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Molecular Actions of Smoking Cessation Drugs at Α4β2 Nicotinic Receptors Defined in Crystal Structures of a Homologous Binding Protein

Molecular Actions of Smoking Cessation Drugs at Α4β2 Nicotinic Receptors Defined in Crystal Structures of a Homologous Binding Protein

Molecular actions of cessation drugs at α4β2 nicotinic receptors defined in crystal structures of a homologous binding protein

Bert Billena, Radovan Spurnya, Marijke Bramsa, René van Elkb, Soledad Valera-Kummerc, Jerrel L. Yakeld, Thomas Voetse, Daniel Bertrandc, August B. Smitb, and Chris Ulensa,1

aLaboratory of Structural Neurobiology, KU Leuven, 3000 Leuven, Belgium; bDepartment of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, 1081 HV, Amsterdam, The Netherlands; cHiQScreen, 1211 Geneva, Switzerland; dLaboratory of Neurobiology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 22709; and eLaboratory of Ion Channel Research, KU Leuven, 3000 Leuven, Belgium

Edited by Palmer Taylor, University of California at San Diego, La Jolla, CA, and accepted by the Editorial Board April 26, 2012 (received for review October 5, 2011) Partial of the α4β2 nicotinic receptor domain in complex with α- (16). However, signifi- (nAChR), such as , are therapeutically used in smoking cant progress has been made since the discovery of water-soluble cessation treatment. These drugs derive their therapeutic effect pentameric acetylcholine binding proteins (AChBPs) from snails from fundamental molecular actions, which are to desensitize and the subsequent elucidation of their high-resolution ligand- α4β2 nAChRs and induce channel opening with higher affinity, bound crystal structures. AChBPs are homologs of the extracel- but lower efficacy than a full at equal receptor occupancy. lular domain of nAChRs and are the best-studied structural Here, we report X-ray crystal structures of a unique acetylcholine models of ligand recognition by the extracellular domain of binding protein (AChBP) from the annelid Capitella teleta, Ct- nAChRs (17–19). AChBP, in complex with varenicline or , which are both In this study, we report the high-resolution X-ray crystal partial agonists. These structures highlight the architecture for structures of an AChBP from the marine worm Capitella teleta

