Molecular Recognition of the Neurotransmitter Acetylcholine by an Acetylcholine Binding Protein Reveals Determinants of Binding to Nicotinic Acetylcholine Receptors

<div><p>Despite extensive studies on nicotinic acetylcholine receptors (nAChRs) and homologues, details of acetylcholine binding are not completely resolved. Here, we report the crystal structure of acetylcholine bound to the receptor homologue acetylcholine binding protein from <i>Lymnaea stagnalis</i>. This is the first structure of acetylcholine in a binding pocket containing all five aromatic residues conserved in all mammalian nAChRs. The ligand-protein interactions are characterized by contacts to the aromatic box formed primarily by residues on the principal side of the intersubunit binding interface (residues Tyr89, Trp143 and Tyr185). Besides these interactions on the principal side, we observe a cation-π interaction between acetylcholine and Trp53 on the complementary side and a water-mediated hydrogen bond from acetylcholine to backbone atoms of Leu102 and Met114, both of importance for anchoring acetylcholine to the complementary side. To further study the role of Trp53, we mutated the corresponding tryptophan in the two different acetylcholine-binding interfaces of the widespread α4β2 nAChR, <i>i.e.</i> the interfaces α4(+)β2(−) and α4(+)α4(−). Mutation to alanine (W82A on the β2 subunit or W88A on the α4 subunit) significantly altered the response to acetylcholine measured by oocyte voltage-clamp electrophysiology in both interfaces. This shows that the conserved tryptophan residue is important for the effects of ACh at α4β2 nAChRs, as also indicated by the crystal structure. The results add important details to the understanding of how this neurotransmitter exerts its action and improves the foundation for rational drug design targeting these receptors.</p></div

Structure of ACh bound to <i>Ls</i>-AChBP.

<p>(<b>A</b>) The structure of acetylcholine (ACh). (<b>B</b>) Displacement of tritium-labeled epibatidine (³H-Epi) bound to <i>Ls</i>-AChBP by ACh was used to determine the IC₅₀ value of ACh. The data points shown are from one determination of the IC₅₀ value. The average of three such experiments were converted to the K_i value of ACh by the Cheng-Prusoff equation. (<b>C</b>) Top-view of a cartoon representation of the structure of one <i>Ls</i>-AChBP pentamer with an ACh molecule bound in each interface. (<b>D</b>) Side-view of a cartoon representation of the <i>Ls</i>-AChBP with ACh shown in green stick representation. The ACh molecule is located between two colored subunits: the green subunit forms the principal side of the binding pocket, (+) interface, while the orange subunit forms the complementary side, (−) interface.</p

Concentration-response relationships at mutated nAChRs measured by two-electrode voltage clamp on <i>X. laevis</i> oocytes.

<p>(<b>A</b>) α4β2 nAChRs with 3α:2β stoichiometry have three binding sites for ACh (black arrows): two with high sensitivity (HS) and one with low sensitivity (LS). Point-mutation of a central tryptophan residue in the α4 subunit will change the complementary (−) side of the LS site (red circle and arrow). Point-mutation of the corresponding tryptophan in the β2 subunit will change the complementary side of both HS binding sites (blue circles and arrows). (<b>B</b>) Concentration-response relationships (CRRs) of ACh at α4^(W88A)β2 and α4β2^(W82A) receptors. The black curve is drawn from previously published data for the ACh CRR at type α4β2 nAChRs with 3α:2β stoichiometry <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091232#pone.0091232-Harpse1" target="_blank">[22]</a>, with EC₅₀ and fraction values listed below the figure. ‘Fraction’ describes the fraction of the maximum response that is elicited by the high-sensitivity phase. Numbers in parenthesis refer to 95% confidence intervals. ‘n’ is the range of the number of measurements that were made of each point on a curve. An F test was carried out in GraphPad Prism 4 against the null hypothesis of a monophasic fit, which was rejected for α4^(W88A)β2 (F = 120, DFnN = 2, DFnD = 288) and accepted for α4β2^(W82A) (F = 0.73, DFnN = 2, DFnD = 72), where DFnN and DFnD are the degrees of freedom of the numerator and denominator in the F test, respectively.</p

Two conformations of Trp53, Leu112 and Met114.

