NMR Structure of μ-Conotoxin GIIIC: Leucine 18 Induces Local Repacking of the N-Terminus Resulting in Reduced NaV Channel Potency

Abstract

μ-Conotoxins are potent and highly specific peptide blockers of voltage-gated sodium channels. In this study, the solution structure of μ-conotoxin GIIIC was determined using 2D NMR spectroscopy and simulated annealing calculations. Despite high sequence similarity, GIIIC adopts a three-dimensional structure that differs from the previously observed conformation of μ-conotoxins GIIIA and GIIIB due to the presence of a bulky, non-polar leucine residue at position 18. The side chain of L18 is oriented towards the core of the molecule and consequently the N-terminus is re-modeled and located closer to L18. The functional characterization of GIIIC defines it as a canonical μ-conotoxin that displays substantial selectivity towards skeletal muscle sodium channels (Na_V), albeit with ~2.5-fold lower potency than GIIIA. GIIIC exhibited a lower potency of inhibition of Na_V1.4 channels, but the same Na_V selectivity profile when compared to GIIIA. These observations suggest that single amino acid differences that significantly affect the structure of the peptide do in fact alter its functional properties. Our work highlights the importance of structural factors, beyond the disulfide pattern and electrostatic interactions, in the understanding of the functional properties of bioactive peptides. The latter thus needs to be considered when designing analogues for further applications.

Keywords: NMR; protein structure; voltage-gated sodium channel blocker; μ-conotoxins.

Figure 1

Structural overview of μ-conotoxins. (A) Sequence alignment of GIIIA (C. geographus), PIIIA (C. purpurascens), SmIIIA (C. stercusmuscarum) and BuIIIB (C. bullatus). Standard one-letter codes are used for amino acids, except for hydroxyproline (O) and pyroglutamate (Z). (B) 3D structures of GIIIA (PDB ID: 1TCG) [14], PIIIA (PDB ID: 1R9I) [18], SmIIIA (PDB ID: 1Q2J) [20] and BuIIIB (PDB ID: 2LO9) [21]. Disulfide crosslinks are shown as yellow sticks. (C) Sequence alignment of GIIIA, GIIIB and GIIIC. Lines indicate cysteine connectivity. The most significant difference between GIIIC and GIIIA, GIIIB is at position 18 where a leucine residue substitutes for glutamine (GIIIA) and methionine (GIIIB).

Figure 2

NMR and HPLC analysis of synthetic GIIIC. (A) Folding and disulfide formation of GIIIC monitored by RP-HPLC. The grey trace corresponds to the fully reduced polypeptide, whereas the black trace corresponds to the crude folding mixture. (B) Natural abundance ¹H-¹⁵N HSQC spectrum of synthetic GIIIC. C20 and K11 could not be assigned unambiguously (boxed correlations).

Figure 3

Three-dimensional structure of μ-conotoxin GIIIC. (A) Backbone superimposition of the 20 lowest energy structures of GIIIC. (B) Cartoon representation of GIIIC (lowest energy structure) and (C) highlighting residues important for interaction with VGSCs. The disulfide bonds are shown in yellow.

Figure 4

Pharmacological evaluation of GIIIC inhibition of Na_v channels. (A) Representative current traces of rNa_V1.4 in control (black) and after 300 nM GIIIC application (red) (holding potential: −120 mV, test potential, −10 mV, 1Hz); Scale bars: 500 pA, 5 ms. Representative I-V relationship shown in the inset. (B) Concentration-response curves for GIIIC and GIIIA inhibition of rNa_V1.4-mediated Na⁺ currents (n = 3–8 for each concentration). (C) Fractional block of various Na_V1.x channel isoforms exposed to 1 μM GIIIC (n = 4–8). Hashed bars correspond to fractional block by GIIIA.

Figure 5

Structural comparison of GIIIC with other C. geographus μ-conotoxins. Backbone superimposition of GIIIA (A, red) or GIIIB (B, red) with GIIIC (blue). Disulfides are shown in yellow and the side chain of residue 18 is indicated in stick representation. (C) Distance between Cα atoms of Arg1 and residue 18 in each of the three NMR-derived structures. Data is represented as dot plot (n = 20 for GIIIB and GIIIC, n = 10 for GIIIA) and the mean and 95% confidence intervals are indicated. (D) Density of NOE connectivities for GIIIA (upper panel) and GIIIC (lower panel).