molecular recognition of these ligands, indicating the contact res- (20), a unique nonmolluscan AChBP, in complex with two partial PHARMACOLOGY idues that potentially mediate their molecular actions in α4β2 agonists, lobeline and the aid varenicline. nAChRs. We then used structure-guided mutagenesis and electro- These structures offer detailed insight into high-affinity inter- physiological recordings to pinpoint crucial interactions of vareni- actions of lobeline and varenicline in the binding pocket of Ct- cline with residues on the complementary face of the binding site AChBP. We then used structure-guided mutagenesis and elec- in α4β2 nAChRs. We observe that residues in loops D and E are trophysiological recordings of α4β2 nAChRs to reveal the key molecular determinants of desensitization and channel opening interactions of varenicline with the complementary face of the with limited efficacy by the varenicline. Together, binding pocket that account for receptor desensitization and this study analyzes molecular recognition of smoking cessation channel opening with limited efficacy. By defining structural drugs by nAChRs in a structural context. determinants of these molecular actions at the α4β2 nAChR, we open avenues for the rational design of smoking cessation aids. | cys-loop receptor | ligand-gated ion channel Results obacco smoking is a major cause of premature death world- Pharmacological Characterization of Ct-AChBP. In this study, we Twide. To a large extent, the reinforcing effects of result characterized the pharmacological properties of an AChBP from from the direct activation of neuronal α4β2 nicotinic acetylcholine the marine worm Capitella teleta, which is a unique nonmolluskan receptors (nAChRs), which triggers downstream events such as AChBP (20). To investigate the validity of Ct-AChBP as a poten- increased release in the mesolimbic system (1–4). tial model for the nAChR, we compared the binding properties of Therefore, the α4β2 nAChR subtype has become a key target for varenicline and various other ligands with two well-described development of therapeutic agents for smoking cessation (5, 6). In molluskan AChBPs, namely AChBP from Aplysia californica (Ac- particular, high-affinity partial agonists for α4β2 nAChRs, such as AChBP) and Lymnaea stagnalis (Ls-AChBP). Using competitive 3 lobeline (7), (8), and varenicline (8), are molecules of in- binding assays with H-, we calculated Ki values for terest because they exhibit the unique property of acting as a mixed these ligands (Table 1). When Ct-AChBP is compared with the agonist/antagonist. High-affinity varenicline binding competes other AChBPs, we conclude that it has a distinct pharmacological fi fi with nicotine at the α4β2 nAChR and, thereby, antagonizes the pro le. Ct-AChBP shows a low af nity for acetylcholine and a high fi fi reward sensation of smoking (9, 10). However, varenicline acti- af nity for nicotine. The af nity of varenicline for Ct-AChBP is vates α4β2 nAChRs with low efficacy and desensitizes, leaving more than 10-fold higher than for Ac-AChBP and is close to the channels unopened even at high binding occupancy, thereby minimizing withdrawal symptoms and increasing the success rate of smoking cessation attempts (9, 10). Understanding the molec- Author contributions: B.B., R.v.E., S.V.-K., D.B., A.B.S., and C.U. designed research; B.B., R.S., M.B., R.v.E., S.V.-K., D.B., and C.U. performed research; M.B. and J.L.Y. contributed ular mechanism of partial agonism at the nAChR has great po- new reagents/analytic tools; B.B., R.S., M.B., R.v.E., S.V.-K., T.V., D.B., A.B.S., and C.U. tential for designing novel smoking cessation compounds but, as analyzed data; and B.B., M.B., R.v.E., T.V., D.B., A.B.S., and C.U. wrote the paper. yet, is hampered by the absence of the high-resolution structure of The authors declare no conflict of interest. a eukaryote nicotinic receptor. This article is a PNAS Direct Submission. P.T. is a guest editor invited by the Editorial Board. Current structural insight is derived from medium-resolution Freely available online through the PNAS open access option. electron microscopy images of the nAChR from Torpedo mar- Data deposition: The structural coordinates and structure factors reported in this paper have morata (11), X-ray structures from prokaryotic nAChR homologs been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4AFG and 4AFH). GLIC (12, 13) and ELIC (14), the Caenorhabditis elegans gluta- 1To whom correspondence should be addressed. E-mail: [email protected]. mate-activated chloride channel GluCl (15) and an X-ray struc- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ture from the monomeric mouse nAChR α-subunit extracellular 1073/pnas.1116397109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1116397109 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 Table 1. Binding properties of Ct-AChBP in comparison with Ac-AChBP, Ls-AChBP, and α4β2 nAChRs Ligand Ct-AChBP Ac-AChBP Ls-AChBP α4β2 nAChR Ligand action

Lobeline 0.29 ± 0.06 1.2 ± 0.4 1,000 ± 64 7 ± 1 Partial agonist Epibatidine 4.6 ± 0.3 11.4 ± 0.7 2.5 ± 0.2 0.05 ± 0.01 Agonist Varenicline 5 ± 172± 83± 1 0.18 ± 0.01 Partial agonist 6 ± 138± 3223± 26 819 ± 90 Antagonist d-tubocurarine 140 ± 6509± 38 171 ± 18 2 ± 1 Antagonist Cytisine 247 ± 31 206 ± 52 219 ± 66 0.9 ± 0.1 Partial agonist 358 ± 66 15 ± 415± 6 ND Antagonist Nicotine 423 ± 59 583 ± 84 1,110 ± 231 6 ± 2 Partial agonist α- ImI > 1mM 4± 2 > 1 mM ND Antagonist 4.6 ± 0.8 μM 6.0 ± 0.4 μM27± 3 μM ND Agonist Acetylcholine 57 ± 6 μM35± 5 μM29± 3 μM28± 4 μM Agonist Serotonine 171 ± 76 μM319± 143 μM893± 364 μM8± 3 μM Antagonist GABA > 1mM > 1mM > 1mM ——

All measurements are in nanomolars unless otherwise noted. Binding constants (Ki values) were determined by using a competitive binding assay with 3H-epibatidine (n ≥ 3). ND, not determined.

