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Structure of the pentameric ligand-gated ion channel ELIC cocrystallized with its competitive antagonist acetylcholine.

Pan J, Chen Q, Willenbring D, Yoshida K, Tillman T, Kashlan OB, Cohen A, Kong XP, Xu Y, Tang P - Nat Commun (2012)

Bottom Line: The side chain of the pore-lining residue F247 reorients and the pore size consequently enlarges, but the channel remains closed.We attribute the inability of acetylcholine to activate ELIC primarily to weak cation-π and electrostatic interactions in the pocket, because an acetylcholine derivative with a simple quaternary-to-tertiary ammonium substitution activates the channel.This study presents a compelling case for understanding the structural underpinning of the functional relationship between agonism and competitive antagonism in the Cys-loop receptors, providing a new framework for developing novel therapeutic drugs.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, 2057 Biomedical Science Tower 3, 3501 Fifth Avenue, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260, USA.

ABSTRACT
ELIC, the pentameric ligand-gated ion channel from Erwinia chrysanthemi, is a prototype for Cys-loop receptors. Here we show that acetylcholine is a competitive antagonist for ELIC. We determine the acetylcholine-ELIC cocrystal structure to a 2.9-Å resolution and find that acetylcholine binding to an aromatic cage at the subunit interface induces a significant contraction of loop C and other structural rearrangements in the extracellular domain. The side chain of the pore-lining residue F247 reorients and the pore size consequently enlarges, but the channel remains closed. We attribute the inability of acetylcholine to activate ELIC primarily to weak cation-π and electrostatic interactions in the pocket, because an acetylcholine derivative with a simple quaternary-to-tertiary ammonium substitution activates the channel. This study presents a compelling case for understanding the structural underpinning of the functional relationship between agonism and competitive antagonism in the Cys-loop receptors, providing a new framework for developing novel therapeutic drugs.

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Comparison of aromatic residues lining the ligand-binding pocket.(a) ELIC determined in this study (PDB code: 3RQW); (b) the muscle-type nAChR between α- and γ-subunits (PDB code: 2BG9); (c) LS-AChBP (PDB code: 1UV6); and (d) GLIC (PDB code: 3EAM). The principal subunit is coloured in yellow with side chains in orange and the complementary subunit is coloured in marine with side chains in purple.
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f4: Comparison of aromatic residues lining the ligand-binding pocket.(a) ELIC determined in this study (PDB code: 3RQW); (b) the muscle-type nAChR between α- and γ-subunits (PDB code: 2BG9); (c) LS-AChBP (PDB code: 1UV6); and (d) GLIC (PDB code: 3EAM). The principal subunit is coloured in yellow with side chains in orange and the complementary subunit is coloured in marine with side chains in purple.

