Structural mechanisms for α-conotoxin activity at the human α3β4 nicotinic acetylcholine receptor

Abstract

Nicotinic acetylcholine receptors (nAChR) are therapeutic targets for a range of human diseases. α-Conotoxins are naturally occurring peptide antagonists of nAChRs that have been used as pharmacological probes and investigated as drug leads for nAChR related disorders. However, α-conotoxin interactions have been mostly characterised at the α7 and α3β2 nAChRs, with interactions at other subtypes poorly understood. This study provides novel structural insights into the molecular basis for α-conotoxin activity at α3β4 nAChR, a therapeutic target where subtype specific antagonists have potential to treat nicotine addiction and lung cancer. A co-crystal structure of α-conotoxin LsIA with Lymnaea stagnalis acetylcholine binding protein guided the design and functional characterisations of LsIA analogues that identified the minimum pharmacophore regulating α3β4 antagonism. Interactions of the LsIA R10F with β4 K57 and the conserved -NN- α-conotoxin motif with β4 I77 and I109 conferred α3β4 activity to the otherwise inactive LsIA. Using these structural insights, we designed LsIA analogues with α3β4 activity. This new understanding of the structural basis of protein-protein interactions between α-conotoxins and α3β4 may help rationally guide the development of α3β4 selective antagonists with therapeutic potential.

Figure 1. LsIA/ Ls-AChBP co-crystal structure.

(a) LsIA-NH2 was co-crystallised with Ls-AChBP. Clear electron density for the ligand was seen in all five binding pockets.(2Fo-Fc) map for the ligand countoured to 1.0 σ is shown. (b) LsIA binds to the orthosteric binding pocket with the α-helical backbone buried deep within the pocket, the N-terminus oriented to the bottom and C-terminus to the top of the pocket. Within the binding pocket LsIA adopts the typical α-conotoxin binding orientation, as can be seen from the superimposition of LsIA backbone with that of previously co-crystallised α-conotoxins PnIA(A10L,D14K), ImI and TxIA. (c) The receptor ligand interactions are characterised by several hydrogen bonds and some hydrophobic interactions (dotted lines indicate hydrogen bonds). Interactions LsIA R10 and N12 (*) were investigated in this study. These interactions were found to be important for LsIA activity at the α3β4 subtype.

Figure 2. LsIA R10 interactions at the human α7, α3β2 and α3β4 nAChR subtypes.

(a) The homology model of the α7 receptor was generated based on the Ls-AChBP/LsIA co-crystal structure. The residues constituting the surface interacting with LsIA R10 are similar in both the α7 and the Ls-AChBP. Therefore, it is likely that the LsIA R10 engages in interactions similar to those seen in the crystal structure. (b) Residues on the human α3β2 and α3β4 subtype forming the surface that interacts with the LsIA R10 are shown. The interacting surface on α3β2 consists of hydrophobic residues with the exception of T57 and S166, which are outside of hydrogen bonding distance. On the α3β4 interacting surface residue K57 lies in close proximity (2.6 Å) to LsIA R10. This is thought to contribute to the inactivity of LsIA at this subtype.

Figure 3. LsIA N12 interactions at Ls-AChBP and the human α7, α3β2 and α3β4.

Residues constituting the interacting surface for LsIA N12 on Ls-AChBP as seen in the structure and the corresponding residues in α7, α3β2 and α3β4 are shown. The more extensive hydrophobic patch on α3β4 contributes to the enhanced affinity of the N12L analogue at this subtype.

Figure 4. Functional characterisation of LsIA analogues at AChBPs, α7 and α3β4 nAChRs and Q55K mutant AChBP.

(a,b) Displacement of ³H-epibatidine from Ac and Ls-AChBP by R10 and N12 analogues of LsIA. (c,d) Concentration response curves for LsIA analogues at the α7 and α3β4 nAChRs. (e) Displacement of ³H-epibatidine from Q55K mutant Ls-AChBP by LsIA and LsIA-R10 analogues. Data represent the mean ± S.E.M of triplicate data from three independent experiments.

Figure 5. α3β4 pharmacophore.

(a) α3β4 activity was successfully introduced into LsIA through systematic modification of interactions at position 10 and 12. The [R10F][N12L]-LsIA provided a > 250-fold selectivity for α3β4 over α7 nAChR. Data represent the mean ± S.E.M of triplicate data from three independent experiments. (b) The highly conserved aromatic cage involved in ligand recognition at the nAChRs is shown in black. Using α-conotoxin LsIA we have identified residues (boxed) that lie outside this conserved aromatic cage and contribute to ligand recognition at the α3β4.