high affinity for Ls-AChBP. The high affinity of lobeline and transmembrane domain in integral Cys-loop receptors. The Cys varenicline for Ct-AChBP resembles the high-affinity binding to loop in Ct-AChBP comprises only 8 amino acids (C137–C146), α4β2 nAChR (7 ± 1 nM for lobeline and 0.18 ± 0.01 nM for var- compared with 12 amino acids in molluskan AChBPs and 13 in enicline; Table 1). Lobeline and varenicline both display partial membrane integral Cys-loop receptors. The β1-β2 loop in Ct- agonist action at α4β2 nAChR (7, 9). The pharmacology of α4β2 AChBP is 4 amino acids longer than in Ac-AChBP (M53–N56). nAChRs is complex because these receptors can occur in alternate Finally, Ct-AChBP has different conformations in the β5 strand, stoichiometries and contain ligand binding sites with high and low which lines the pentamer vestibule, and loop F, which forms part sensitivity (21). In addition, these receptors can occur in different of the ligand binding site. Although subtle differences exist, these allosteric conformations and display cooperativity upon ligand binding, which are properties lacking in AChBPs. Despite these limitations, our binding data demonstrate high-affinity binding of lobeline and varenicline and suggest that Ct-AChBP is a useful addition to the family of AChBPs as a suitable model for structural studies.

