form the E172 salt bridge, and the two complexes differ by an RMSD of 0.439Å. W136 packs against F83, A92 and T109 of the adjacent monomer. Prior experiments found that replacing W136 with glycine impairs multimerization. · Ligand-protein contacts; W136 emerges as a key determinant of…; Figure legends

5. SuggestedCorrelation network analysis found W136 to be the main hub in both complexes. Haloperidol gave a more cohesive network and coherent communication paths from E172 to W169 across the protomer interface. With (+)-pentazocine, the paths split apart at the interface.

   W136 centrality was markedly higher in S1R-Hal. S1R-PnT had 24 communities vs 14 in S1R-Hal (edges kept at |cij| ≥ 0.75). In S1R-Hal, all 100 computed paths ran through T109 and A110 to W169; in S1R-PnT the paths diverged after crossing between monomers. · Correlation network analysis (CNA); Ligands alter S1R interprotomer interaction and…