Conformational Space of the Translocation Domain of Botulinum Toxin: Atomistic Modeling and Mesoscopic Description of the Coiled-Coil Helix Bundle

Abstract

The toxicity of botulinum multi-domain neurotoxins (BoNTs) arises from a sequence of molecular events, in which the translocation of the catalytic domain through the membrane of a neurotransmitter vesicle plays a key role. A recent structural study of the translocation domain of BoNTs suggests that the interaction with the membrane is driven by the transition of an α helical switch towards a β hairpin. Atomistic simulations in conjunction with the mesoscopic Twister model are used to investigate the consequences of this proposition for the toxin-membrane interaction. The conformational mobilities of the domain, as well as the effect of the membrane, implicitly examined by comparing water and water-ethanol solvents, lead to the conclusion that the transition of the switch modifies the internal dynamics and the effect of membrane hydrophobicity on the whole protein. The central two α helices, helix 1 and helix 2, forming two coiled-coil motifs, are analyzed using the Twister model, in which the initial deformation of the membrane by the protein is caused by the presence of local torques arising from asymmetric positions of hydrophobic residues. Different torque distributions are observed depending on the switch conformations and permit an origin for the mechanism opening the membrane to be proposed.

Keywords: Clostridium botulinum; botulinum toxin; hydrophobicity; mesoscopic Twister model; molecular dynamics; residue protonation; water–ethanol solvent.

Figure 1

(A) X-ray crystallographic structures determined for the translocation domain (PDB entries: 6MHJ determined at pH 8.5 and 6DKK determined at pH 5.1 [8]) are represented as grey ribbons. The tubes generated by Bendix [9] on the helices 1 (cyan) and 2 (yellow) are shown for the two PDB structures. The hydrophobic residues in these helices are represented by green spheres. The limits of the helices are defined as in Table 1. The switch is colored in magenta, and the N-terminal domain and the C-terminal $\alpha$ helix in orange. In helices 1 and 2, the helix halves H11, H12, H21, and H22 are indicated with labels. (B) Structure 6DKK represented as a grey surface. The hydrophobic residues GLY, ALA, VAL, LEU, ILE, PRO, PHE, MET, and TRP are colored in green. This image was prepared using pymol [10].

Figure 2

Coordinate root-mean-square deviation (RMSD, Å) along the molecular dynamics (MD) trajectories recorded in water and in 50/50 water–ethanol mixture at the two pH values. Different colors correspond to different replicates of the trajectory.

Figure 3

Coordinate root-mean-square fluctuations (RMSFs, Å) calculated on the MD trajectories recorded on the two protein systems. Different colors correspond to different replicates of the trajectory.

Figure 4

Cylinder radius r calculated as $r=\sqrt{2I/m}$, where I is the moment of inertia with respect to the axis of the cylinder and m is the total molar mass of the C$\alpha$ atoms. Different colors correspond to different replicates of the trajectory.

Figure 5

Isosurfaces of the spatial density function of water and ethanol atoms around the protein. Cyan isosurface: water oxygen atoms; red and green: oxygen and methyl carbon atoms, respectively. The isosurfaces are represented at the same isodensity level (0.0115) for both water and ethanol. The protein regions are colored as in Figure 1. Data were collected from the three replicates of the trajectory. This image was prepared using VMD [28].

Figure 6

Local torque values ${\tilde{T}}_{kl}$ along helix 1. Values averaged along the first replicate of the trajectory and their standard deviations are plotted as a function of the residue number. Standard deviations are smaller than the size of the data points except for residue ALA-719.

Figure 7

Bending angles of helices 1 and 2. The analysis was performed using the package Bendix [9] along the first replicate of the trajectory.

Figure 8

Torque values averaged along the residues 695–713 (H11: orange), 730–748 (H12: cyan), 766–784 (H21: green), and 801–819 (H22: magenta) and plotted along the trajectory time. H12 and H21 are interacting through a coiled-coil motif, as well as H11 and H22. For pH 7, these values were, respectively, calculated on the following sets of hydrophobic residues: I695, A698, L699, W706, V709, and I713 for H11, M732, A735, L736, A740, A742, A745, I746, and I747 for H12, I766, L769, L773, I777, A780, M781, I782, and I784 for H21, I801, P802, G804, V805, L808, F811, A813, L815, A818, and L819 for H22. For pH 4.7, the protonated residue E809 should be added to the list of H22. Note that the curves of H11 and H21 superimpose on each other. Dashed lines indicate the zero line.

Figure 9

Geometry of the coarse-grained $\alpha$ helix describing the calculation of the torque $T_{kl}$ (in purple). The $\alpha$ helix is represented by a transparent cylinder, the helix axis is parallel to the torque. The two vectors $V_k$ and $V_l$ are perpendicular to the helix axis and connect the axis to the C$\alpha$ atoms of the two consecutive hydrophobic residues k and l (in green). The projection of $V_l$ next to $V_k$ (dashed vector) is drawn in order to illustrate the definition of ${\theta }_{kl}$.