Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism

Abstract

Spirolide and gymnodimine macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Analysis of 13-desmethyl spirolide C and gymnodimine A by binding and voltage-clamp recordings on muscle-type alpha1(2)betagammadelta and neuronal alpha3beta2 and alpha4beta2 nicotinic acetylcholine receptors reveals subnanomolar affinities, potent antagonism, and limited subtype selectivity. Their binding to acetylcholine-binding proteins (AChBP), as soluble receptor surrogates, exhibits picomolar affinities governed by diffusion-limited association and slow dissociation, accounting for apparent irreversibility. Crystal structures of the phycotoxins bound to Aplysia-AChBP ( approximately 2.4A) show toxins neatly imbedded within the nest of ar-omatic side chains contributed by loops C and F on opposing faces of the subunit interface, and which in physiological conditions accommodates acetylcholine. The structures also point to three major features: (i) the sequence-conserved loop C envelops the bound toxins to maximize surface complementarity; (ii) hydrogen bonding of the protonated imine nitrogen in the toxins with the carbonyl oxygen of loop C Trp147 tethers the toxin core centered within the pocket; and (iii) the spirolide bis-spiroacetal or gymnodimine tetrahydrofuran and their common cyclohexene-butyrolactone further anchor the toxins in apical and membrane directions, along the subunit interface. In contrast, the se-quence-variable loop F only sparingly contributes contact points to preserve the broad receptor subtype recognition unique to phycotoxins compared with other nicotinic antagonists. These data offer unique means for detecting spiroimine toxins in shellfish and identify distinctive ligands, functional determinants and binding regions for the design of new drugs able to target several receptor subtypes with high affinity.

Fig. 1.

Chemical structures of the spirolides (Left) and gymnodimines (Right). The seven- and six-membered cyclic imines common to the phycotoxins are highlighted with a gray background, and the chemical groups specific to each are boxed. Locations of group substitutions that differ among the various spirolides and gymnodimines are indicated. SPX (13-desmethyl spirolide C): R₁ = CH₃, R₂ = H, R₃ = CH₃. GYM (gymnodimine A): R₁ = CH₃. Substitutions found in SPX and GYM congeners are described in the SI Appendix.

Fig. 2.

Inhibition of ACh-evoked currents from neuronal and muscle-type nAChRs by the phycotoxins. SPX with human α4β2 expressed in oocytes (A and B); GYM with Torpedo α1₂βγδ incorporated into the oocyte membrane (C and D). (A and C) Typical inward nicotinic currents evoked by a 5-s perfusion of 150 μM ACh (EC₅₀ value for α4β2) before and after application of 1.5 nM SPX (A), and a 7-s perfusion of 25 μM ACh (EC₅₀ value for α1₂βγδ) before and after application of 1.5 nM GYM (C). The desensitization component typical of α1₂βγδ is not modified in presence of the toxin. (B and D) SPX (B) and GYM (D) concentration-to-current inhibition relationships. The amplitudes of the ACh-induced currents recorded in the presence of SPX and GYM (mean ± SEM; 3–4 oocytes per concentration) were normalized to control currents and fitted to the Hill equation. SPX on α4β2: IC₅₀ = 3.87 ± 1.1 nM; nH = 1.9 ± 0.33; GYM on α1₂βγδ: IC₅₀ = 2.8 ± 1.15 nM; nH = 0.96 ± 0.15 (compare Table 2).

Fig. 3.

Overall views of the A-AChBP pentameric complexes and subunit interfaces with bound phycotoxins. (A) Ribbon diagrams of the pentamer (Left) and subunit interface (Center) with bound SPX (Upper; orange bonds and surface, red oxygens, blue nitrogen) and GYM (Lower; green bonds and surface, red oxygens, blue nitrogen) viewed from the “membrane” side (Left) and radial perspectives with the apical side at top and the membrane side at bottom (Center). The main and side chains at the (+) and (-) faces of the interface are displayed in yellow and cyan, respectively. The bound toxins (Right) are perfectly ordered, as shown by their 2.5/2.4 Å resolution 2F_o-F_c electron density maps contoured at 1.2σ (blue). (B) Close-up views of the bound toxins in their aromatic nest at the subunit interface, showing details of the cyclic imine environment. Bound SPX (Upper) is shown alone (Left) and overlaid with nicotine as bound to L-AChBP (pink bonds; Right). Bound GYM (Lower) is shown alone. The dashed lines denote H bonds.

Fig. 4.

Structural comparisons of the SPX and GYM complexes with other antagonist complexes. (A) Superimposition of SPX (orange toxin) and GYM (green) (Left) and SPX (orange) and MLA (pink) (Right) bound to A-AChBP. The molecular surface of SPX and key side chains within the binding pocket are displayed. (B Left and Right) Molecular surfaces buried at the A-AChBP subunit interfaces by bound SPX (orange toxin and surface), GYM (green toxin and surface), MLA (pink toxin and surface), and the peptidic α-conotoxin ImI (blue toxin with green disulfides, mauve surface), viewed radially from the pentamer outer periphery. (B Center) Overlay of loop C in the SPX (orange loop), GYM (green), MLA (magenta), ImI (blue) and nicotine (white) complexes as viewed from the pentamer apical side, also showing overlaid bound SPX and GYM molecules.