AChR activation.

A. Traditional scheme. Vertical is ‘gating’ and horizontal is ‘binding’; red, main physiological pathway. The isomerization between closed-channel/low-affinity (CL) and open-channel/high-affinity (OH) conformations occurs with or without agonists (equilibrium constant Ln; n, number of bound ligands), and is spontaneous (depends only on temperature) and global (on a ∼μs time scale). Agonists (A) bind weakly to CL (equilibrium association constant KL, free energy change ΔGL) and strongly to OH (KH, ΔGH). The 2 orthosteric sites of adult AChRs are approximately equivalent and independent and there is no significant external energy, so L2/L0 =(KH/KL)2 (Nayak & Auerbach, 2017). B. Expansions of binding (top, ends with catch) and gating (bottom, starts with hold). The agonist diffuses to and contact the target (’touch’) to form an encounter complex (A-C); a local ‘catch’ rearrangement establishes the low affinity complex (ACL); a local ‘hold’ rearrangement establishes the high affinity complex (ACH); the remaining protein domain ‘isomerize’ without a further change in affinity to generate a conducting channel (AOH). Gray arrows, steps that incur the same energy change for all agonists used in this study; black arrows, agonist-dependent free energy changes occur in catch (ΔGL) and in hold (ΔGH-ΔGL). 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 H conformation, 3 aromatic groups in the α subunit (149-190-198) surround the agonist’s cationic center (+) together provide most of the ACh binding energy (Purohit et al., 2014); the agonist’s tail points away from the α 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 with docked agonists (top 3 poses). Resting-C, 6UVW.pdb minus toxin (red): loop C is up and agonist is cis; 200 ns, after simulation and removal of CCh (blue): loop C is down and agonist is trans. B. Bottom, for all 4 agonists the docking scores (mean±SD, n=3) were more favorable after simulation. C. Cα RMSD (mean+SD, triplicates) are stable after ∼120 ns (ACh, cyan; CCh, green; Ebt, orange; Ebx, purple). D. close-up of the CCh-occupied pocket. Red, resting-C; orange, equilibrated (0 ns MD); blue, after 200 ns MD. IN the simulations, loop C flops down (arrow), loop F moves in, the agonist flips cis→trans (circled inset).

Principal Component Analysis (PCA).

Left, for each agonist a plot of PC-1 versus PC-2, 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 (darkest red) - m1, m2, and m3 - that correspond to different conformations of the neurotransmitter site. Right, ‘porcupine’ plots indicating that the direction and magnitude of changes PC-1 versus PC-2 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) we hypothesize that m1 represent state ACL, m3 represents state ACH, and m2 is an intermediate state in the L→H, hold transition (Figure 1B).

Binding free energies and pocket properties.

A. Calculated (yellow) versus experimental (blue) binding free energies for 4 agonists (structures in 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 (1-ΔGL/ΔGH). The agreement in efficiencies supports the hypothesis that m1 represents ACL and m3 represents ACH B. In L→H (red→blue), VdW interactions (left) increase, pocket volume (center) decreases, and the number of water molecules in the pocket (right) decreases. Overall, the pocket stabilizes, compacts and de-wets.

Agonist and loop movements in hold (flip and flop).

A. Left, superimposed cartoons of ACL (m1; orange) and ACH (m3; blue). Loop C is upper left and loop F is lower right. In L→H (orange→blue) there is a cis→trans reorientation of the agonist (flip) and a downward movement of loop C (flop, arrow). Right, agonist structure m1 (red) versus m2 (yellow) versus m3 (blue). Degree pertains to the m1→m3 rotation angle.

Representative snapshots in L→H (hold). Left, rearrangements of loop C, loop F and the ligand (red, m1; yellow, m2; blue, m3); right, residue and ligand orientations. m1 is ACL, m2 is an intermediate state, m3 is ACH. (FIgure 1B). 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.