Conotoxin Φ‐MiXXVIIA from the Superfamily G2 Employs a Novel Cysteine Framework that Mimics Granulin and Displays Anti‐Apoptotic Activity

Jin, Aihua; Dekan, Zoltan; Smout, Michael J.; Wilson, David T.; Dutertre, Sébastien; Vetter, Irina; Lewis, Richard J.; Loukas, Alex; Daly, Norelle L.; Alewood, Paul F.

doi:10.1002/ange.201708927

Cited by 6 publications

(8 citation statements)

References 33 publications

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“…As a consequence, the third disulfide bond (Cys III -Cys VI ) of VxXXB-C(21-50), which typically threads the loop formed by the first two disulfide bonds to make a knot in the ICK fold (Figure 2B), instead links the β3 loop with the unstructured C-terminal region (Figure 2A). This tertiary structure resembles the N-terminal of the cell proliferation regulator human granulin A (Tolkatchev et al, 2008) and has been previously reported in conotoxins N ext H-Vc7.2 and ϕ-MiXXVIIA (Jin et al, 2017;Nielsen et al, 2019). Indeed, with a large second β-hairpin, VxXXB-C(21-50) superimposes the N-terminal of human granulin A (RMSD 2.23 Å), N ext H-Vc7.2 (RMSD 3.82 Å) and ϕ-MiXXVIIA (RMSD 2.79 Å).…”

Section: The Vxxxb-c(21-50)/ls-achbp Complexsupporting

confidence: 77%

“…Granulins are ancestral ~55-residue growth factors responsible for development and wound healing. The N-terminal ~30 residues of granulins includes the characteristic core granulin fold identified previously in conotoxins Φ-MiXXVIIA and N ext H-Vc7.2 as a mini-granulin fold distinct from the ICK toxin fold (Tolkatchev et al, 2008;Palfree et al, 2015;Jin et al, 2017;Nielsen et al, 2019). Indeed, mini-granulin-fold proteins (Sheng et al, 2008) were used by AlphaFold to model VxXXB from human and Drosophila E3 ubiquitin-protein ligase protein (Ho et al, 2022).…”

Section: Discussionmentioning

confidence: 99%

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Unravelling the allosteric binding mode of αD-VxXXB at nicotinic acetylcholine receptors

Abraham²,

Lewis³

2023

Front. Pharmacol.

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αD-conotoxins are 11 kDa homodimers that potently inhibit nicotinic acetylcholine receptors (nAChRs) through a non-competitive (allosteric) mechanism. In this study, we describe the allosteric binding mode of the granulin-like C-terminal (CTD) of VxXXB bound to Lymnea stagnalis acetylcholine binding protein (Ls-AChBP), a soluble homologue of the extracellular ligand-binding domain of nAChRs. This co-crystal complex revealed a novel allosteric binding site for nAChR antagonists outside the C-loop that caps the orthosteric site defined by the nAChR agonist nicotine and the antagonist epibatidine. Mutational and docking studies on Ls-AChBP supported a two-site binding mode for full-length VxXXB, with the first CTD binding site located outside the C-loop as seen in the co-crystal complex, with a second CTD binding site located near the N-terminal end of the adjacent subunit of AChBP. These results provide new structural insight into a novel allosteric mechanism of nAChR inhibition and define the cooperative binding mode of the N-terminal domain linked granulin core domains of αD-conotoxins.

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Section: The Vxxxb-c(21-50)/ls-achbp Complexsupporting

confidence: 77%

Section: Discussionmentioning

confidence: 99%

Unravelling the allosteric binding mode of αD-VxXXB at nicotinic acetylcholine receptors

Abraham²,

Lewis³

2023

Front. Pharmacol.

