Limits...
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 SXN101959 and comparison with LHn/D.(A) The crystal structure of SXN101959, in dark red, was superposed with LHn/D, in cyan. The disulphide bridge between LC and Hn is highlighted in orange. The zinc ion of SXN101959 is shown as a grey sphere. (B) Close-up view of the domain interface in SXN101959 and LHn/D. The C-terminal end of LC and N-terminus of Hn are labelled. The location of the targeting ligand is represented with the missing part of the linker shown as a dashed line. (C) The visible part of the linker region of SXN101959 is highlighted with the 2Fo-Fc electron density map countered at 1σ level shown in blue. (D) Temperature factor (B-factor) analysis. Ribbon diagram representation of LHn/D and SXN101959 structures, with a gradient colouring from low (cyan) to high (red) B-factors. Gradients were adjusted independently to highlight the less ordered area for each structure. (E) Activation of the human GHRH receptor. The receptor activation was detected by the intracellular accumulation of cAMP in CHO-K1-hGHRH-R cells incubated with hGHRH(1–44) (•) or SXN101959 (). Data are mean ± sem of triplicate samples from one experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4555039&req=5

f5: Crystal structure of SXN101959 and comparison with LHn/D.(A) The crystal structure of SXN101959, in dark red, was superposed with LHn/D, in cyan. The disulphide bridge between LC and Hn is highlighted in orange. The zinc ion of SXN101959 is shown as a grey sphere. (B) Close-up view of the domain interface in SXN101959 and LHn/D. The C-terminal end of LC and N-terminus of Hn are labelled. The location of the targeting ligand is represented with the missing part of the linker shown as a dashed line. (C) The visible part of the linker region of SXN101959 is highlighted with the 2Fo-Fc electron density map countered at 1σ level shown in blue. (D) Temperature factor (B-factor) analysis. Ribbon diagram representation of LHn/D and SXN101959 structures, with a gradient colouring from low (cyan) to high (red) B-factors. Gradients were adjusted independently to highlight the less ordered area for each structure. (E) Activation of the human GHRH receptor. The receptor activation was detected by the intracellular accumulation of cAMP in CHO-K1-hGHRH-R cells incubated with hGHRH(1–44) (•) or SXN101959 (). Data are mean ± sem of triplicate samples from one experiment.

Mentions: The main components of the translocation domain are the two coiled-coil helices of approximately 105 Å (Fig. 1). While the secondary α-helical structure is conserved across serotypes, the two helices all have a central bending point which curves to various degrees (Fig. 4). Of the four serotypes (with known Hn structures thus far), D appears the straightest. It should also be noticed that Hn/D presented particularly high B-factor at the extremities of these helices (Fig. 5D), with weak electron density for the random-coiled loop linking them. This is likely an indication of flexibility within these regions. The smaller helices flanking the main frame are also well conserved and similarly positioned across serotypes. Additionally the visible C-terminal end forms a small α-helix which is involved in crystal packing with a symmetry-related molecule and appears in a different orientation to the similar segment in LHn/A and /B.


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 SXN101959 and comparison with LHn/D.(A) The crystal structure of SXN101959, in dark red, was superposed with LHn/D, in cyan. The disulphide bridge between LC and Hn is highlighted in orange. The zinc ion of SXN101959 is shown as a grey sphere. (B) Close-up view of the domain interface in SXN101959 and LHn/D. The C-terminal end of LC and N-terminus of Hn are labelled. The location of the targeting ligand is represented with the missing part of the linker shown as a dashed line. (C) The visible part of the linker region of SXN101959 is highlighted with the 2Fo-Fc electron density map countered at 1σ level shown in blue. (D) Temperature factor (B-factor) analysis. Ribbon diagram representation of LHn/D and SXN101959 structures, with a gradient colouring from low (cyan) to high (red) B-factors. Gradients were adjusted independently to highlight the less ordered area for each structure. (E) Activation of the human GHRH receptor. The receptor activation was detected by the intracellular accumulation of cAMP in CHO-K1-hGHRH-R cells incubated with hGHRH(1–44) (•) or SXN101959 (). Data are mean ± sem of triplicate samples from one experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Crystal structure of SXN101959 and comparison with LHn/D.(A) The crystal structure of SXN101959, in dark red, was superposed with LHn/D, in cyan. The disulphide bridge between LC and Hn is highlighted in orange. The zinc ion of SXN101959 is shown as a grey sphere. (B) Close-up view of the domain interface in SXN101959 and LHn/D. The C-terminal end of LC and N-terminus of Hn are labelled. The location of the targeting ligand is represented with the missing part of the linker shown as a dashed line. (C) The visible part of the linker region of SXN101959 is highlighted with the 2Fo-Fc electron density map countered at 1σ level shown in blue. (D) Temperature factor (B-factor) analysis. Ribbon diagram representation of LHn/D and SXN101959 structures, with a gradient colouring from low (cyan) to high (red) B-factors. Gradients were adjusted independently to highlight the less ordered area for each structure. (E) Activation of the human GHRH receptor. The receptor activation was detected by the intracellular accumulation of cAMP in CHO-K1-hGHRH-R cells incubated with hGHRH(1–44) (•) or SXN101959 (). Data are mean ± sem of triplicate samples from one experiment.
Mentions: The main components of the translocation domain are the two coiled-coil helices of approximately 105 Å (Fig. 1). While the secondary α-helical structure is conserved across serotypes, the two helices all have a central bending point which curves to various degrees (Fig. 4). Of the four serotypes (with known Hn structures thus far), D appears the straightest. It should also be noticed that Hn/D presented particularly high B-factor at the extremities of these helices (Fig. 5D), with weak electron density for the random-coiled loop linking them. This is likely an indication of flexibility within these regions. The smaller helices flanking the main frame are also well conserved and similarly positioned across serotypes. Additionally the visible C-terminal end forms a small α-helix which is involved in crystal packing with a symmetry-related molecule and appears in a different orientation to the similar segment in LHn/A and /B.

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