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Title: A viral-fusion-peptide-like molecular switch drives membrane insertion of botulinum neurotoxin A1

Abstract

Botulinum neurotoxin (BoNT) delivers its protease domain across the vesicle membrane to enter the neuronal cytosol upon vesicle acidification. This process is mediated by its translocation domain (H N), but the molecular mechanism underlying membrane insertion of H N remains poorly understood. Here, we report two crystal structures of BoNT/A1 H N that reveal a novel molecular switch (termed BoNT-switch) in H N, where buried α-helices transform into surface-exposed hydrophobic β-hairpins triggered by acidic pH. Locking the BoNT-switch by disulfide trapping inhibited the association of H N with anionic liposomes, blocked channel formation by H N, and reduced the neurotoxicity of BoNT/A1 by up to ~180-fold. Single particle counting studies showed that an acidic environment tends to promote BoNT/A1 self-association on liposomes, which is partly regulated by the BoNT-switch. Furthermore, these findings suggest that the BoNT-switch flips out upon exposure to the acidic endosomal pH, which enables membrane insertion of H N that subsequently leads to LC delivery.

Authors:
 [1];  [2];  [3];  [4];  [5];  [3]; ORCiD logo [3];  [2]; ORCiD logo [1]
  1. Univ. of California, Irvine, CA (United States)
  2. Stony Brook Univ., Stony Brook, NY (United States)
  3. Medizinische Hochschule Hannover, Hannover (Germany)
  4. SLAC National Accelerator Lab., Menlo Park, CA (United States); Stanford Univ., Menlo Park, CA (United States)
  5. Cornell Univ., Argonne, IL (United States)
Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1490651
Grant/Contract Number:  
AC02-76SF00515
Resource Type:
Accepted Manuscript
Journal Name:
Nature Communications
Additional Journal Information:
Journal Volume: 9; Journal Issue: 1; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Lam, Kwok-ho, Guo, Zhuojun, Krez, Nadja, Matsui, Tsutomu, Perry, Kay, Weisemann, Jasmin, Rummel, Andreas, Bowen, Mark E., and Jin, Rongsheng. A viral-fusion-peptide-like molecular switch drives membrane insertion of botulinum neurotoxin A1. United States: N. p., 2018. Web. doi:10.1038/s41467-018-07789-4.
Lam, Kwok-ho, Guo, Zhuojun, Krez, Nadja, Matsui, Tsutomu, Perry, Kay, Weisemann, Jasmin, Rummel, Andreas, Bowen, Mark E., & Jin, Rongsheng. A viral-fusion-peptide-like molecular switch drives membrane insertion of botulinum neurotoxin A1. United States. doi:10.1038/s41467-018-07789-4.
Lam, Kwok-ho, Guo, Zhuojun, Krez, Nadja, Matsui, Tsutomu, Perry, Kay, Weisemann, Jasmin, Rummel, Andreas, Bowen, Mark E., and Jin, Rongsheng. Tue . "A viral-fusion-peptide-like molecular switch drives membrane insertion of botulinum neurotoxin A1". United States. doi:10.1038/s41467-018-07789-4. https://www.osti.gov/servlets/purl/1490651.
@article{osti_1490651,
title = {A viral-fusion-peptide-like molecular switch drives membrane insertion of botulinum neurotoxin A1},
author = {Lam, Kwok-ho and Guo, Zhuojun and Krez, Nadja and Matsui, Tsutomu and Perry, Kay and Weisemann, Jasmin and Rummel, Andreas and Bowen, Mark E. and Jin, Rongsheng},
abstractNote = {Botulinum neurotoxin (BoNT) delivers its protease domain across the vesicle membrane to enter the neuronal cytosol upon vesicle acidification. This process is mediated by its translocation domain (HN), but the molecular mechanism underlying membrane insertion of HN remains poorly understood. Here, we report two crystal structures of BoNT/A1 HN that reveal a novel molecular switch (termed BoNT-switch) in HN, where buried α-helices transform into surface-exposed hydrophobic β-hairpins triggered by acidic pH. Locking the BoNT-switch by disulfide trapping inhibited the association of HN with anionic liposomes, blocked channel formation by HN, and reduced the neurotoxicity of BoNT/A1 by up to ~180-fold. Single particle counting studies showed that an acidic environment tends to promote BoNT/A1 self-association on liposomes, which is partly regulated by the BoNT-switch. Furthermore, these findings suggest that the BoNT-switch flips out upon exposure to the acidic endosomal pH, which enables membrane insertion of HN that subsequently leads to LC delivery.},
doi = {10.1038/s41467-018-07789-4},
journal = {Nature Communications},
number = 1,
volume = 9,
place = {United States},
year = {2018},
month = {12}
}

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Figures / Tables:

Fig. 1 Fig. 1: Biochemical characterization of tHNA. a The structure of BoNT/A1 (PDB code 3BTA). LC, the “belt”, tHN, and HC are colored green, marine, orange, and cyan, respectively. The disulfide linkage between LC and HN is shown as salmon sphere and the BoNT-switch is highlighted in magenta. b, c Cosedimentationmore » of tHNA with liposomes. tHNA was incubated with asolectin liposomes at pH 7.5, 5.0 or 4.4 (b); or incubated with liposomes containing 60/40mol% PC/cholesterol, 30/30/40mol% PC/PS/cholesterol, or 60/40mol% PS/cholesterol at pH 4.4 (c). After liposomes were pelleted, the proteins in input, supernatant, and pellet fractions were analyzed by SDS-PAGE. These experiments were performed in triplicate and quantification of protein band intensities are shown in Supplementary Fig. 11. Uncropped images of gels are shown in Supplementary Fig. 13. d Calcein dye release assay. tHNA was tested with four different liposomes loaded with 50mM calcein at pH 4.6 or pH 7.0, whereas liposomes were composed of DOPC alone, 80/20 mol% DOPC/DOPS, 60/40 mol% DOPC/DOPS, or asolectin. The rate of calcein dye release was determined based on the increase of fluorescence at 525 nm during excitation at 493 nm. Error bars indicate SD of triplicate measurements. e Membrane depolarization assay. Liposomes composed of 70/20/10 mol% DOPC/DOPS/cholesterol were polarized at a positive internal voltage by adding valinomycin in the presence of a transmembrane KCl gradient. Membrane potential was measured using the voltage-sensitive fluorescence dye ANS. After 3 min, tHNA was added at the indicated buffer pH. The data are presented as mean ± S.D., n =3« less

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