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“…The crude linear peptide was purified by preparative RP-HPLC (Vydac C18). For the one-pot regioselective formation of the 1–3 and 2–4 (or 1–4, 2–3) disulfide bonds, the method has been described before [ 45 ]. Briefly, the crude dithiol product was first subjected to a 5 min I 2 treatment in 90% AcOH/MeOH to form the Cys 2–4 or Cys 2–3 disulfide bond.…”

Section: Methodsmentioning

confidence: 99%

The Allosteric Activation of α7 nAChR by α-Conotoxin MrIC Is Modified by Mutations at the Vestibular Site

Gulsevin

Papke

Stokes

et al. 2021

Toxins

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α-conotoxins are 13–19 amino acid toxin peptides that bind various nicotinic acetylcholine receptor (nAChR) subtypes. α-conotoxin Mr1.7c (MrIC) is a 17 amino acid peptide that targets α7 nAChR. Although MrIC has no activating effect on α7 nAChR when applied by itself, it evokes a large response when co-applied with the type II positive allosteric modulator PNU-120596, which potentiates the α7 nAChR response by recovering it from a desensitized state. A lack of standalone activity, despite activation upon co-application with a positive allosteric modulator, was previously observed for molecules that bind to an extracellular domain allosteric activation (AA) site at the vestibule of the receptor. We hypothesized that MrIC may activate α7 nAChR allosterically through this site. We ran voltage-clamp electrophysiology experiments and in silico peptide docking calculations in order to gather evidence in support of α7 nAChR activation by MrIC through the AA site. The experiments with the wild-type α7 nAChR supported an allosteric mode of action, which was confirmed by the significantly increased MrIC + PNU-120596 responses of three α7 nAChR AA site mutants that were designed in silico to improve MrIC binding. Overall, our results shed light on the allosteric activation of α7 nAChR by MrIC and suggest the involvement of the AA site.

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“… a The linear peptide comprised four different sets of S -protected Cys residues. (a) I 2 /AcOH, MeOH (9:1), 5 min, (b) I 2 /45% AcOH, 5% MeOH and 0.5% TFA, 90 min, (c) HF/ p -cresol, 0 °C, 1h followed by I 2 /0.1% TFA/50% MeCN/H 2 O, 5 min, (d) NH 4 I/DMS/TFA, 0 °C, 30 min (* = the arrangement of the disulfide bonds doesn’t pertain to a specific conformation) (ref ). …”

Section: Safety Catch Protecting Groupsmentioning

confidence: 99%

Ready to Use Cysteine Thiol Protecting Groups in SPPS, A Practical Overview

Chakraborty,

Mthembu,

de la Torre

et al. 2024

Org. Process Res. Dev.

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Protecting group chemistry plays a pivotal role during peptide synthesis. For the synthesis of cysteinyl peptides, this is more significant as cysteine (Cys) is involved in the formation of two key peptide bonds�the amide and the disulfide. Cys also poses itself as one of the most sensitive amino acid residues during peptide synthesis, especially for the occurrence of side reactions such as racemization, elimination, alkylation, etc. The magnitude of these side reactions is dependent on several factors including the choice of appropriate protecting groups for Cys thiol. The regioselective synthesis of multidisulfide peptides is a sequential process and it requires different sets of Cys thiol protections with unique removal conditions for each set of protecting groups used. Here, the selection of Cys side-chain protections is significant in obtaining the final peptide with the desired conformation and a good yield. With the proper knowledge of the chemistry of Cys thiol protecting groups trouble-free synthesis of multicysteinyl peptides can be achieved with minimization/elimination of a plethora of side reactions related to Cys thiol. In this current review, ready to use/commercialized Cys thiol protecting groups have been discussed with a focus on their use for the synthesis of multidisulfide peptides.

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Conotoxin Φ‐MiXXVIIA from the Superfamily G2 Employs a Novel Cysteine Framework that Mimics Granulin and Displays Anti‐Apoptotic Activity

Cited by 6 publications

References 33 publications

Unravelling the allosteric binding mode of αD-VxXXB at nicotinic acetylcholine receptors

Unravelling the allosteric binding mode of αD-VxXXB at nicotinic acetylcholine receptors

The Allosteric Activation of α7 nAChR by α-Conotoxin MrIC Is Modified by Mutations at the Vestibular Site

Ready to Use Cysteine Thiol Protecting Groups in SPPS, A Practical Overview

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