1. |
- Davies, Jonathan R., et al.
(author)
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High-resolution crystal structures of the botulinum neurotoxin binding domains from subtypes A5 and A6
- 2020
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In: FEBS Open Bio. - : Wiley. - 2211-5463. ; 10:8, s. 1474-1481
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Journal article (peer-reviewed)abstract
- Clostridium botulinumneurotoxins (BoNTs) cause flaccid paralysis through inhibition of acetylcholine release from motor neurons; however, at tiny doses, this property is exploited for use as a therapeutic. Each member of the BoNT family of proteins consists of three distinct domains: a binding domain that targets neuronal cell membranes (H-C), a translocation domain (H-N) and a catalytic domain (LC). Here, we present high-resolution crystal structures of the binding domains of BoNT subtypes/A5 (H-C/A5) and/A6 (H-C/A6). These structures show that the core fold identified in other subtypes is maintained, but with subtle differences at the expected receptor-binding sites.
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2. |
- Elliott, Mark, et al.
(author)
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Engineered botulinum neurotoxin B with improved binding to human receptors has enhanced efficacy in preclinical models
- 2019
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In: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 5:1
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Journal article (peer-reviewed)abstract
- Although botulinum neurotoxin serotype A (BoNT/A) products are common treatments for various disorders, there is only one commercial BoNT/B product, whose low potency, likely stemming from low affinity toward its human receptor synaptotagmin 2 (hSyt2), has limited its therapeutic usefulness. We express and characterize two full-length recombinant BoNT/B1 proteins containing designed mutations E1191M/S1199Y (rBoNT/B1(MY)) and E1191Q/S1199W (rBoNT/B1(QW)) that enhance binding to hSyt2. In preclinical models including human-induced pluripotent stem cell neurons and a humanized transgenic mouse, this increased hSyt2 affinity results in high potency, comparable to that of BoNT/A. Last, we solve the cocrystal structure of rBoNT/B1(MY) in complex with peptides of hSyt2 and its homolog hSyt1. We demonstrate that neuronal surface receptor binding limits the clinical efficacy of unmodified BoNT/B and that modified BoNT/B proteins have promising clinical potential.
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3. |
- Rollauer, Sarah E., et al.
(author)
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Structure of the TatC core of the twin-arginine protein transport system
- 2012
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In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 492:7428, s. 210-
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Journal article (peer-reviewed)abstract
- The twin-arginine translocation (Tat) pathway is one of two general protein transport systems found in the prokaryotic cytoplasmic membrane and is conserved in the thylakoid membrane of plant chloroplasts. The defining, and highly unusual, property of the Tat pathway is that it transports folded proteins, a task that must be achieved without allowing appreciable ion leakage across the membrane. The integral membrane TatC protein is the central component of the Tat pathway. TatC captures substrate proteins by binding their signal peptides. TatC then recruits TatA family proteins to form the active translocation complex. Here we report the crystal structure of TatC from the hyperthermophilic bacterium Aquifex aeolicus. This structure provides a molecular description of the core of the Tat translocation system and a framework for understanding the unique Tat transport mechanism.
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4. |
- Tao, Liang, et al.
(author)
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Engineered botulinum neurotoxin B with improved efficacy for targeting human receptors
- 2017
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 8
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Journal article (peer-reviewed)abstract
- Botulinum neurotoxin B is a Food and Drug Administration-approved therapeutic toxin. However, it has lower binding affinity toward the human version of its major receptor, synaptotagmin II (h-Syt II), compared to mouse Syt II, because of a residue difference. Increasing the binding affinity to h-Syt II may improve botulinum neurotoxin B's therapeutic efficacy and reduce adverse effects. Here we utilized the bacterial adenylate cyclase two-hybrid method and carried out a saturation mutagenesis screen in the Syt II-binding pocket of botulinum neurotoxin B. The screen identifies E1191 as a key residue: replacing it with M/C/V/Q enhances botulinum neurotoxin B binding to human synaptotagmin II. Adding S1199Y/W or W1178Q as a secondary mutation further increases binding affinity. Mutant botulinum neurotoxin B containing E1191M/S1199Y exhibits similar to 11-fold higher efficacy in blocking neurotransmission than wild-type botulinum neurotoxin B in neurons expressing human synaptotagmin II, demonstrating that enhancing receptor binding increases the overall efficacy at functional levels. The engineered botulinum neurotoxin B provides a platform to develop therapeutic toxins with improved efficacy.
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