X-Ray Crystal Structures of Ct-AChBP in Complex with Lobeline and Varenicline. The lobeline-bound structure of Ct-AChBP (1.9 Å, crystallographic details in Table S1) is shown along the fivefold symmetry axis with the C terminus of the protein pointing toward the viewer in Fig. 1A and away from the viewer in Fig. 1B. The Ct- AChBP monomer has an architectural fold that closely resembles Ac-AChBP and can be superimposed with an r.m.s. deviation of 1.6 Å for 190 Cα atoms. Difference electron density at the subunit interfaces unambiguously revealed the occupancy of lobeline at all five binding sites (Fig. 1A, Inset). In addition, we observed electron density for an N-linked glycosyl chain at N122 (side view in Fig. 1C and Inset), which likely corresponds to paucimannose commonly found in insect cells (22) and partially wraps around the water- accessible part of the binding pocket at the subunit interface. Treatment with PNGase had no significant effect on the Ki value of nicotine, suggesting that the glycosyl chain attached to N122 does Fig. 1. X-ray crystal structures of AChBP from Capitella teleta (Ct-AChBP). not affect the pharmacological properties of Ct-AChBP (Fig. S1). Cartoon representation of Ct-AChBP in complex with lobeline, seen along the The varenicline-bound structure of Ct-AChBP (2.0 Å) is shown in fivefold symmetry axis with the N terminus pointing away from the viewer (A) fi Fig. 1D. The Ct-AChBP structures in complex with varenicline and and pointing toward the viewer (B). Each of the ve subunits is displayed in lobeline are almost identical, and pentamers can be superimposed a different color. Lobeline molecules are shown as spheres. Oxygen, red; ni- α trogen, blue; sulfur, green. Insets show a detail of 2Fo-Fc electron density with an r.m.s. deviation of 0.4 Å for 1,053 C atoms. contoured around lobeline (ball-and-stick representation) at a σ level of 1.5. The architectural fold of the annelid Ct-AChBP is very similar (C) Side view of Ct-AChBP in complex with lobeline with a subunit interface to molluskan AChBPs, but there are notable structural differences positioned toward the viewer. The principal face is shown in yellow; the in certain loops and β-strands. A structure-based sequence complementary face is shown in blue. Lobeline molecules are shown as alignment of Ct-AChBP and Ac-AChBP is shown in Fig. S2A. spheres, and the glycosyl chain N-linked to N122 is shown in magenta ball- Cartoon representations with magnified insets of superimposed and-stick representation. Inset shows a detail of 2Fo-Fc electron density con- σ monomers are shown for Ct-AChBP (magenta) and Ac-AChBP toured around the glycosyl chain at a level of 1.0. The paucimannose chain is composed of N-acetylglucosamine (NAG), α-D-mannose (α-MAN), and β-D- (yellow) in Fig. S2B. Notable differences between the two struc- mannose (β-MAN). (D) Cartoon representation of Ct-AChBP in complex with tures (highlighted in blue boxes) include a longer turn between the varenicline, in the same orientation as Ct-AChBP in A. Varenicline is shown as α β N-terminal -helix and the 1-strand, F27-L28-D29, and the Cys- spheres, and Inset shows a detail of 2Fo-Fc electron density contoured around loop and β1-β2 loop, which form part of the interface between the varenicline (ball-and-stick representation) at a σ level of 2.0.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1116397109 Billen et al. Downloaded by guest on September 28, 2021 data show that the overall protein architecture is well conserved A remarkable difference between Ct-AChBP and Ac-AChBP despite the relatively low sequence identity between Ct-AChBP binding pockets is the lack of the highly conserved aromatic and Ac-AChBP. Next, we investigated whether some of the residue in loop D (Y53 in Ac-AChBP and I64 in Ct-AChBP). In structural differences at the ligand binding site could explain the addition, superposition of Ct-AChBP and Ac-AChBP binding observed changes in pharmacological properties in Ct-AChBP pockets reveals that the lobeline molecule adopts different compared with other AChBPs (Table 1). configurations in Ct-AChBP and Ac-AChBP (Fig. 2C). In Ct- AChBP, the lobeline ring adopts a chair conforma- Lobeline and Varenicline Recognition in the Ct-AChBP Binding Pocket. tion, versus a half-chair conformation in Ac-AChBP. In addition, The ligand binding site of Ct-AChBP is found at the interface the hydroxyl group is in the (S)-configuration in Ct-AChBP, between two subunits and ligands interact with amino acids from versus the (R)-configuration in Ac-AChBP. As a result, the lo- both the principal face (containing loops A, B, and C) and beline configuration observed in Ct-AChBP corresponds to the complementary face (containing loops D, E, and F). Lobeline α-lobeline configuration, whereas the configuration observed in contacts residues from the principal and complementary face of Ac-AChBP is the opposite stereochemical configuration. These the binding pocket in Ct-AChBP resembling interactions in the results demonstrate that subtle differences exist in the binding complex with Ac-AChBP (Fig. 2 A and B and Table S2). In the pocket architecture of Ct-AChBP and other AChBPs, which complex of Ct-AChBP, the tertiary amine of the piperidine ring might account for different ligand binding properties (Table 1). fi fl of lobeline is stabilized through cation–π interactions with con- In addition, lobeline is suf ciently exible to adapt to subtle served aromatic residues of the principal face, including W153 differences in the AChBP binding pocket architecture and sug- (loop B) and Y201 (loop C). Similar to the complex in Ac- gests it might also do so in different Cys-loop receptors. AChBP (23), lobeline exposes a subpocket in Ct-AChBP by Interactions of varenicline with residues of the principal and changing the rotameric state of F102 (loop A) from the g-tot- complementary binding site in Ct-AChBP are shown in Fig. 3A conformation (homologous to Y91 in Ac-AChBP; ref. 24). Lo- and compared with interactions of nicotine (25) [Protein Data beline forms four hydrogen bonds that converge upon residues of Bank (PDB) ID code 1UW6; Fig. 3B] and carbamylcholine (25) (PDB ID code 1UV6; Fig. 3C) in complex with Ls-AChBP loop B (shown as dashed lines in Fig. 2A). The lobeline hydroxyl (Table S2). In the X-ray crystal structure of Ct-AChBP, vareni- group interacts with the S152 and W153 backbone oxygens. The cline forms interactions on the principal face of the binding site tertiary amine of the piperidine ring binds with the W153 with aromatic amino acids, which are highly conserved among backbone oxygen, and the lobeline carbonyl oxygen interacts