<p>(<b>A</b>) On the complementary side of the interface, three residues near ACh adopt two distinct sets of conformations (shown in purple and light-blue sticks, respectively), at some interfaces occurring separately and at other interfaces with both conformations occurring as shown here. (<b>B</b>) <b>and</b> (<b>C</b>) Trp53 is shown in stick representation in the two different orientations observed, in interfaces where distinct orientations are seen. Principle side carbon atoms are colored green, while complementary side carbon atoms are orange. A mesh is shown in each case, corresponding to a partial omit map shown at 1ó and carved at 2 Å around Trp53. The partial omit map was generated using PHENIX by refining the structure after changing all Trp53 residues to alanine, thus alleviating side-chain orientation bias for this residue. (<b>B</b>) In one possible orientation, the Trp53 side-chain nitrogen atom is pointing “away” from Met114 with Trp53 and Trp143 aligned for T-type ππstacking. (<b>C</b>) In the other conformation, which is favored when a PEG400 molecule is present nearby, the Trp53 side-chain nitrogen atom is pointing towards Met114 and can form a hydrogen bond to the backbone carbonyl oxygen atom of this residue.</p

Data collection and refinement statistics.

a<p>AU: asymmetric unit of the crystal.</p>b<p>Numbers in parenthesis correspond to the outer resolution bin.</p>c<p>A measure of agreement among multiple measurements of the same reflections. R_merge is calculated as follows: I_i(hkl) is the intensity of an individual measurement of the reflection with Miller indices hkl, and I(hkl) is the intensity from multiple observations:R_merge = ∑_hkl∑_i|I_i(hkl)−I(hkl)|/∑_hkl∑_i|I_i(hkl)|.</p>d<p>R(work) = ∑_hkl| F_obs−F_calc |/∑_hkl|F_obs|, where F_obs and F_calc are the observed and calculated structure factor amplitudes, respectively. The free R-factor, R(free), is computed in the same manner as R(work), but using only a small set (5%) of randomly chosen reflections not used in the refinement of the model.</p>e<p>The Ramachandran plot was calculated using PHENIX.</p

Saz-A and TC-2559 act selectively at the α4-β2 interface.

<p><b>A</b> Schematic showing α4-β2 (black arrow) and α-α (grey arrow) binding sites for ACh at the 3α4:2β2, 2α4:3β2 and 3α4:2β2^HQT receptors. <b>B.</b> Current traces of Saz-A (1μM) and <b>C.</b> TC-2559 (10μM) for 3α4:2β2, 2α4:3β2 and 3α4:2β2^HQT receptor compared to maximum ACh currents on the same oocyte. Peak currents elicited by <b>D</b> Saz-A and <b>E</b> TC-2559 were normalized to the saturating concentration of ACh and fitted to non-linear curve fits. The injection ratios of α4:β2 that correspond to the 3α4:2β2 (blue) and the 2α4:3β2 (red) receptors are 4:1 and 1:4 respectively. Saz-A and TC-2559 do not activate the 3α4:2β2^HQT (green) receptor that has three α4-α4-like interfaces. Dots represent the mean ± s.e.m.</p

Ligand Binding at the α4-α4 Agonist-Binding Site of the α4β2 nAChR Triggers Receptor Activation through a Pre-Activated Conformational State - Fig 2

<p>Saz-A and TC-2559 co-application with NS9283: <b>A</b> Schematic of the 3α4:2β2 receptor with the binding sites of Saz-A and TC-2559 at the α4-β2 interface, and NS9283 at the α4-α4 interface. Representative current trace of <b><i>B</i>.</b> Saz-A and <b><i>C</i>.</b> TC-2559, co-applied with NS9283 (10μM) (Green) compared to max ACh (1mM) current on 3α4:2β2 receptors <b><i>D</i></b> Saz-A and <b><i>E</i></b> TC-2559 concentration-response curves in the absence (blue) and presence (green) of 10 μM NS9283 normalized to 1 mM ACh at 3α4:2β2 receptors. Dots represent the mean ± s.e.m.</p

Parameters derived from fitting of data to the Hill Equation with a Hill co-efficient of one.

<p>Parameters derived from fitting of data to the Hill Equation with a Hill co-efficient of one.</p

Kinetic equilibrium constants derived from three-step models proposed.

<p>Kinetic equilibrium constants derived from three-step models proposed.</p

Estimation of open probability.

<p><b><i>A</i></b> Schematic depicting the hypothesized areas for NS206 binding [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161154#pone.0161154.ref034" target="_blank">34</a>]. <b><i>B</i></b> Representative current trace of the response of the 3α4:2β2 receptor to 1 mM ACh (blue) and 1 mM ACh co-applied with 10 μM NS206 to estimate open channel probability. <b><i>C</i></b> The level of NS206 (10μM) positive modulation of ACh currents for the WT. <b><i>D</i></b> Saz-A and <b><i>E</i></b> TC-2559 concentration-response curves in the absence (blue) and presence (green) of 10 μM NS9283 normalized to 1 mM ACh co-applied with 10 μM NS206 (Est <i>P</i>_{<i>o</i>,<i>max</i>}) at 3α4:2β2 receptors. Dots represent the mean ± s.e.m.</p