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Structural analysis of Clostridium botulinum neurotoxin type D as a platform for the development of targeted secretion inhibitors.

Masuyer G, Davies JR, Moore K, Chaddock JA, Ravi Acharya K - Sci Rep (2015)

Bottom Line: Furthermore, structural information from small-angle X-ray scattering of LHn/D is compared among serotypes A, B, and D.Taken together, these results demonstrate the robustness of the 'LHn fold' across serotypes and its use in engineering additional polypeptide components with added functionality.Our study demonstrates the suitability of botulinum neurotoxin, and serotype D in particular, as a basis for engineering novel secretion inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK.

ABSTRACT
The botulinum neurotoxin type D is one of seven highly potent toxins produced by Clostridium botulinum which inhibit neurotransmission at cholinergic nerve terminals. A functional fragment derived from the toxin, LHn, consisting of the catalytic and translocation domains, has been heralded as a platform for the development of targeted secretion inhibitors. These secretion inhibitors are aimed at retargeting the toxin towards a specific cell type to inhibit vesicular secretion. Here we report crystal structures of LHn from serotype D at 2.3 Å, and that of SXN101959 at 3.1 Å resolution. SXN101959, a derivative that combines LHn from serotype D with a fragment of the growth hormone releasing hormone, has previously revealed promising results in inhibiting growth hormone release in pituitary somatotrophs. These structures offer for the first time insights into the translocation domain interaction with the catalytic domain in serotype D. Furthermore, structural information from small-angle X-ray scattering of LHn/D is compared among serotypes A, B, and D. Taken together, these results demonstrate the robustness of the 'LHn fold' across serotypes and its use in engineering additional polypeptide components with added functionality. Our study demonstrates the suitability of botulinum neurotoxin, and serotype D in particular, as a basis for engineering novel secretion inhibitors.

No MeSH data available.


Related in: MedlinePlus

Crystal structure of LHn/D.Ribbon diagram representation of LHn/D structure, LC in cyan, belt region in grey and Hn in blue. C-terminus of LC and N-terminus of Hn are marked with a cyan and grey asterisk, respectively. The disordered region of the belt is presented as a grey-dashed line. The disulphide bond between LC and Hn is highlighted in orange.
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f1: Crystal structure of LHn/D.Ribbon diagram representation of LHn/D structure, LC in cyan, belt region in grey and Hn in blue. C-terminus of LC and N-terminus of Hn are marked with a cyan and grey asterisk, respectively. The disordered region of the belt is presented as a grey-dashed line. The disulphide bond between LC and Hn is highlighted in orange.

Mentions: This is a mutant endo-negative form of LHn/D in which the catalytic domain is inactivated by a two amino acid substitution in the zinc-binding site (H233Y, E230Q). The crystal of LHn/D diffracted at 2.3 Å resolution in space group P6422 with cell dimensions a = b = 173, c = 222 Å; α = β = 90, γ = 120° (Table 1). Despite the large unit cell, a single molecule was present in the asymmetric unit corresponding to an unusually high solvent content of 75%. The X-ray crystal structure presents the two domains, LC and Hn, in a close interaction. No electron density was observed for regions 442–457 (between the two domains), residues 494–510, and the C-terminal poly-histidine tag (last fifteen residues), all in solvent-accessible areas (Fig. 1).


Structural analysis of Clostridium botulinum neurotoxin type D as a platform for the development of targeted secretion inhibitors.

Masuyer G, Davies JR, Moore K, Chaddock JA, Ravi Acharya K - Sci Rep (2015)

Crystal structure of LHn/D.Ribbon diagram representation of LHn/D structure, LC in cyan, belt region in grey and Hn in blue. C-terminus of LC and N-terminus of Hn are marked with a cyan and grey asterisk, respectively. The disordered region of the belt is presented as a grey-dashed line. The disulphide bond between LC and Hn is highlighted in orange.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555039&req=5

f1: Crystal structure of LHn/D.Ribbon diagram representation of LHn/D structure, LC in cyan, belt region in grey and Hn in blue. C-terminus of LC and N-terminus of Hn are marked with a cyan and grey asterisk, respectively. The disordered region of the belt is presented as a grey-dashed line. The disulphide bond between LC and Hn is highlighted in orange.
Mentions: This is a mutant endo-negative form of LHn/D in which the catalytic domain is inactivated by a two amino acid substitution in the zinc-binding site (H233Y, E230Q). The crystal of LHn/D diffracted at 2.3 Å resolution in space group P6422 with cell dimensions a = b = 173, c = 222 Å; α = β = 90, γ = 120° (Table 1). Despite the large unit cell, a single molecule was present in the asymmetric unit corresponding to an unusually high solvent content of 75%. The X-ray crystal structure presents the two domains, LC and Hn, in a close interaction. No electron density was observed for regions 442–457 (between the two domains), residues 494–510, and the C-terminal poly-histidine tag (last fifteen residues), all in solvent-accessible areas (Fig. 1).

Bottom Line: Furthermore, structural information from small-angle X-ray scattering of LHn/D is compared among serotypes A, B, and D.Taken together, these results demonstrate the robustness of the 'LHn fold' across serotypes and its use in engineering additional polypeptide components with added functionality.Our study demonstrates the suitability of botulinum neurotoxin, and serotype D in particular, as a basis for engineering novel secretion inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK.

ABSTRACT
The botulinum neurotoxin type D is one of seven highly potent toxins produced by Clostridium botulinum which inhibit neurotransmission at cholinergic nerve terminals. A functional fragment derived from the toxin, LHn, consisting of the catalytic and translocation domains, has been heralded as a platform for the development of targeted secretion inhibitors. These secretion inhibitors are aimed at retargeting the toxin towards a specific cell type to inhibit vesicular secretion. Here we report crystal structures of LHn from serotype D at 2.3 Å, and that of SXN101959 at 3.1 Å resolution. SXN101959, a derivative that combines LHn from serotype D with a fragment of the growth hormone releasing hormone, has previously revealed promising results in inhibiting growth hormone release in pituitary somatotrophs. These structures offer for the first time insights into the translocation domain interaction with the catalytic domain in serotype D. Furthermore, structural information from small-angle X-ray scattering of LHn/D is compared among serotypes A, B, and D. Taken together, these results demonstrate the robustness of the 'LHn fold' across serotypes and its use in engineering additional polypeptide components with added functionality. Our study demonstrates the suitability of botulinum neurotoxin, and serotype D in particular, as a basis for engineering novel secretion inhibitors.

No MeSH data available.


Related in: MedlinePlus