different nAChR subtypes. On the complementary face of the PHARMACOLOGY with the W153 indole nitrogen. The interactions on the com- binding site, varenicline forms mostly hydrophobic interactions plementary face are predominantly hydrophobic and include with residues that are weakly conserved. In Ct-AChBP, these contacts with I118, L126, and I128 (loop E). The lobeline car- residues on the complementary face of the binding site are I118, bonyl oxygen also interacts with the backbone nitrogen of I128 L126, and I128 in loop E and I64 in loop D (Fig. 3A). In- through a water molecule that occupies the same position as terestingly, varenicline also exposes a subpocket in Ct-AChBP by observed in the lobeline complex with Ac-AChBP (Fig. 2B). changing the rotameric state of F102 (loop A) from the g-tot- conformation, which we also observed in the lobeline-bound structure (F102 is homologous to Y89 in Ls-AChBP; Fig. 3 B and C). Also similar to lobeline, varenicline forms cation–π inter- actions and multiple hydrogen bonds that converge on residues of loop B, one of which involves a water molecule that forms a bridge between the varenicline nitrogen and the W153 carbonyl oxygen. A second water molecule forms hydrogen bonds between the varenicline pyrazino nitrogen and the back- bone nitrogen and oxygen of I128 and Q116, respectively. Re- markably, this water molecule occupies a position that is almost identical to the water molecule observed between nicotine and residues L102 and M114 in the complex with Ls-AChBP, and the water molecule in the lobeline complexes with Ct-AChBP (Fig. 2A) and Ac-AChBP (Fig. 2B). This result indicates that lobeline, varenicline, and nicotine, which act as partial agonists at α4β2 nAChRs, share a common molecular recognition that involves water molecules acting as a bridge with residues of the binding pocket. In contrast, carbamylcholine (Fig. 3C), which acts as a full agonist at α4β2 nAChRs, forms interactions with the resi- dues L102, L112, and M114 on the complementary face that are mainly hydrophobic and lack the contribution of water molecules to these interactions. These results confirm that the nature of ligand interactions with residues of the complementary face possibly contribute to discrimination between full and partial agonists in different Cys-loop receptors (26). Fig. 2. Molecular recognition of lobeline. (A) Amino acid interactions of We have analyzed conformational changes in loop C upon lobeline in Ct-AChBP. The principal face is shown in yellow; the comple- binding of agonists, partial agonist, and antagonist in more than mentary face is shown in blue. Side chains are shown in ball-and-stick rep- 30 AChBP crystal structures solved to date (27). Consistent with resentation (yellow for principal face; pink for the complementary face). this analysis, we find that varenicline produces a contraction of Dashed lines indicate hydrogen bonds. A water molecule between lobeline ± and I128 is represented as a red sphere. (B) Amino acid interactions of lo- loop C (7.79 0.08 Å; Fig. 3D), which is intermediate between full agonists (<7 Å distance) and the extended state observed for beline in Ac-AChBP (PDB ID code 2BYS). (C) Lobeline adopts different con- > figurations in the Ct-AChBP and Ac-AChBP pocket. The configuration in Ct- antagonists ( 11 Å distance) (27). For example, the previously AChBP (yellow) corresponds to α-lobeline, whereas the configuration in characterized partial agonist in complex with Ac- Ac-AChBP (white) is the opposite stereochemical configuration. AChBP (26) produces a contraction of loop C of 7.96 ± 0.30 Å

