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

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

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

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

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

Crystal Structure of <em>Lymnaea stagnalis</em> AChBP Complexed with the Potent nAChR Antagonist DH<em>β</em>E Suggests a Unique Mode of Antagonism

<div><p>Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that belong to the Cys-loop receptor superfamily. These receptors are allosteric proteins that exist in different conformational states, including resting (closed), activated (open), and desensitized (closed) states. The acetylcholine binding protein (AChBP) is a structural homologue of the extracellular ligand-binding domain of nAChRs. In previous studies, the degree of the C-loop radial extension of AChBP has been assigned to different conformational states of nAChRs. It has been suggested that a closed C-loop is preferred for the active conformation of nAChRs in complex with agonists whereas an open C-loop reflects an antagonist-bound (closed) state. In this work, we have determined the crystal structure of AChBP from the water snail <em>Lymnaea stagnalis</em> (<em>Ls</em>) in complex with dihydro-<em>β</em>-erythroidine (DH<em>β</em>E), which is a potent competitive antagonist of nAChRs. The structure reveals that binding of DH<em>β</em>E to AChBP imposes closure of the C-loop as agonists, but also a shift perpendicular to previously observed C-loop movements. These observations suggest that DH<em>β</em>E may antagonize the receptor via a different mechanism compared to prototypical antagonists and toxins.</p> </div

The structure of DH<i>β</i>E and <i>Ls</i>-AChBP complexed with DH<i>β</i>E.

<p>(<i>a</i>) Structure of DH<i>β</i>E. (<i>b</i>) Cartoon diagram showing homopentameric <i>Ls</i>-AChBP viewed along the five-fold symmetry axis. The five subunits are shown in different colors and DH<i>β</i>E in red spheres representation. (<i>c</i>) Ligand-binding pocket at the interface of two monomers formed by the highly conserved aromatic residues Tyr89, Trp143, Tyr185, and Tyr192 from the principal side of the interface (yellow) and Trp53 from the complementary side (limon). DH<i>β</i>E is shown in red and an omit 2Fo-Fc map is shown at 1<i>σ</i>. Hydrogen bonds between DH<i>β</i>E and its surroundings are shown as stippled lines. A blow-up of DH<i>ß</i>E and the omit 2Fo-Fc map shown at 1<i>σ</i> is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040757#pone.0040757.s002" target="_blank">Fig. S2</a>.</p

Comparison of DH<i>β</i>E-bound and nicotine-bound structures of <i>Ls</i>-AChBP.

<p>(<i>a,b</i>) Comparison of the ligand-binding site of the DH<i>β</i>E-bound structure (<i>a</i>, red) with the nicotine-bound structure (<i>b</i>, green). DH<i>β</i>E and nicotine are colored in cyan and purple, respectively. Hydrogen bonds between ligand and its surroundings are shown as stippled lines. The location of the residues is identical except for the residues from the C-loop (residues 185–192). Also, the conformation of the Met114 side chain from the complementary side is different between the two structures. (<i>c</i>) Conformational change of the C-loop due to DH<i>β</i>E binding to <i>Ls</i>-AChBP. The DH<i>β</i>E-bound structure (red) has been superimposed onto the nicotine-bound <i>Ls</i>-AChBP structure (green). (<i>d</i>) The projection vectors belonging to the nicotine-bound and DH<i>β</i>E-bound <i>Ls</i>-AChBP structures are shown in green and red, respectively. The angle between the two projection vectors is 21.4°. Angles between projection vectors of <i>Ls</i>-AChBP co-crystallized with nAChR agonists are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040757#pone-0040757-t002" target="_blank">Table 2</a> for comparison. For further details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040757#pone.0040757.s003" target="_blank">Fig. S3</a>.</p

Quantification of the C-loop conformational change.

a<p>Quantification of C-loop closure by the method of Brams <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040757#pone.0040757-Brams2" target="_blank">[21]</a>.</p>b<p>For explanation on projection vectors, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040757#s4" target="_blank">Materials and Methods</a>.</p><p>PDB ID (chain A):</p>c<p>1uw6;</p>d<p>1uv6;</p>e<p>3u8l (chain B);</p>f<p>2zju;</p>g<p>3u8k;</p>h<p>3u8m;</p>i<p>3u8n;</p>j<p>3u8j;</p>k<p>2zjv;</p>l<p>4alx.</p

Data collection and refinement statistics of the DH<i>β</i>E-bound <i>Ls</i>-AChBP structure.

a<p>Numbers in parentheses represent the last resolution shell values.</p