Unique substrate recognition mechanism of the botulinum neurotoxin D light chain

Abstract

Botulinum neurotoxins are the most potent protein toxins in nature. Despite the potential to block neurotransmitter release at the neuromuscular junction and cause human botulism, they are widely used in protein therapies. Among the seven botulinum neurotoxin serotypes, mechanisms of substrate recognition and specificity are known to a certain extent in the A, B, E, and F light chains, but not in the D light chain (LC/D). In this study, we addressed the unique substrate recognition mechanism of LC/D and showed that this serotype underwent hydrophobic interactions with VAMP-2 at its V1 motif. The LC/D B3, B4, and B5 binding sites specifically recognize the hydrophobic residues in the V1 motif of VAMP-2. Interestingly, we identified a novel dual recognition mechanism employed by LC/D in recognition of VAMP-2 sites at both the active site and distal binding sites, in which one site of VAMP-2 was recognized by two independent, but functionally similar LC/D sites that were complementary to each other. The dual recognition strategy increases the tolerance of LC/D to mutations and renders it a good candidate for engineering to improve its therapeutic properties. In conclusion, in this study, we identified a unique multistep substrate recognition mechanism by LC/D and provide insights for LC/D engineering and antitoxin development.

Keywords: Bacterial Toxins; Enzyme Catalysis; Enzyme Kinetics; Metalloprotease; Neurotoxin.

FIGURE 1.

Overall view of the modeled LC/D·VAMP-2 complex structure. Left panel, view of the active site side; right panel, view after a 90° clockwise turn. LC/D is shown as a surface structure, and VAMP-2 is shown as a ribbon structure. The active site recognition and binding site interactions are highlighted. Negatively charged residues are shown in red, positively charged residues are shown in blue, hydrophobic residues are shown in gray, and polar residues are shown in green.

FIGURE 2.

CD spectroscopy analysis of LC/D and its derivatives. Far-UV CD (200–250 nm) data were obtained for LC/D and its derivatives with a JASCO J-810 spectropolarimeter at room temperature. The data for the most representative LC/D derivatives are shown in different colors.

FIGURE 3.

Specific recognition of VAMP-2 by LC/D pockets. Shown are surface representations of the recognition of different P sites of VAMP-2 by the S pockets and recognition of VAMP-2-binding sites by the B1–B5 binding sites of LC/D. Negatively charged residues are shown in red, positively charged residues are shown in blue, hydrophobic residues are shown in gray, and polar residues are shown in green. a, recognition of the P sites of VAMP-2 by the active site pockets of LC/D. The P2′ site (Ser⁶¹) of VAMP-2 is recognized by the S2′ pocket (Arg³⁷²). The P1′ site (Leu⁶⁰) of VAMP-2 is recognized by the S1′ pocket (Tyr¹⁶⁸ and Leu²⁰⁰) of LC/D. The P1 site (Lys⁵⁹) of VAMP-2 interacts with the oxygen atom of Pro⁶⁴ of LC/D. The P3 site (Asp⁵⁷) of VAMP-2 is recognized by the S3 pocket (Arg⁶³) of LC/D. b, recognition of Val⁵³ by the B1 binding site (Phe⁵⁰ and Ile¹⁹¹) of LC/D. c, recognition of Asn⁴⁹ of VAMP-2 by the B2 binding site (Arg²³ and His¹³²) of LC/D and recognition of Met⁴⁶ of VAMP-2 by the B3 binding site (Val¹⁴⁸ and Ile¹⁵¹) of LC/D. d, recognition of Val⁴² by the B4 binding site (Trp³¹⁵) of LC/D and recognition of Val³⁹ by the B5 binding site (Trp⁴⁴, Ile¹⁵², and Pro¹⁵⁴) of LC/D.

FIGURE 4.

Mechanism of substrate recognition by LC/D. After internalization to the cytoplasm of neuronal cells, LC/D attacks the free form of VAMP-2 through interaction with and recognition of hydrophobic residues in the V1 motif of VAMP-2, including Val³⁹, Val⁴², and Met⁴⁶, by the substrate-binding regions B5, B4, and B3 of LC/D on the substrate-binding cleft, respectively. In particular, binding of Met⁴⁶ of VAMP-2 to the LC/D B3 binding site was suggested to be very important for LC/D substrate recognition. This binding facilitates further binding of VAMP-2 Asn⁴⁹ and Val⁵³ to the B2 and B1 binding sites located at the active site surface of LC/D. The recognition of VAMP-2 Val⁵³ by the LC/D Phe⁵⁰/Ile¹⁹¹ pocket further orientates and stabilizes VAMP-2 for subsequent recognition of different P sites of VAMP-2 by the corresponding S pockets in the active site of LC/D. Active P site recognition includes the formation of a salt bridge between P3 (Asp⁵⁷) of VAMP-2 and S3 (Arg⁶³) of LC/D, a hydrogen bond interaction between P1 (Lys⁵⁸) of VAMP-2 and the main chain oxygen atom of Pro⁶⁴, recognition of P1′ (Leu⁶⁰) of VAMP-2 by the S1′ pocket (Tyr¹⁶⁸ and Leu²⁰⁰) of LC/D, and finally a hydrogen bond interaction between P2′ (Ser⁶¹) of VAMP-2 and the S2′ pocket (Arg³⁷²) of LC/D. The anchoring of VAMP-2 P sites to different S pockets in the active site of LC/D aligns the VAMP-2 scissile bond close enough to the active site zinc ion to facilitate peptide bond cleavage.