Synthesis of full-length homodimer αD-VxXXB that targets human α7 nicotinic acetylcholine receptors

Abstract

αD-conotoxin VxXXB is a pseudo-homodimer that allosterically inhibits nicotinic acetylcholine receptors (nAChRs) with high potency and selectivity. However, challenges in synthesizing αD-conotoxins have hindered further structure-function studies on this novel...

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

“…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). 5Ai) of the four (W53, Y185, Y192, and W143) aromatic residues forming the orthosteric binding site, and thus avoid overlapping with the binding of orthosteric ligands both agonists (Figure 4A) and antagonists (Figure 4B) that typically position deep within this aromatic cage.…”

“…The observations from the VxXXB-C(21–50)/ Ls -AChBP co-crystal structure, together with functional data previously reported ( Ho et al, 2022 ), were used to formulate the potential binding mode of native homodimeric VxXXB ( Table 2 ). The comparable potency of VxXXB-C(19–50) and VxXXB-NC, and the 2-fold enhance potency of VxXXB-CNC suggests that NTD mainly acts to facilitate cooperative binding between the two CTDs.…”

“…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 ). Unlike Φ-MiXXVIIA and N ext H-Vc7.2, which have few residues in loop III and more residues in loop IV, VxXXB-C(21–50) has most residues in its negatively charged loop III, which allows closer alignment to the core motif of granulin A.…”

“…Previously, we successfully synthesised the NTD, CTD and full-length synthetic VxXXB using the α-ketoacidhydroxylamine (KAHA) ligation strategy ( Ho et al, 2022 ). To characterize the structural basis for allosteric inhibition by αD-conotoxins, we obtained the co-crystal structure of the C-terminal domain VxXXB-C (21–50) ( Figure 1 ) with Lymnea stagnalis acetylcholine binding protein ( Ls -AChBP).…”

The Impact of Neurotransmitters on the Neurobiology of Neurodegenerative Diseases

“…nAChRs are formed by the assembly of five transmembrane subunits. Here, we mainly discuss neuronal nAChRs, which are assembled either as homo-pentamers of α7, α8, and α9 or hetero-pentamers of α2-α6 in combination with β2-β4 or α9 with α10 subunits [54,55].…”