Light chain separated from the rest of the type a botulinum neurotoxin molecule is the most catalytically active form

Abstract

Botulinum neurotoxins (BoNT) are the most potent of all toxins. The 50 kDa N-terminal endopeptidase catalytic light chain (LC) of BoNT is located next to its central, putative translocation domain. After binding to the peripheral neurons, the central domain of BoNT helps the LC translocate into cytosol where its proteolytic action on SNARE (soluble NSF attachment protein receptor) proteins blocks exocytosis of acetyl choline leading to muscle paralysis and eventual death. The translocation domain also contains 105 Å -long stretch of ∼100 residues, known as "belt," that crosses over and wraps around the LC to shield the active site from solvent. It is not known if the LC gets dissociated from the rest of the molecule in the cytosol before catalysis. To investigate the structural identity of the protease, we prepared four variants of type A BoNT (BoNT/A) LC, and compared their catalytic parameters with those of BoNT/A whole toxin. The four variants were LC + translocation domain, a trypsin-nicked LC + translocation domain, LC + belt, and a free LC. Our results showed that K(m) for a 17-residue SNAP-25 (synaptosomal associated protein of 25 kDa) peptide for these constructs was not very different, but the turnover number (k(cat)) for the free LC was 6-100-fold higher than those of its four variants. Moreover, none of the four variants of the LC was prone to autocatalysis. Our results clearly demonstrated that in vitro, the LC minus the rest of the molecule is the most catalytically active form. The results may have implication as to the identity of the active, toxic moiety of BoNT/A in vivo.

Figure 1. Schematic presentation of LC as it occurs in BoNT/A (residues M1-L1296) (A), LC+Hn (residues (M1-Q861) (B), trypsin-cleaved LC+Hn' (residues M1-Q861) (C), LC+Belt residues M1-F552) (D), and LC alone (residues M1-K449) (E).

The numbers represent the sequence stretch of each construct.

Figure 2. Ribbon diagram of the five versions of the BoNT/A LC used in this study.

These diagrams are based on the original 3-dimensional structure of BoNT/A dichain determined by Stevens et al . The constructs used in this study and represented by the structures A, B, and D however are single polypeptide chains. A, Structure of BoNT/A whole toxin. The three major structural domains, the n-terminal LC (red), the central translocation Hn (blue), and the C-terminal binding Hc (yellow) are shown. A stretch of ∼115 residues belonging to the translocation domain Hn that wraps around the LC and known as belt, is shown in green. B, LC+Hn; C, LC+Hn that was nicked by trypsin. Here Figure B is slightly rotated to visualize the tryptic cleavge site indicated by an arrow; D, LC+Belt, and E, LC. Although LC and Hn are shown separated in C to distinguish it from B, these domains are in fact still connected by a disulfide bond (see Figure 1) in addition to other ionic and hydrophobic interactions. Figures B–E were generated by simple truncations from the C-terminus of A.

Figure 3. Reducing (R) and non reducing (NR) SDS-PAGE of various forms of the LC used.

Samples (0.15 mg/ml) were heated at >95°C for 5 min without (NR) and with (R) 5% β-mercaptoethanol in the SDS-load buffer before electrophoresis. Molecular mass in kDa of the marker proteins in the right lane are shown at right.

Figure 4. Far-UV CD spectra of LC variants at 20°C.

Average of five spectra were collected for 0.2 mg/ml of each of LC (closed circle), LC+Belt (open triangle), LC+Hn (closed triangle), and LC+Hn' (crossed hatch, X) in 50 mM sodium-phosphate, pH 6.5.

Figure 5. Tryptophan fluorescence spectra of the LC variants at 20°C.

Fluorescence emission of each protein collected at 0.20–0.33 mg/ml in 50 mM sodium phosphate, pH 6.5, was adjusted for 1 µM protein/tryptophan. LC (closed circle), LC+Belt (open triangle), LC+Hn (closed triangle), and LC+Hn' (crossed X).

Figure 6. Time course of reactions catalyzed by LC and LC+Hn.

LC (0.4 µg, closed circle) or LC+Hn (1.8 µg, closed triangle) and 1 mM peptide substrate in a 30 µl of reaction mixture containing ZnCl₂ and DTT was incubated at 37°C. At the indicated times, products in two vials was analyzed by HPLC as described in the EXPERIMENTAL PROCEDURES.

Figure 7. Lineweaver-Burke plots of reaction velocity versus substrate concentration of reactions catalyzed by three LC variants.

The bars accompanying the data points are standard deviations of three to five assays. LC (closed circle), LC+Belt (open triangle), LC+Hn (closed triangle), and BoNT/A (open circle).

Figure 8. Reducing SDS-PAGE of LC variants before (C), and after 48-h incubation at 22°C in the absence (−) and presence (+) of 5 mM DTT.

Samples (0.15 mg/ml) were heated at >95°C for 5 min in the SDS-load buffer containing 5% β-mercaptoethanol before electrophoresis. Molecular mass in kDa of the marker proteins in the right lane are shown at right.