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Neuronal entry and high neurotoxicity of botulinum neurotoxin A require its N-terminal binding sub-domain.

ABSTRACT: Botulinum neurotoxins (BoNTs) are the most toxic proteins known, due to inhibiting the neuronal release of acetylcholine and causing flaccid paralysis. Most BoNT serotypes target neurons by binding to synaptic vesicle proteins and gangliosides via a C-terminal binding sub-domain (H_CC). However, the role of their conserved N-terminal sub-domain (H_CN) has not been established. Herein, we created a mutant form of recombinant BoNT/A lacking H_CN (rA?H_CN) and showed that the lethality of this mutant is reduced 3.3?×?10⁴-fold compared to wild-type BoNT/A. Accordingly, low concentrations of rA?H_CN failed to bind either synaptic vesicle protein 2C or neurons, unlike the high-affinity neuronal binding obtained with ¹²⁵I-BoNT/A (K_d?=?0.46?nM). At a higher concentration, rA?H_CN did bind to cultured sensory neurons and cluster on the surface, even after 24?h exposure. In contrast, BoNT/A became internalised and its light chain appeared associated with the plasmalemma, and partially co-localised with vesicle-associated membrane protein 2 in some vesicular compartments. We further found that a point mutation (W985L) within H_CN reduced the toxicity over 10-fold, while this mutant maintained the same level of binding to neurons as wild type BoNT/A, suggesting that H_CN makes additional contributions to productive internalization/translocation steps beyond binding to neurons.

SUBMITTER: Wang J

PROVIDER: S-EPMC5353748 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Neuronal entry and high neurotoxicity of botulinum neurotoxin A require its N-terminal binding sub-domain.

Wang Jiafu J Meng Jianghui J Nugent Marc M Tang Minhong M Dolly J Oliver JO

Scientific reports 20170315

Botulinum neurotoxins (BoNTs) are the most toxic proteins known, due to inhibiting the neuronal release of acetylcholine and causing flaccid paralysis. Most BoNT serotypes target neurons by binding to synaptic vesicle proteins and gangliosides via a C-terminal binding sub-domain (HCC). However, the role of their conserved N-terminal sub-domain (HCN) has not been established. Herein, we created a mutant form of recombinant BoNT/A lacking HCN (rAΔHCN) an ...[more]

PMID: 28295026

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Project description:Botulinum neurotoxin (BoNT), the causative agent of the deadly neuroparalytic disease botulism, is the most poisonous protein known for humans. Produced by different strains of the anaerobic bacterium Clostridium botulinum, BoNT effects cellular intoxication via a multistep mechanism executed by the three modules of the activated protein. Endocytosis, the first step of cellular intoxication, is triggered by the ~50 kDa, heavy-chain receptor-binding domain (HCR) that is specific for a ganglioside and a protein receptor on neuronal cell surfaces. This dual receptor recognition mechanism between BoNT and the host cell's membrane is well documented and occurs via specific intermolecular interactions with the C-terminal sub-domain, Hcc, of BoNT-HCR. The N-terminal sub-domain of BoNT-HCR, Hcn, comprises ~50% of BoNT-HCR and adopts a ?-sheet jelly roll fold. While suspected in assisting cell surface recognition, no unambiguous function for the Hcn sub-domain in BoNT has been identified. To obtain insights into the potential function of the Hcn sub-domain in BoNT, the first crystal structure of a BoNT with an organic ligand bound to the Hcn sub-domain has been obtained. Here, we describe the crystal structure of BoNT/CD-HCR determined at 1.70 Å resolution with a tetraethylene glycol (PG4) moiety bound in a hydrophobic cleft between ?-strands in the ?-sheet jelly roll fold of the Hcn sub-domain. The PG4 moiety is completely engulfed in the cleft, making numerous hydrophilic (Y932, S959, W966, and D1042) and hydrophobic (S935, W977, L979, N1013, and I1066) contacts with the protein's side chain and backbone that may mimic in vivo interactions with the phospholipid membranes on neuronal cell surfaces. A sulfate ion was also observed bound to residues T1176, D1177, K1196, and R1243 in the Hcc sub-domain of BoNT/CD-HCR. In the crystal structure of a similar protein, BoNT/D-HCR, a sialic acid molecule was observed bound to the equivalent residues suggesting that residues T1176, D1177, K1196, and R1243 in BoNT/CD may play a role in ganglioside binding.

Dataset Information

Neuronal entry and high neurotoxicity of botulinum neurotoxin A require its N-terminal binding sub-domain.

Publications

Neuronal entry and high neurotoxicity of botulinum neurotoxin A require its N-terminal binding sub-domain.

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