doi:10.1016/j.jmb.2006.09.002 
Copyright © 2006 Elsevier Ltd All rights reserved.
Structure and Action of the Binary C2 Toxin from Clostridium botulinum
Christian Schleberger1, Henrike Hochmann2, Holger Barth3, Klaus Aktories2 and Georg E. Schulz1, 
, 
1Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Albertstr. 21, D-79104 Freiburg im Breisgau, Germany
2Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität, Albertstr. 25, D-79104 Freiburg im Breisgau, Germany
3Institut für Pharmakologie und Toxikologie, Universität Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany
Received 18 July 2006;
revised 25 August 2006;
accepted 1 September 2006.
Edited by I. Wilson.
Available online 5 September 2006.
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Abstract
C2 toxin from Clostridium botulinum is composed of the enzyme component C2-I, which ADP-ribosylates actin, and the binding and translocation component C2-II, responsible for the interaction with eukaryotic cell receptors and the following endocytosis. Three C2-I crystal structures at resolutions of up to 1.75 Å are presented together with a crystal structure of C2-II at an appreciably lower resolution and a model of the prepore formed by fragment C2-IIa. The C2-I structure was determined at pH 3.0 and at pH 6.1. The structural differences are small, indicating that C2-I does not unfold, even at a pH value as low as 3.0. The ADP-ribosyl transferase activity of C2-I was determined for α and β/γ-actin and related to that of Iota toxin and of mutant S361R of C2-I that introduced the arginine observed in Iota toxin. The substantial activity differences between α and β/γ-actin cannot be explained by the protein structures currently available. The structure of the transport component C2-II at pH 4.3 was established by molecular replacement using a model of the protective antigen of anthrax toxin at pH 6.0. The C-terminal receptor-binding domain of C2-II could not be located but was present in the crystals. It may be mobile. The relative orientation and positions of the four other domains of C2-II do not differ much from those of the protective antigen, indicating that no large conformational changes occur between pH 4.3 and pH 6.0. A model of the C2-IIa prepore structure was constructed based on the corresponding assembly of the protective antigen. It revealed a surprisingly large number of asparagine residues lining the pore. The interaction between C2-I and C2-IIa and the translocation of C2-I into the target cell are discussed.
Keywords: actin; ADP-ribosylation; endosomal compartment; membrane pore; pH-dependent conformation
Abbreviations: C2-I, enzyme component of C2 toxin; C2-II, binding and translocation component of C2 toxin; C2-IIa, activated fragment of C2-II after removal of 181 N-terminal residues; PA, protective antigen; VIP, vegetative insecticidal protein; GST, glutathione-S-transferase
Figure 1. Stereoview of a ribbon plot of the enzymatic component C2-I. (a) The underlying colors of the N and C-terminal domains are blue and green, respectively. The structurally highly conserved central quartets of β-strands are light green. Helix α1 involved in translocation11 is red and the chain segment known to be involved in binding to the C2-IIa prepore is gold.11 The crystallization mutants K20E and Q77R are marked by blue spheres and Mut-S361R by a pink sphere. The loops mentioned in the text are labeled with red numbers. NAD+ is modeled as transferred from the homologous enzymes VIP2 (black) and IotaA (red) using a superposition based on the central β-strand quartets. Some residues important for NAD+ binding and catalysis are drawn out. (b) Magnification of the NAD+ binding site showing all residues of C2-I that are within a 5 Å distance to either of the two NAD+ models.
Figure 2. Structure-based sequence alignment of C2-I with the related toxins VIP2 and IotaA. The secondary structure is from C2-I and labeled sequentially except for 310-helices. The colors correspond to those of Figure 1. The domain border is between β8 and α7 and marked (
). Every tenth residue of C2-I is marked (●). Conserved residues are highlighted in yellow. The N-terminal methionine of C2-I is absent.
Figure 3. Stereoview of C2-I (carrying NAD+ transferred from IotaA) and β/γ-actin (top, accession code 2BTF) in the proposed reaction geometry. Both models are shown in an inflated-stick-mode and opened by a total angle of 32° towards the viewer so that the contacting surfaces can be visualized. A dotted straight line between the C1′-carbon of NAD+ and the modified Arg177 of actin indicates the ADP-ribosyl transfer. The three α versus β/γ-actin substitutions on the interaction surface as well as Mut-S361R of C2-I are colored pink and labeled (black).
Figure 4. Alignment of C2-II with the related transport component PA of anthrax toxin. Those parts lacking structure are given in lower case letters. The secondary structure is labeled sequentially except for 310-helices. The domain borders () and the proteolytic cuts in the activation loops considered as domain borders (
, 
) are indicated. Conserved residues are gray. Residues that are also conserved in the transport components VIP1 and IotaB are marked (*).
Figure 5. Stereoview of the transport component C2-II given as a ribbon plot. The domains C2-II20 (red), D1′ (green), D2 (orange) and D3 (blue) are closely related to the respective domains of the transport component PA from anthrax toxin.4 The dotted parts of the chain have no defined structure. The C-terminal domain D4 is represented by a transparent sphere that was placed at a position corresponding to that observed in PA. The vertical black line is the central axis of the putative heptamer of C2-IIa as derived from the prepore formed by PA63.4
Figure 6. Stereoview of a model of the C2-IIa heptamer as derived from a superposition with the established prepore structure of PA63 from anthrax toxin.4 (a) Model of the C2-IIa prepore shown in an inflated-stick-mode. The expected docking site of the enzymic component C2-I as derived from experimental data for anthrax toxin is yellow.19,20 (b) Ribbon plot of two adjacent subunits of the C2-IIa model as viewed from the lumen of the prepore. All residues pointing into the prepore lumen are drawn out and numbered. The mobile loop containing Phe428 is labeled. The blue sphere marks position 319, which was suggested to form the tip of the putative α-hemolysin-like β-barrel inserted into the membrane.4
Table 1.
Data collection and phasing
a The crystals contained the wild-type or Mut-S361R at pH 3.0 with the surface mutation K20E in a large packing contact. The space group was
P6
322 with axes
a =
b = 115.3 Å,
c = 162.6 Å, one molecule per asymmetric unit and 61% solvent.
b The crystal contained the wild-type enzyme at pH 6.1 with the surface mutation Q77R participating in a large packing contact. The space group was
C222
1 with axes
a = 93.4 Å,
b = 272.9 Å,
c = 246.3 Å, six molecules per asymmetric unit and 54% solvent.
c The crystal contained the full chain of C2-II at pH 4.3. The space group was
P4
32
12 with axes
a =
b = 104.4 Å,
c = 153.2 Å, one molecule per asymmetric unit and 52% solvent.
d Beamline X06SA of SLS.
e Beamline BL14 of BESSY.
f Beamline X11 of DESY.
g SAD phasing power of program SHARP,
28 the overall figure of merit was 0.34 before density modification and 0.82 thereafter.
Table 2.
Refinement statistics
a See
Table 1 for the abbreviations. Refined using program REFMAC5.
35 Mut-S361R showed a particularly high α-actin activity (
Table 3).
b Refined with program BUSTER.
38
Table 3.
Enzymatic activity of C2-I, Mut-S361R and IotaA
a All data are relative
kcat values. The
kcat value for IotaA modifying β/γ-actin was 0.63 s
− 1.
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