AChR activation.

A. Cyclic, thermalized activation scheme (vertical, gating; horizontal, binding; red, main physiological pathway). Receptors isomerize globally between closed-channel/low-affinity (CLA) and open-channel/high-affinity (OHA) conformations (equilibrium constant Ln; n, number of bound ligands). Agonists (A) bind weakly to CLA (equilibrium association constant KLA, free energy change ΔGLA) and strongly to OHA (KHA, ΔGHA). In adult AChRs the 2 orthosteric sites are approximately equivalent and independent, and there is no significant external energy: L2/L0 =(KHA/KLA)2 (Nayak & Auerbach, 2017). B. Top, binding and gating. Bottom, catch and hold. The agonist diffuses to the target to form an encounter complex (A-C), a local ‘catch’ rearrangement establishes the LA complex (ACLA), a local ‘hold’ rearrangement establishes the HA complex (ACHA), the rest of the protein isomerizes without a further change in affinity to generate a conducting pore (AO). Gray, steps that incur the same energy change for all agonists used in this study; black, agonist-dependent free energy changes in catch is ΔGLA and in hold (ΔGHA-ΔGLA). C. α-δ subunit extracellular domains, red, after toxin removal (6UWZ.pdb) and blue, apo (7QKO.pdb). There are no major deviations (Cα RMSD = 0.3 Å). D. Closeup of the desensitized Torpedo α-δ subunit neurotransmitter site occupied by carbamylcholine (CCh, blue) (7QL6.pdb; (Zarkadas et al., 2022)). In this is HA conformation, 3 aromatic groups in the α subunit (149-190-198) surround the agonist’s cationic center (+) together provide ~90% of the binding energy for ACh (Purohit et al., 2014), and the tail points toward he δ subunit (trans orientation).

Agonist docking and loop dynamics.

A. Top, agonists (blue, cationic center): carbamylcholine (CCh), acetylcholine (ACh), epibatidine (Ebt) and epiboxidine (Ebx). Bottom, α−δ site (6UVW.pdb minus toxin, resting-C) with docked agonists (top 3 poses). docked into resting-C (red): loop C is up, agonist is cis. docked after 200 ns simulation and removing CCh (blue): loop C is down, agonist is trans. B. Bottom, docking scores (mean±SD, n=3). red, resting-C; blue, 200 ns. C. Cα RMSD (mean+SD, triplicates) (ACh, cyan; CCh, green; Ebt, orange; Ebx, purple). D. close-up of the CCh-occupied pocket. Red, resting-C; orange, equilibrated (0 ns); blue, 200 ns. After 200 ns loop C has flopped, loop F has moved inward, and the agonist has flipped cis→trans.

Principal Component Analysis (PCA).

For each agonist, the left panel plots PC1 vs PC2, the first two principal components that capture the maximum variance in the trajectory (Figure 3-Source Data 1). Colors represent free energy value in kcal/mol (scale, upper left-bottom). For all agonists there are 3 energy minima (dark red), m1, m2, and m3, that correspond to different conformations of the protein. The right panels are ‘porcupine’ plots indicating that the direction and magnitude of changes between PC1 and PC2 is in loops C and F. From energy comparisons (Figure 4, Figure 4-Source Data 1) and temporal sequences (Figure 3-figure supplement 2, Figure 4-Source Data 1): m1 is ACLA, m3 is ACHA, and m2 is an intermediate configuration.

Binding free energies and pocket properties.

A. Calculated (yellow) versus experimental (blue) binding free energies for 4 agonists (Figure 2A, top) (Figure 4-Source Data 1). PBSA calculations were done on clusters selected from m1 and m3 minima of PCA plots (Figure 3; Figure 3-figure supplement 2). Left, absolute ΔG and right, efficiency. The agreement in efficiencies supports the hypothesis that m1 represents ACLA and m3 represents ACHA. B. In ACLA→ACHA, VdW interactions increase (left), pocket volume decreases (center) and the number of water molecules decreases (right). Overall, the pocket stabilizes, compacts and de-wets.

Agonist and loop movements in hold.

In each panel left, superimposed cartoons of ACLA (m1; orange) and ACHA (m3; blue). Loop C is upper left and loop F is lower right. In ACLA→ACHA (red→blue) there is a cis→trans reorientation of the agonist (a flip) and a downward movement of loop C (a flop, orange arrow); right, flip from m1 (red) to m2 (yellow) to m3 (blue); degree, m1→m3.

Representative snapshots in ACLA→ACHA (hold).

left, rearrangements of loop C, loop F and the ligand (red, m1; yellow, m2; blue, m3); right, residue and ligand orientations. m1 is ACLA, m2 is an intermediate state, m3 is ACHA. In m1, a functional group in the agonist tail interacts with αY93 (all agonists) and αD200 (only CCh and Ebt). The position and orientation of αW149 relative to N+ of the agonist remains nearly unchanged m1→m2→m3 and serves as a fulcrum for the cis→trans flip (see Figure 5). In m2, the functional nitrogen at the agonist tail (CCh, Ebt and Ebx) interacts with the hydroxyl group of αY198. For all ligands, αY190 repositioning and loop C flop (m1→m3) are correlated. In m3, the agonist fully flips to trans, facilitating VdW interactions, de-wetting, and the formation of water-mediated hydrogen bonds with the reactive group at its tail with δN109/δL121 backbone (loop E) via a structural water.