A gating mechanism proposed from a simulation of a human α7 nicotinic acetylcholine receptor Richard J. Law, Richard H. Henchman, and J. Andrew McCammon, PNAS, May 10, 2005

nAChR Part of LGIC family Pentameric cationic channel four transmembrane regions (M1- M4) in the TM domain 10 β sheets in the LB domain with one short helix at the N-terminus AChBP has upto 23% sequence identity with the LB domain, and most similar to the human α7 receptor M2 M3 M4 M1 LB TM involved in Parkinson’s disease, Alzheimer’s disease, myasthenia gravis, frontal lope epilepsy, etc. plays a role in nicotine addiction activation, desensitization, and gating mechanisms are not understood

Aim Using a 15ns apo MD simulation, they look at the direction of motions that hint at the complete motions involved in the gating, activation, and desensitization of the human α7 nAChR

Method Production of a homology model using AChBP at 2.7 Å and Unwin’s EM structure of the TM domain of the Torpedo marmorata at 4.0 Å (MODELLER 4.0) Combining the two domains at residue T231 (termini residue) Model checked with procheck and WHATCHECK Simulation of this homology model Model was inserted into a POPC bilayer CHARMM forcefield in NAMD was used 500ps equilibration followed by 15 ns production run at 310K PME electrostatics and SHAKE were used Essential dynamics using John Mongan’s IED 2.0 add-on for VMD

Results LB domain is relatively rigid compared to the TM domain Only substantial motions of the LB domain are those of the loops

Focus on functionally important loops of the receptor rmsf of one subunit showing less mobility of the LB domain

C loop A part of the LB domain and thought to be functionally important, covers the binding pocket In two subunits the C loop moves away from the binding pocket (B and D) Previous studies indicate that the C loop is more mobile in the absence of a ligand, so an open C loop correlates with the closed state of the receptor It is thought that contact between the ligand and the C loop helps stabilize the binding pocket Increase in the radius of gyration as C loop moves out

C loop of two non-adjacent subunits are most mobile and move outwards The loop that connects β8 and β9 moves most in the complementary subunits (C and E) of those with the most mobile C loops (B and D) The closure of the pore along with the outward motion of the C loop: pore closure: a counterclockwise twisting motion of the helices in subunits B and D

No M2 kinking was observed…could be the short time of the simulation Significant motion of the M2-M3 loop, but not much for the Cys loop and the β1-β2 loop (Unwin, 2004), therefore they suggest that their motion is not coupled Rotation in TM domains happens in two ways: 1 – rotation of the whole subunit relative to adjacent subunits and 2 – rotation of individual helices of a subunit relative to adjacent helices * majority of these rotations in subunits B and D

Essential dynamics First eigenvector shows the twisting/closing of the pore and opening of the C loop Second eigenvector shows the motion of the M2-M3 loop…not equal in all subunits

Conclusions The motion of a single subunit (LB domain to TM domain) are more correlated that between adjacent subunits of either domain consistent with the lack of sequence conservation in residues at the subunit interfaces in the LGIC family The different domains pivot around the CYS loop (static) The rigidity of the LB domain acts as a lever that transmits the motion of loop C to the TM domain

Rotations in the LB domain are not stable or consistent Spacial restrictions may only allow two TM domain subunits to move into the pore Only 2 non-adjacent subunits undergo substantial displacement. This asymmetrical functionality is also seen in the muscular α 2 β 2 δγ receptor and the neuronal α 4 β 2 Conclusions continued..