Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs)

Abraham, Nikita; Paul, Blessy; Ragnarsson, Lotten; Lewis, Richard J.

doi:10.1371/journal.pone.0157363

Cited by 9 publications

(13 citation statements)

References 56 publications

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“…Ls-AChBP and Ac-AChBP was over-expressed and purified as described previously 39 . Ls-AChBP was concentrated to 5 mg/mL for crystallisation trials.…”

Section: Methodsmentioning

confidence: 99%

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

Abraham

Healy

Ragnarsson

et al. 2017

Sci Rep

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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.

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“…Ls-AChBP and Ac-AChBP was over-expressed and purified as described previously 39 . Ls-AChBP was concentrated to 5 mg/mL for crystallisation trials.…”

Section: Methodsmentioning

confidence: 99%

“…Competitive radioligand binding assay with 3 H-epibatidine (specific activity 1.11–2.59 TBq/mmol) were performed as described previously 39 .…”

Section: Methodsmentioning

confidence: 99%

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

Abraham

Healy

Ragnarsson

et al. 2017

Sci Rep

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“…Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that are therapeutic targets for a range of human diseases . Acetylcholine binding protein (AChBP) is the structural and functional homologue of the extracellular ligand-binding domain of nAChRs that has been used as a pharmacological tool to identify novel drug leads for AChR-related disorders . A number of co-crystal structures of Aplysia californica AchBP in complex with different α-conotoxin cyclic peptides have been reported. − A representative α-conotoxin IMI is described here (PDB 2C9T, 2.25 Å).…”

Section: Cyclic Peptide-bound Proteinsmentioning

confidence: 99%

Crystal Structures of Protein-Bound Cyclic Peptides

et al. 2019

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Cyclization is an important post-translational modification of peptides and proteins that confers key advantages such as protection from proteolytic degradation, altered solubility, membrane permeability, bioavailability, and especially restricted conformational freedom in water that allows the peptide backbone to adopt the major secondary structure elements found in proteins. Non-ribosomal synthesis in bacteria, fungi, and plants or synthetic chemistry can introduce unnatural amino acids and non-peptidic constraints that modify peptide backbones and side chains to fine-tune cyclic peptide structure. Structures can be potentially altered further upon binding to a protein in biological environments. Here we analyze three-dimensional crystal structures for 211 bioactive cyclic peptides bound to 65 different proteins. The protein-bound cyclic peptides were examined for similarities and differences in bonding modes, for main-chain and side-chain structure, and for the importance of polarity, hydrogen bonds, hydrophobic effects, and water molecules in interactions with proteins. Many protein-bound cyclic peptides show backbone structures like those (strands, sheets, turns, helices, loops, or distorted variations) found at protein−protein binding interfaces. However, the notion of macrocycles simply as privileged scaffolds that primarily project side-chain substituents for complementary interactions with proteins is dispelled here. Unlike small-molecule drugs, the cyclic peptides do not rely mainly upon hydrophobic and van der Waals interactions for protein binding; they also use their main chain and side chains to form polar contacts and hydrogen bonds with proteins. Compared to small-molecule ligands, cyclic peptides can bind across larger, polar, and water-exposed protein surface areas, making many more contacts that can increase affinity, selectivity, biological activity, and ligand−receptor residence time. Cyclic peptides have a greater capacity than small-molecule drugs to modulate protein−protein interfaces that involve large, shallow, dynamic, polar, and water-exposed protein surfaces.

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“…The over-expression of Ls -AChBP was performed as previously described ( Abraham et al, 2016 ). Ubiquitin (Ub)-tagged AChBPs were used for radioligand binding assay.…”

Section: Methodsmentioning

confidence: 99%

Unique Pharmacological Properties of α-Conotoxin OmIA at α7 nAChRs

Abraham

Lewis

2021

Front. Pharmacol.

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OmIA, isolated from Conus omaria venom, is a potent antagonist at α7 nAChRs. We determined the co-crystal structure of OmIA with Lymnae stagnalis acetylcholine binding protein (Ls-AChBP) that identified His5, Val10 and Asn11 as key determinants for the high potency of OmIA at α7 nAChRs. Remarkably, despite a competitive binding mode observed in the co-crystal structure, OmIA and analogues displayed functional insurmountable antagonism at α7 and α3β4 nAChRs, except OmIA analogues having long side chain at position 10 ([V10Q]OmIA and [V10L]OmIA), which were partial insurmountable antagonist at α7 nAChRs in the presence of type II positive allosteric modulators (PAMs). A “two-state, two-step” model was used to explain these observations, with [V10Q]OmIA and [V10L]OmIA co-existing in a fast reversible/surmountable as well as a tight binding/insurmountable state. OmIA and analogues also showed biphasic-inhibition at α7 nAChRs in the presence of PNU120596, with a preference for the high-affinity binding site following prolonged exposure. The molecular basis of binding and complex pharmacological profile of OmIA at α7 nAChRs presented in here expands on the potential of α-conotoxins to probe the pharmacological properties of nAChRs and may help guide the development novel α7 modulators.

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Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs)

Cited by 9 publications

References 56 publications

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

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

Crystal Structures of Protein-Bound Cyclic Peptides

Unique Pharmacological Properties of α-Conotoxin OmIA at α7 nAChRs

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