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Structure of alpha-conotoxin BuIA: influences of disulfide connectivity on structural dynamics.
Jin AH, Brandstaetter H, Nevin ST, Tan CC, Clark RJ, Adams DJ, Alewood PF, Craik DJ, Daly NL.
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Alpha-conotoxins have exciting therapeutic potential based on their high selectivity and affinity for nicotinic acetylcholine receptors. The spacing between the cysteine residues in alpha-conotoxins is variable, leading to the classification of sub-families. BuIA is the only alpha-conotoxin containing a 4/4 cysteine spacing and thus it is of significant interest to examine the structure of this conotoxin. In the current study we show the native globular disulfide connectivity of BuIA displays multiple conformations in solution whereas the non-native ribbon isomer has a single well-defined conformation. Despite having multiple conformations in solution the globular form of BuIA displays activity at the nicotinic acetylcholine receptor, contrasting with the lack of activity of the structurally well-defined ribbon isomer. These findings are opposite to the general trends observed for alpha-conotoxins where the native isomers have well-defined structures and the ribbon isomers are generally disordered. This study thus highlights the influence of the disulfide connectivity of BuIA on the dynamics of the three-dimensional structure.
Figure 1. Disulfide isomers of BuIA. (A) A schematic representation of the ribbon, globular and beads disulfide connectivities of BuIA, a conotoxin having the unique 4/4 loop spacing. (B) HPLC traces of ribbon and globular BuIA. Analytical HPLC was carried out using a C18 Phenomenex analytical column (250 × 4.6 mm) with 90% acetonitrile and 0.043% trifluroacetic acid as the eluting solvent B and 1% B per minute as the gradient. Ribbon BuIA elutes earlier than the globular form but has a slightly broadened peak under the conditions utilized.
Figure 2. NMR analysis of ribbon and globular BuIA. The upper panels show the amide regions of the TOCSY spectra at 600 MHz of globular (A) and ribbon BuIA (B) at 282 K in 95% H2O/5% 2H2O at pH 3. The one letter code and the residue numbers are used for labeling. For globular BuIA (A) stretches of assigned residues are labeled, as a or b to indicate conformers A and B. The lower panels (C & D) show a secondary shift analysis of conformers A and B from globular BuIA and ribbon BuIA compared to AuIB. For each residue, represented by the one letter code, the differences between the actual chemical shifts for the α-protons and their respective random coil shifts are graphed. (C) Native AuIB (black bars), conformer A (white bars), conformer B (grey bars). (D) Ribbon AuIB (black bars), ribbon BuIA (white bars).
Figure 3. Three-dimensional structure of the ribbon isomer of BuIA. (top) Stereoview of the 20 NMR-derived lowest energy structures of ribbon BuIA. For clarity only the backbone (N, Cα, C') is shown and the two disulfide bonds are omitted. The structures are superimposed over the whole molecule. N refers to the N-terminus; C refers to the C-terminus. Residues were labeled with the one letter code. (bottom) Solvent accessible surface of ribbon BuIA. The two views are rotated by 180° about the vertical axis. Hydrophobic residues (Pro, Ala, Val, Leu, Tyr) are green, hydrophilic residues (Ser, Thr) are blue, Gly is grey and the Cys residues are yellow. Selected residues are labeled with the one letter code.
Figure 4. Effect of globular and ribbon isomers of α-conotoxin BuIA at nAChR subtypes expressed in Xenopus oocytes. (A)Representative ACh-evoked currents mediated by α3β2 and α3β4 nAChR subtypes obtained in the absence (control) and presence of 10 nM and 100 nM BuIA-globular, respectively. Complete recovery was observed after 10 min washout (broken line). (B)Concentration-response curves for the inhibition of α3β2 and α3β4 nAChRs by globular (filled symbols) and ribbon (open symbols) isomers of BuIA. Best fit of the data gave IC50 values of 4.8 ± 0.4 nM (nH = 1.3) and 59.1 ± 2.3 nM (nH = 1.2) for α3β2 and α3β4 nAChRs, respectively, whereas the ribbon isomer of BuIA exhibited no inhibition at 1 μM. Responses are shown as a percentage of ACh (100 μM)-induced peak current amplitude after a 5 min incubation of the BuIA isomers with respect to control (ACh alone). Error bars are SEM with n = 4–7 for each data point.
Figure 5. Proline geometries of the BuIA isomers and the sunflower trypsin inhibitor SFTI-1 [26]. The proline residues with trans geometry are marked as t and those with cis as c. The three-dimensional structures of a classic α-conotoxin, ribbon BuIA and SFTI-1 are shown to highlight the structural differences between the various peptides.
Figure 6. Backbone trace of the structures of ribbon BuIA (A), globular AuIB (B), ribbon AuIB (C), and ribbon MrIA (D). Disulfide bonds are shown as balls and sticks. The helices are shown with thickened ribbons and β-strands are indicated with arrows.
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