Billen et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 alignments of AChBPs seeded with human β2 subunit nAChR sequences to identify homologous residues of varenicline contact sites (I118, L126, and I128 in loop E and I64 in loop D) in Ct- AChBP (Fig. 3E). These residues likely contribute to αβ and ββ interfaces in α4β2 nAChRs (21, 28, 29). In the human β2 subunit, these residues on the complementary face of the binding site correspond to V111, F119, and L121 (loop E) and W57 (loop D). Because the smoking cessation action through α4β2 nAChRs has been best described for varenicline (9), we compared relative affinity and efficacy for varenicline and desensitization by vare- nicline and the full agonist acetylcholine. Using two-electrode voltage-clamp recordings from oocytes, we measured ligand-ac- tivated currents of nAChRs composed of wild-type α4 subunits coexpressed with wild-type β2 subunits or four different β2 mutants, namely W57A, V111F, F119A, and L121F. To in- vestigate changes in the volume of the binding pocket and im- portance of aromaticity, we mutated residues to if nonaromatic and to alanine if aromatic. Examples of current responses evoked by various concentrations of varenicline are shown in Fig. 4 A–D. In agreement with previous studies, varenicline activates α4β2 nAChRs with an EC50 value of 1.0 ± 0.3 μM(n =3–11) and low efficacy (14.7 ± 0.6%, n =3–11) compared with the full agonist acetylcholine (EC50 =56.4± 9.1 μM, efficacy = 100%, n =4–10). Consistent with previous studies (9, 30), we observe that vareni- cline concentrations between 3 and 300 nM desensitize ACh-in- duced responses of wild-type α4β2 nAChRs (Fig. 4A), without causing significant channel opening when applied alone in this concentration range (9, 30). This effect on desensitization of α4β2 nAChRs is thought to occur at clinically effective concentrations of varenicline and, likely, contributes to smoking cessation relief Fig. 3. Molecular recognition of varenicline and comparison with nicotine by reduction of craving during nicotine abstinence (9). In contrast, and carbamylcholine. Amino acid interactions observed for varenicline at concentrations more than 300 nM, varenicline alone produces bound to Ct-AChBP (A), nicotine bound to Ls-AChBP (B; PDB ID code 1UW6), channel opening with high affinity, but low efficacy relative to the and carbamylcholine bound to Ls-AChBP (C; PDB ID code 1UV6). (D) Super- response obtained with a saturating concentration of the full ag- position and different C loop conformations observed for AChBP complexes onist acetylcholine (Fig. 4B). This effect likely reduces nicotine with varenicline (blue; Ct-AChBP), nicotine (green; Ls-AChBP) and α-con- reinforcement during smoking lapses, because varenicline binds otoxin ImI (red; Ac-AChBP). Varenicline is shown in white sticks. (E) Residues fi α β of the complementary face of Ct-AChBP involved in ligand interactions with with higher af nity to 4 2 nAChRs than nicotine (9). varenicline were mapped onto a structure-based sequence alignment of When wild-type α4 subunits are coexpressed with W57A β2 AChBPs from different species that was seeded with human nAChR subunits, we observe that the mutation abolishes varenicline- sequences. Residues are colored in shades of blue by using an identity mediated desensitization of ACh responses of these mutant α4β2 threshold of 50%. Residues involved in critical contacts with varenicline are nAChRs (Fig. 4 C and F). This result suggests that the loop D indicated in black. Ct, C. teleta;Ac,Aplysia californica; Ls, Lymnaea stagnalis; residue W57, which is homologous to I64 in Ct-AChBP, forms Bt, Bulinus truncatus;Hs,Homo sapiens. Numbers above the sequence a critical interaction with varenicline to mediate transitions to alignment correspond to amino acid numbering for Ct-AChBP; numbers a densensitized state of the α4β2 nAChRs. In addition, we ob- below the alignment correspond to the human β2 nAChR. serve that the W57A mutation eliminates the relative difference in efficacy of channel opening compared with the full agonist α β (27), which is very similar to varenicline in complex with Ct- acetylcholine (Fig. 4 D and F). Three other mutations in 4 2 AChBP. This result indicates that, similar to other partial agonists nAChRs at positions homologous to those involved in vareni- cline contacts in Ct-AChBP have similar, but less pronounced cocrystallized with AChBPs, varenicline causes an incomplete fi contraction of loop C around the binding site, which may con- effects on varenicline ef cacy compared with the W57A muta- tion. L121F produces channel opening by varenicline with an tribute to limited efficacy of channel opening in α4β2 nAChRs. efficacy of 81.9 ± 4.4% of ACh (n =7–15), for V111F the effi- Similar to the lobeline complex with Ac-AChBP, we find that cacy is 39.2 ± 0.5% (n =4–13), and for F119A the efficacy is lobeline produces an exceptionally strong contraction, usually 39.2 ± 0.5% (n =4–13) (summarized in Fig. 4E). Remarkably, associated with full agonists, of loop C around the ligand binding these point mutations have little effect on the affinity of vare- site in Ct-AChBP. The distance measured between the carbonyl nicline compared with the pronounced changes in efficacy (Table oxygen of the conserved loop B aromatic residue (W153 in Ct- S3). W57A (EC50 =2.0± 0.3 μM, n =4–11) and L121F AChBP and W145 in Ac-AChBP) and the tip of loop C (γSatom (EC50 = 2.6 ± 0.5 μM, n =7–15) result in a small decrease in of C196 in Ct-AChBP and C188 in Ac-AChBP) is 6.54 ± 0.06 Å affinity for varenicline, whereas F119A exhibits a higher affinity ± in the lobeline complex with Ct-AChBP and 6.74 0.34 Å in the (EC50 = 0.6 ± 0.2 μM, n =4–9). V111F does not significantly Ac-AChBP complex. Together, these results indicate that for alter the affinity of varenicline (EC50 = 1.3 ± 0.1 μM, n =4–13). most ligands, the extent of C-loop contraction correlates to their Together, these results illustrate the predictive value of the co- mode of action at nAChRs, but exceptions exist, such as lobeline. crystal structure of Ct-AChBP with varenicline. The pronounced effects of homologous mutations in the β2 nAChR subunit on Structure-Guided Mutagenesis Analysis of Varenicline Interactions in receptor desensitization and efficacy of channel opening by var- Human α4β2 nAChRs. To investigate the importance of varenicline enicline indicate that the binding mode in α4β2 nAChRs is likely interactions in α4β2 nAChRs, we used structure-based sequence similar to the one observed in the crystal structure. In particular,

