Modeling neuronal nicotinic and GABA receptors: important interface salt-links and protein dynamics

Abstract

Protein motions in the Cys-loop ligand-gated ion receptors that govern the gating mechanism are still not well understood. The details as to how motions in the ligand-binding domain are translated to the transmembrane domain and how subunit rotations are linked to bring about the cooperative movements involved in gating are under investigation. Homology models of the alpha4beta2 nicotinic acetylcholine (nACh) and beta2alpha1gamma2 GABA receptors were constructed based on the torpedo neuromuscular-like nicotinic receptor structure. The template constructed for the full electron microscopy structure must be considered more reliable for structure-function studies due to the preservation of the E45-R209 salt-link. Many other salt-links are seen to transiently form, including switching off of the E45-R209 link, within a network of potential salt-links at the binding domain to the transmembrane domain interface region. Several potentially important intersubunit salt-links form in both the nAChR and GABAR structures during the simulation and appear conserved across many subunit combinations, such as the salt-link between alpha4.E262 and beta2.K255 in nAChR (beta2.E262 and alpha1.K263 in GABAR), at the top of the pore-lining M2 helices, and the intersubunit link of R210 on the M1-linker to E168 on the beta8-sheet of the adjacent subunit in the GABA receptor (E175-K46 being the structurally equivalent link in the nAChR, with reversed polarity). A network of other salt-links may be vital for transmitting the cooperative gating motions between subunits that become biased upon ligand binding. The changes seen in the simulations suggest that this network of salt-links helps to set limits and specific states for the conformational changes involved in gating of the receptor. We hope that these hypotheses will be tested experimentally in the near future.

Figure 1

(A) RMSD for Cα atoms in the α4β2 nAChR model simulations, demonstrating the difference in mobility between the two models. This added mobility is almost completely due to two small regions of the complex, as shown in B. This DSSP plot shows the mobility and loss of secondary structure in the vestibule helix and the peripheral binding domain helix. The vestibule helix was not present in the chimeric template, and therefore does not contribute to the RMSD. (C) Plot of the RMSF, averaged over five subunits, of the α4β2 full-EM template model in simulation. The inset is an image of a single subunit of the channel structure, with the ribbons colored based on these RMSF values. It shows three “hot” areas: the binding domain helix, the C-loop, and the vestibule segment/domain.

Figure 2

Panel A illustrates the difference in the position of the E45–R209 salt-link in the α4β2 nAChR model built from the chimeric versus the full-EM templates (i.e., the salt-link is formed in the full-EM based models, but not in the chimera-based models). The protein ribbons are colored according to a red-white-blue scale based on residue number; therefore, the binding domain β1–β2 loop, early in the sequence, appears red in the images, whereas the TM helices appear as darkening shades of blue. (B) A plot showing the side-chain center of mass (COM) distance between four E45 and R209 pairings in the four simulations. Salt-links were selected with a 3.2 Å cutoff between N-O atoms, but the side-chain COM is plotted to reduce noise in the plot. E45–R209 are bridged to each other in the EM-template model, but not in the chimeric-template models. For the chimeric-template based models, these two plot lines are representative of the typical lack of this salt-link in all the subunits within these two models (shown here for an nAChR α-subunit and a GABA β-subunit). In the EM-template-based models, the salt-link exists in all the subunits where the residues are conserved. Three of these E45–R209 salt-links, in the EM-template model subunits (two GABA β subunits and one nAChR α-subunit; the one shown here is from the GABA β-subunit) demonstrate a temporary switching whereby other nearby ionizable residues act as surrogate salt-bridge partners, and in this case switch back again.

Figure 3

Network of potential salt-links exists at the interface between the LBD and TMD, involving the five loop sections of the structure that exist there in the models. The residue numbering is for the nicotinic α-subunit, and is kept consistent across the sequence alignment for clarity in making comparisons between the different structures. The black connecting lines show a salt-link present for >50% of the simulation, and the gray connecting lines indicate transient salt-links that are present for <50% of the simulation. E45–R209 is the longest lived of these salt-links and the best conserved across the CL-LGIC family. In the nAChR model, R209 transiently switches to E175. In GABAR, E/D45 transiently switches to either R136 or K272. Within the β-subunits of nAChR, an extra salt-link exists between R46 and D271, with R/K46 transiently forming a link to E/D132. R/K208–D139 in the nAChR and GABAR subunits represents another potentially conserved salt-link that is seen transiently in the simulations. Many of the charged residues at the interface are unpaired.

Figure 4

Only a small number of intersubunit salt-links appear to exist in the initial models or to even form during the simulation. In the nAChR BD, the salt-link between E175(β) and K46(α) seems to be equivalent to the salt-link E168(α) to R210(β) in the GABAR. On the pore-lining M2 helices of the TMDs, the link from K255(β) to E262(α) in the nAChR seems to be equivalent to the link from K263(α) to E262(β) in the GABAR. These salt-links are not present throughout the simulation. The graph in the lower panel shows their transient nature.