Mentions: Figure 3 depicts the atomic details of the binding pocket. ACh is in direct contact (within 4 Å) with residues from loop A (E77, I79), loop B (E131, P132, F133) and loop C (Y175, L178, F188) on the principal side, and residues from loop D (Y38), loop E (N103) and loop G (F19) on the complementary side. ACh is framed in an aromatic cage, which is a general feature found in the structures of AChBPs and the ligand-binding domain of nAChRs121314151617181920. Figure 4 exhibits a few examples of residues comprising the aromatic cage. ELIC, nAChR and AChBP have four to five aromatic residues to comprise an aromatic cage. In contrast, GLIC has only one aromatic residue in the principal side. The necessity of having adequate aromatic residues for the ligand binding was also corroborated by our attempt to cocrystallize GLIC with ACh. Although we used the same ACh concentration as used for ELIC crystallization and solved the GLIC structure to a resolution of 3.1 Å (data not shown), no bound ACh was detected. The overall pocket-lining residues in ELIC more closely resemble those in the glycine and GABAA receptors. A sequence alignment of ELIC with several Cys-loop receptors is provided in Supplementary Fig. S1. The negatively charged residues do not exist in the ligand pocket of AChBPs and nAChRs, but are present in some subtypes of glycine and GABAA receptors. E131 in ELIC is equivalent to a conserved glutamate residue (β2-E155) in the GABAA receptors, which is critical in coupling ligand binding to channel gating26. E131 and E77 are close to the positively charged quaternary ammonium of ACh, forming favourable electrostatic interactions (Fig. 3). Aromatic residues lining the pocket on the principal side are close enough to the choline group for potential cation-π interactions. F19 and Y38 on the complementary side provide hydrophobic contacts for the acetyl group of ACh. All these interactions contribute to the stabilization of ligand binding in ELIC. Several specific features in the ELIC ligand pocket, however, may account for the inability of ACh to activate the channel. First, the residue equivalent to I79 in loop A of ELIC is a tyrosine residue in the nAChR family (Supplementary Fig. S1). The hydroxyl of this tyrosine in AChBPs and the α7-AChBP chimera provides a hydrogen-bond interaction with ligands131419. Mutation of an aromatic residue to a non-aromatic or different aromatic residue in the orthosteric ligand pocket of nAChRs resulted in dramatic reductions in the apparent ligand affinities and channel activation2728. Second, the carbonyl oxygen of F133 in the ELIC structure is further away from the C2 atom of the positively charged choline group (3.6 Å) than the distance (3.1 Å) for the CH–O hydrogen bond found in the ligand-bound AChBPs13, indicative of a weaker hydrogen bond. Third, while the distances (4.7–4.8 Å) between the quaternary ammonium of ACh and the aromatic rings of Y175 and F188 are similar to those found in the structure of the ACh-bound AChBP17, F133's aromatic ring is much further away from ACh (5.7 Å) than is the homologous residue W143 (4.5 Å) in ACh–AChBP17, presenting an overall weaker cation-π interaction. Moreover, having a tryptophan residue at the equivalent F133 position was suggested to be much more critical than other aromatic positions to ensure a strong cation-π interaction and a high ligand-binding affinity2829. Taken together, our high-resolution structural data explain why the ACh binding to the homologous orthosteric ligand site cannot activate ELIC.


Structure of the pentameric ligand-gated ion channel ELIC cocrystallized with its competitive antagonist acetylcholine.

Pan J, Chen Q, Willenbring D, Yoshida K, Tillman T, Kashlan OB, Cohen A, Kong XP, Xu Y, Tang P - Nat Commun (2012)

Comparison of aromatic residues lining the ligand-binding pocket.(a) ELIC determined in this study (PDB code: 3RQW); (b) the muscle-type nAChR between α- and γ-subunits (PDB code: 2BG9); (c) LS-AChBP (PDB code: 1UV6); and (d) GLIC (PDB code: 3EAM). The principal subunit is coloured in yellow with side chains in orange and the complementary subunit is coloured in marine with side chains in purple.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3316889&req=5