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1116397109 Billen et al. Downloaded by guest on September 28, 2021 channel opening with lower probability than full agonists at equal levels of receptor occupancy. Partial agonists open these chan- nels with low efficacy through a mechanism that is common to different members of the Cys-loop receptor family. Using single channel recordings from the muscle nAChR and GlyR, Lape et al. (31) demonstrated that partial and full agonists have sim- ilar kinetics for the closed to open transition of the channel pore, but that the response to partial agonists is limited by an early conformational change, termed flipping, that precedes channel opening. At the structural level, flipping possibly corresponds to an early conformational change after ligand recognition by the extracellular ligand-binding domain, whereas the ion channel domain is still closed, similar to domain closure observed for glutamate receptors (32, 33). Despite these insights, the precise interactions of partial agonists with the receptor binding site are not understood. From new crystal structures of lobeline and varenicline in complex with Ct-AChBP, together with a comparative analysis of more than 30 existing AChBP structures (27), common structural and functional properties of partial agonists start to emerge. Typically, partial agonists stabilize loop C in a conformation that is intermediate between the fully contracted state observed for full agonists and the extended state observed for antagonists (26, 27). It is likely that low efficiency of flipping by partial agonists for the muscle nAChR and GlyR as described by Lape et al. (31) can be understood in terms of incomplete contraction of loop C as described in crystal structures of AChBP.

In combination with the conformational stabilization of loop PHARMACOLOGY C, the detailed molecular interactions of partial agonists with residues of the binding pocket appear equally important. Be- cause most of the residues on the principal subunit are highly conserved among different nAChR subtypes, we focused our attention to the variable residues on the complementary subunit, which likely mediates partial agonist effects of varenicline in certain nAChR subtypes, including the important α4β2 subtype. In the crystal structure of Ct-AChBP in complex with varenicline, a water molecule that occupies a common position in nicotine Fig. 4. Mutation of homologous residues in α4β2 nAChRs affects de- fi and lobeline complexes with AChBP plays a key role in estab- sensitization and ef cacy of channel opening. (A) Example traces from lishing hydrogen bonds with the backbone atoms of hydrophobic oocytes expressing wild-type α4β2 and activated with a fixed concentration μ residues of loop E. In addition, a highly conserved aromatic of acetylcholine (30 M) and desensitized by increasing concentrations of α β fi varenicline (0.01–300 nM). (B) When applied alone, concentrations of vare- residue of loop D, W57 in 4 2, also plays a key role in de ning nicline more than 300 nM cause channel opening, but with low efficacy the architecture of the complementary subunit. The critical compared with 1 mM acetylcholine. (C) Desensitization of mutant α4β2 contribution of this residue has been demonstrated in other Cys- α β nAChRs, obtained by coexpression of wild-type 4-subunits and W57A 2- loop receptors, including α7 nicotinic receptors (34), GABAA subunits, is abolished. (D) The W57A mutation eliminates the relative dif- receptors (35), 5-HT3 receptors (36–38), and glycine receptors ference in efficacy of channel opening between the partial agonist vareni- (39). Consistent with earlier observations (26), we took the as- cline and the full agonist acetylcholine. (E) Bar graph summarizes the effect fi fi sumption that these speci c contacts of ligand and binding site of mutations on the relative ef cacy of channel opening by varenicline residues might be of key importance for discriminating agonists normalized against the saturating response with acetylcholine on each mutant α4β2 nAChRs (*P < 0.01). (F) Concentration-activation (solid lines) from partial agonists. We determined the relative contribution of and concentration-inhibition (dashed lines) curves for varenicline on wild- these interactions to partial agonist effects of varenicline by type (red) and W57A receptors (green). The activation responses are nor- mutating homologous positions of the complementary subunit at malized to the maximum response to 1 mM acetylcholine (=1). Inhibition the high-affinity α4/β2 interface in α4β2 nAChRs and comparing responses are normalized to the half-maximal response to 30 μM acetyl- the effect of mutations on the efficacy of channel opening rela- (=0.5). (G) Models illustrate characteristic differences between tive to the full agonist acetylcholine. We found that mutations antagonists (red), partial agonists (blue), and full agonists (green). Together W57A (loop D) and L121F (loop E) have the most pronounced with an intermediate contraction of loop C (magenta), the interactions with effects and almost completely eliminate the difference in efficacy W57 and L121 (solid lines for varenicline), critically determine the rate of between varenicline and acetylcholine. These results indicate desensitization and limit the efficacy of channel opening by varenicline. that both interactions with loop D and E contribute to efficiency of channel opening. Less pronounced effects were observed for the interactions of varenicline with W57 and L121 on the com- mutations V111F and F119A in loop E, indicating that these plementary binding site of the β2 nAChR subunit possibly have interactions are less critical for this. essential contributions to mediate transitions to a desensitized The molecular property of varenicline to mediate channel receptor state and to limit the efficacy of channel opening by the opening of α4β2 nAChRs with high affinity but low efficacy is partial agonist varenicline. believed to contribute to the clinical effects during smoking cessation relief because it limits the effects of nicotine during Discussion moments of smoking relapse. In addition, at concentrations even Partial agonism at pentameric ligand-gated ion channels arises lower than those required for channel opening, varenicline also from a fundamental molecular property, which is to trigger causes pronounced receptor desensitization, an effect that

