Basic Residues at Position 11 of α-Conotoxin LvIA Influence Subtype Selectivity between α3β2 and α3β4 Nicotinic Receptors via an Electrostatic Mechanism

Yves Haufe, Veeresh Kuruva, Ziyana Samanani, Gonxhe Lokaj, Guy Kamnesky, Pranav Kumar Shadamarshan, Rezvan Shahoei, Dana Katz, Jared M. Sampson, Michael Pusch, Ashraf Brik, Annette Nicke, Abba E. Leffler

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Understanding the determinants of α-conotoxin (α-CTX) selectivity for different nicotinic acetylcholine receptor (nAChR) subtypes is a prerequisite for the design of tool compounds to study nAChRs. However, selectivity optimization of these small, disulfide-rich peptides is difficult not only because of an absence of α-CTX/nAChR co-structures but also because it is challenging to predict how a mutation to an α-CTX will alter its potency and selectivity. As a prototypical system to investigate selectivity, we employed the α-CTX LvIA that is 25-fold selective for the α3β2 nAChR over the related α3β4 nAChR subtype, which is a target for nicotine addiction. Using two-electrode voltage clamp electrophysiology, we identified LvIA[D11R] that is 2-fold selective for the α3β4 nAChR, reversing the subtype preference. This effect is specifically due to the change in charge and not shape of LvIA[D11R], as substitution of D11 with citrulline retains selectivity for the α3β2 nAChR. Furthermore, LvIA[D11K] shows a stronger reversal, with 4-fold selectivity for the α3β4 nAChR. Motivated by these findings, using site-directed mutagenesis, we found that β2[K79A] (I79 on β4), but not β2[K78A] (N78 on β4), largely restores the potency of basic mutants at position 11. Finally, to understand the structural basis of this effect, we used AlphaFold2 to generate models of LvIA in complex with both nAChR subtypes. Both models confirm the plausibility of an electrostatic mechanism to explain the data and also reproduce a broad range of potency and selectivity structure-activity relationships for LvIA mutants, as measured using free energy perturbation simulations. Our work highlights how electrostatic interactions can drive α-CTX selectivity and may serve as a strategy for optimizing the selectivity of LvIA and other α-CTXs.

Original languageEnglish
Pages (from-to)4311-4322
Number of pages12
JournalACS Chemical Neuroscience
Volume14
Issue number24
DOIs
StatePublished - 20 Dec 2023
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2023 American Chemical Society.

Funding

The computational aspects of this investigation were sponsored and financially supported by the authors’ employer, Schrödinger, Inc. (Z.S., R.S., D.K., J.M.S., and A.E.L.). This project has received funding from the Israel Science Foundation (ISF) (grant agreement no. [521/19]). M.P. has been supported by the Fondazione AIRC per la Ricerca sul Cancro (grant IG 21558) and Fondazione Telethon (grant GMR22T1029). The electrophysiological work was supported by a grant from the DFG (Research Training Group GRK2338, P01) and the DAAD (German Academic Exchange Service, PPP Project-ID 57388586) to A.N.

FundersFunder number
Fondazione AIRC per la ricerca sul cancro ETSIG 21558
Deutscher Akademischer AustauschdienstPPP Project-ID 57388586
Deutsche ForschungsgemeinschaftGRK2338
Fondazione TelethonGMR22T1029
Israel Science Foundation521/19

    Keywords

    • AlphaFold2
    • conotoxin
    • free energy perturbation
    • nicotinic acetylcholine receptor
    • peptide synthesis
    • selectivity

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