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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

The belt of BoNT/D.(A) Crystal structure of LHn/D with the belt region represented with grey sticks. The rest of the translocation domain is shown in blue, the light chain in shown as a cyan surface. The structure of LC/F in complex with a VAMP peptide inhibitor (PDB 3FIE25) was superposed to LHn/D and VAMP is represented as a pink ribbon. (B) Superposition of the belt regions of LHn/A (PDB 2W2D17), /B (PDB 2XHL18), /D and BoNT/E (PDB 3FFZ21), in blue, magenta, cyan and grey ribbon, respectively. The amino- and carboxyl-end of the belt are labelled.
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f3: The belt of BoNT/D.(A) Crystal structure of LHn/D with the belt region represented with grey sticks. The rest of the translocation domain is shown in blue, the light chain in shown as a cyan surface. The structure of LC/F in complex with a VAMP peptide inhibitor (PDB 3FIE25) was superposed to LHn/D and VAMP is represented as a pink ribbon. (B) Superposition of the belt regions of LHn/A (PDB 2W2D17), /B (PDB 2XHL18), /D and BoNT/E (PDB 3FFZ21), in blue, magenta, cyan and grey ribbon, respectively. The amino- and carboxyl-end of the belt are labelled.

Mentions: The long substrate requirement of all BoNT serotypes is atypical to other zinc proteases and involves several binding sites distal from the catalytic pocket26. Guo and Chen identified and labelled these exosites B1 to B5 in LC/D24. None of these sites present any conformational changes in LHn/D. While B1 is buried within LC, 15 Å away from the active site, the hydrophobic patches B4 to B5 show the extent of substrate recognition at the accessible surface of the free light chain. In LHn/D these pockets are interacting with the belt (Fig. 3) and implies a potential substrate inhibiting role for this region as also hypothesised for BoNT/A and /B27.


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)

The belt of BoNT/D.(A) Crystal structure of LHn/D with the belt region represented with grey sticks. The rest of the translocation domain is shown in blue, the light chain in shown as a cyan surface. The structure of LC/F in complex with a VAMP peptide inhibitor (PDB 3FIE25) was superposed to LHn/D and VAMP is represented as a pink ribbon. (B) Superposition of the belt regions of LHn/A (PDB 2W2D17), /B (PDB 2XHL18), /D and BoNT/E (PDB 3FFZ21), in blue, magenta, cyan and grey ribbon, respectively. The amino- and carboxyl-end of the belt are labelled.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: The belt of BoNT/D.(A) Crystal structure of LHn/D with the belt region represented with grey sticks. The rest of the translocation domain is shown in blue, the light chain in shown as a cyan surface. The structure of LC/F in complex with a VAMP peptide inhibitor (PDB 3FIE25) was superposed to LHn/D and VAMP is represented as a pink ribbon. (B) Superposition of the belt regions of LHn/A (PDB 2W2D17), /B (PDB 2XHL18), /D and BoNT/E (PDB 3FFZ21), in blue, magenta, cyan and grey ribbon, respectively. The amino- and carboxyl-end of the belt are labelled.
Mentions: The long substrate requirement of all BoNT serotypes is atypical to other zinc proteases and involves several binding sites distal from the catalytic pocket26. Guo and Chen identified and labelled these exosites B1 to B5 in LC/D24. None of these sites present any conformational changes in LHn/D. While B1 is buried within LC, 15 Å away from the active site, the hydrophobic patches B4 to B5 show the extent of substrate recognition at the accessible surface of the free light chain. In LHn/D these pockets are interacting with the belt (Fig. 3) and implies a potential substrate inhibiting role for this region as also hypothesised for BoNT/A and /B27.

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