Billen et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 minimizes reward sensation mediated through signaling path- channel opening by varenicline and the full agonist acetylcholine. ways involving α4β2 nAChRs. We observed that the mutation In combination with an intermediate contraction of loop C, we W57A (loop D) abolishes receptor desensitization of α4β2 at low suggest that these interactions define the molecular recognition of occupancy of varenicline, indicating that the interaction with partial agonists and function as key recognition events that pre- W57 is critical in mediating a transition to a desensitized state. cede channel opening, previously described as flipping. Together, fi Based on our ndings, we propose a model (Fig. 4G) in which these data provide detailed insight into the molecular mechanism the molecular action of the smoking cessation aid varenicline underlying partial agonism at nAChRs. This study opens per- relies on (i) incomplete contraction of loop C around the ligand spectives for the design of new partial agonists to treat disorders binding site, (ii) critical hydrophilic interactions with residues of fi loop E, which occur through a water molecule in the ligand associated with speci c Cys-loop receptor subtypes. binding site, and (iii) interactions with the highly conserved ar- Methods omatic residue of loop D, which play a pivotal role in mediating fi transitions to channel states with low open probability. Ct-AChBP was expressed in Sf21 insect cells and puri ed on nickel Sepharose and Superdex 200 (GE Healthcare). Crystallization trials were carried out by vapor In summary, we show high-resolution crystal structures of a α unique AChBP with structural features and pharmacological diffusion in the presence of 1 mM -lobeline hydrochloride (Sigma-Aldrich) or properties distinct from previously identified AChBPs. The high- 1 mM varenicline hydrochloride. Details on X-ray crystallography, radioligand binding assays, and electrophysiological recordings are described in SI Methods. affinity binding of the smoking cessation aids lobeline and vare- α β nicline, which act as partial agonists at 4 2 nAChRs, suggests ACKNOWLEDGMENTS. We thank Jon Lindstrom for α4β2 cDNAs, Ivy Carroll that Ct-AChBP is a useful model to study atomic interactions for for varenicline hydrochloride, Jan Tytgat for Xenopus oocytes and local con- this receptor subtype. Using structure-guided mutagenesis, we tacts at the European Synchrotron Radiation Facility, and SOLEIL for assis- revealed the importance of key interactions in loop D and E of the tance during data collection. Nathaniel Clark and Ewald Edink contributed α β α β fi to discussion of results. Financial support was from KULeuven OT/08/048 (to 4/ 2 subunit interface of 4 2 nAChRs. We identi ed single C.U.), the European Union Seventh Framework Programme under Grant point mutations in the binding pocket that abolish receptor de- Agreement HEALTH-F2-2007-202088 (to C.U., A.B.S., and D.B.), and Intramu- sensitization and eliminate the relative difference in efficacy of ral Research Program of the National Institutes of Health (J.L.Y.).

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