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Key Structural Determinants in the Agonist Binding Loops of Human ?2 and ?4 Nicotinic Acetylcholine Receptor Subunits Contribute to ?3?4 Subtype Selectivity of ?-Conotoxins.

ABSTRACT: ?-Conotoxins represent a large group of pharmacologically active peptides that antagonize nicotinic acetylcholine receptors (nAChRs). The ?3?4 nAChR, a predominant subtype in the peripheral nervous system, has been implicated in various pathophysiological conditions. As many ?-conotoxins have multiple pharmacological targets, compounds specifically targeting individual nAChR subtypes are needed. In this study, we performed mutational analyses to evaluate the key structural components of human ?2 and ?4 nAChR subunits that determine ?-conotoxin selectivity for ?3?4 nAChR. ?-Conotoxin RegIIA was used to evaluate the impact of non-conserved human ?2 and ?4 residues on peptide affinity. Two mutations, ?3?2[T59K] and ?3?2[S113R], strongly enhanced RegIIA affinity compared with wild-type ?3?2, as seen by substantially increased inhibitory potency and slower off-rate kinetics. Opposite point mutations in ?3?4 had the contrary effect, emphasizing the importance of loop D residue 59 and loop E residue 113 as determinants for RegIIA affinity. Molecular dynamics simulation revealed the side chains of ?4 Lys59 and ?4 Arg113 formed hydrogen bonds with RegIIA loop 2 atoms, whereas the ?2 Thr59 and ?2 Ser113 side chains were not long enough to form such interactions. Residue ?4 Arg113 has been identified for the first time as a crucial component facilitating antagonist binding. Another ?-conotoxin, AuIB, exhibited low activity at human ?3?2 and ?3?4 nAChRs. Molecular dynamics simulation indicated the key interactions with the ? subunit are different to RegIIA. Taken together, these data elucidate the interactions with specific individual ? subunit residues that critically determine affinity and pharmacological activity of ?-conotoxins RegIIA and AuIB at human nAChRs.

SUBMITTER: Cuny H

PROVIDER: S-EPMC5095430 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins.

Cuny Hartmut H Kompella Shiva N SN Tae Han-Shen HS Yu Rilei R Adams David J DJ

The Journal of biological chemistry 20160919 45

α-Conotoxins represent a large group of pharmacologically active peptides that antagonize nicotinic acetylcholine receptors (nAChRs). The α3β4 nAChR, a predominant subtype in the peripheral nervous system, has been implicated in various pathophysiological conditions. As many α-conotoxins have multiple pharmacological targets, compounds specifically targeting individual nAChR subtypes are needed. In this study, we performed mutational analyses to evaluate the key structural components of human β2 ...[more]

PMID: 27646000

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Project description:Nicotinic acetylcholine receptor (nAChR) subtypes are key drug targets, but it is challenging to pharmacologically differentiate between them because of their highly similar sequence identities. Furthermore, α-conotoxins (α-CTXs) are naturally selective and competitive antagonists for nAChRs and hold great potential for treating nAChR disorders. Identifying selectivity-enhancing mutations is the chief aim of most α-CTX mutagenesis studies, although doing so with traditional docking methods is difficult due to the lack of α-CTX/nAChR crystal structures. Here, we use homology modeling to predict the structures of α-CTXs bound to two nearly identical nAChR subtypes, α3β2 and α3β4, and use free-energy perturbation (FEP) to re-predict the relative potency and selectivity of α-CTX mutants at these subtypes. First, we use three available crystal structures of the nAChR homologue, acetylcholine-binding protein (AChBP), and re-predict the relative affinities of twenty point mutations made to the α-CTXs LvIA, LsIA, and GIC, with an overall root mean square error (RMSE) of 1.08 ± 0.15 kcal/mol and an R2 of 0.62, equivalent to experimental uncertainty. We then use AChBP as a template for α3β2 and α3β4 nAChR homology models bound to the α-CTX LvIA and re-predict the potencies of eleven point mutations at both subtypes, with an overall RMSE of 0.85 ± 0.08 kcal/mol and an R2 of 0.49. This is significantly better than the widely used molecular mechanics-generalized born/surface area (MM-GB/SA) method, which gives an RMSE of 1.96 ± 0.24 kcal/mol and an R2 of 0.06 on the same test set. Next, we demonstrate that FEP accurately classifies α3β2 nAChR selective LvIA mutants while MM-GB/SA does not. Finally, we use FEP to perform an exhaustive amino acid mutational scan of LvIA and predict fifty-two mutations of LvIA to have greater than 100X selectivity for the α3β2 nAChR. Our results demonstrate the FEP is well-suited to accurately predict potency- and selectivity-enhancing mutations of α-CTXs for nAChRs and to identify alternative strategies for developing selective α-CTXs.

Dataset Information

Key Structural Determinants in the Agonist Binding Loops of Human ?2 and ?4 Nicotinic Acetylcholine Receptor Subunits Contribute to ?3?4 Subtype Selectivity of ?-Conotoxins.

Publications

Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins.

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