Acetylcholine-Binding Protein Engineered to Mimic the α4-α4 Binding Pocket in α4β2 Nicotinic Acetylcholine Receptors Reveals Interface Specific Interactions Important for Binding and Activity

Abstract

Neuronal a4b2 nicotinic acetylcholine receptors are attractive drug targets for psychiatric and neurodegenerative disorders and smoking cessation aids. Recently, a third agonist binding site between two a4 subunits in the (a4) 3 (b2) 2 receptor subpopulation was discovered. In particular, three residues, H142, Q150, and T152, were demonstrated to be involved in the distinct pharmacology of the a4-a4 versus a4-b2 binding sites. To obtain insight into the three-dimensional structure of the a4-a4 binding site, a surrogate protein reproducing a4-a4 binding characteristics was constructed by introduction of three point mutations, R104H, L112Q, and M114T, into the binding pocket of Lymnaea stagnalis acetylcholine-binding protein (Ls-AChBP). Cocrystallization with two agonists possessing distinct pharmacologic profiles, NS3920 [1-(6-bromopyridin-3-yl)-1,4-diazepane] and NS3573 [1-(5-ethoxypyridin-3-yl)-1,4-diazepane], highlights the roles of the three residues in determining binding affinities and functional properties of ligands at the a4-a4 interface. Confirmed by mutational studies, our structures suggest a unique ligandspecific role of residue H142 on the a4 subunit. In the cocrystal structure of the mutated Ls-AChBP with the high-efficacy ligand NS3920, the corresponding histidine forms an intersubunit bridge that reinforces the ligand-mediated interactions between subunits. The structures further reveal that the binding site residues gain different and ligand-dependent interactions that could not be predicted based on wild-type Ls-AChBP structures in complex with the same agonists. The results show that an unprecedented correlation between binding in engineered AChBPs and functional receptors can be obtained and provide new opportunities for structure-based design of drugs targeting specific nicotinic acetylcholine receptor interfaces.

Ligand Binding at the α4-α4 Agonist-Binding Site of the α4β2 nAChR Triggers Receptor Activation through a Pre-Activated Conformational State

“…We used this information to show the modulator NS9283 that selectively binds at the α4-α4 interface had an agonist-like mechanism by activating via this interface [20]. We further determined the differences in binding affinities between the two sites [11] and studied structure activity relation (SAR) for ligands that have different pharmacological profiles on the two stoichiometries [21]. However, the transitions of the receptor during activation that are perturbed when two, or three, agonist-binding sites are occupied are not understood, along with the transitions that are perturbed by modulators that potentiate receptor activation.…”

Differential α4(+)/(−)β2 Agonist-binding Site Contributions to α4β2 Nicotinic Acetylcholine Receptor Function within and between Isoforms

“…A very recent publication (54) also demonstrated interactions of an ␣4 (Ϫ)-face E-loop engineered into the binding pocket of Lymnaea stagnalis acetylcholinebinding protein with a neighboring subunit. These interactions differed according to which particular ligand was bound (at least for compounds that are exceptionally selective between HS and LS phase activation), and appeared likely to impact receptor activation (54). If somewhat analogous interactions occur at interfaces not bound by agonist, this may provide at least a partial mechanistic basis for E-loop involvement in the neighbor effects noted in this study.…”

Isoform‐specific mechanisms of a3b4*‐nicotinic acetylcholine receptor modulation by the prototoxin lynx1

“…For example, several studies, including our own, demonstrate that binding of both divalent cations (88, 92) and agonists at α(+)/(−)α interfaces can profoundly affect efficacy when measured at the macroscopic (48, 89, 93, 94) and single‐channel levels (92). Second, there is a demonstrated functional role for residues that are located on the conserved agonist‐binding E‐loop of α(−) subunits, both at agonist binding sites and outside of them (74, 89, 95, 96).…”