f4: Comparison of aromatic residues lining the ligand-binding pocket.(a) ELIC determined in this study (PDB code: 3RQW); (b) the muscle-type nAChR between α- and γ-subunits (PDB code: 2BG9); (c) LS-AChBP (PDB code: 1UV6); and (d) GLIC (PDB code: 3EAM). The principal subunit is coloured in yellow with side chains in orange and the complementary subunit is coloured in marine with side chains in purple.
Mentions: Figure 3 depicts the atomic details of the binding pocket. ACh is in direct contact (within 4 Å) with residues from loop A (E77, I79), loop B (E131, P132, F133) and loop C (Y175, L178, F188) on the principal side, and residues from loop D (Y38), loop E (N103) and loop G (F19) on the complementary side. ACh is framed in an aromatic cage, which is a general feature found in the structures of AChBPs and the ligand-binding domain of nAChRs121314151617181920. Figure 4 exhibits a few examples of residues comprising the aromatic cage. ELIC, nAChR and AChBP have four to five aromatic residues to comprise an aromatic cage. In contrast, GLIC has only one aromatic residue in the principal side. The necessity of having adequate aromatic residues for the ligand binding was also corroborated by our attempt to cocrystallize GLIC with ACh. Although we used the same ACh concentration as used for ELIC crystallization and solved the GLIC structure to a resolution of 3.1 Å (data not shown), no bound ACh was detected. The overall pocket-lining residues in ELIC more closely resemble those in the glycine and GABAA receptors. A sequence alignment of ELIC with several Cys-loop receptors is provided in Supplementary Fig. S1. The negatively charged residues do not exist in the ligand pocket of AChBPs and nAChRs, but are present in some subtypes of glycine and GABAA receptors. E131 in ELIC is equivalent to a conserved glutamate residue (β2-E155) in the GABAA receptors, which is critical in coupling ligand binding to channel gating26. E131 and E77 are close to the positively charged quaternary ammonium of ACh, forming favourable electrostatic interactions (Fig. 3). Aromatic residues lining the pocket on the principal side are close enough to the choline group for potential cation-π interactions. F19 and Y38 on the complementary side provide hydrophobic contacts for the acetyl group of ACh. All these interactions contribute to the stabilization of ligand binding in ELIC. Several specific features in the ELIC ligand pocket, however, may account for the inability of ACh to activate the channel. First, the residue equivalent to I79 in loop A of ELIC is a tyrosine residue in the nAChR family (Supplementary Fig. S1). The hydroxyl of this tyrosine in AChBPs and the α7-AChBP chimera provides a hydrogen-bond interaction with ligands131419. Mutation of an aromatic residue to a non-aromatic or different aromatic residue in the orthosteric ligand pocket of nAChRs resulted in dramatic reductions in the apparent ligand affinities and channel activation2728. Second, the carbonyl oxygen of F133 in the ELIC structure is further away from the C2 atom of the positively charged choline group (3.6 Å) than the distance (3.1 Å) for the CH–O hydrogen bond found in the ligand-bound AChBPs13, indicative of a weaker hydrogen bond. Third, while the distances (4.7–4.8 Å) between the quaternary ammonium of ACh and the aromatic rings of Y175 and F188 are similar to those found in the structure of the ACh-bound AChBP17, F133's aromatic ring is much further away from ACh (5.7 Å) than is the homologous residue W143 (4.5 Å) in ACh–AChBP17, presenting an overall weaker cation-π interaction. Moreover, having a tryptophan residue at the equivalent F133 position was suggested to be much more critical than other aromatic positions to ensure a strong cation-π interaction and a high ligand-binding affinity2829. Taken together, our high-resolution structural data explain why the ACh binding to the homologous orthosteric ligand site cannot activate ELIC.

Bottom Line: The side chain of the pore-lining residue F247 reorients and the pore size consequently enlarges, but the channel remains closed.We attribute the inability of acetylcholine to activate ELIC primarily to weak cation-π and electrostatic interactions in the pocket, because an acetylcholine derivative with a simple quaternary-to-tertiary ammonium substitution activates the channel.This study presents a compelling case for understanding the structural underpinning of the functional relationship between agonism and competitive antagonism in the Cys-loop receptors, providing a new framework for developing novel therapeutic drugs.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, 2057 Biomedical Science Tower 3, 3501 Fifth Avenue, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260, USA.

ABSTRACT
ELIC, the pentameric ligand-gated ion channel from Erwinia chrysanthemi, is a prototype for Cys-loop receptors. Here we show that acetylcholine is a competitive antagonist for ELIC. We determine the acetylcholine-ELIC cocrystal structure to a 2.9-Å resolution and find that acetylcholine binding to an aromatic cage at the subunit interface induces a significant contraction of loop C and other structural rearrangements in the extracellular domain. The side chain of the pore-lining residue F247 reorients and the pore size consequently enlarges, but the channel remains closed. We attribute the inability of acetylcholine to activate ELIC primarily to weak cation-π and electrostatic interactions in the pocket, because an acetylcholine derivative with a simple quaternary-to-tertiary ammonium substitution activates the channel. This study presents a compelling case for understanding the structural underpinning of the functional relationship between agonism and competitive antagonism in the Cys-loop receptors, providing a new framework for developing novel therapeutic drugs.

Show MeSH
Related in: MedlinePlus