7kz7
From Proteopedia
Crystals Structure of the Mutated Protease Domain of Botulinum Neurotoxin X (X4130B1).
Structural highlights
FunctionBXX_CLOBO Botulinum toxin causes flaccid paralysis by inhibiting neurotransmitter (acetylcholine) release from the presynaptic membranes of nerve terminals of eukaryotic host skeletal and autonomic nervous system, with frequent heart or respiratory failure. Precursor of botulinum neurotoxin X which has 2 coreceptors; complex polysialylated gangliosides found on neural tissue and specific membrane-anchored proteins found in synaptic vesicles. Receptor proteins are exposed on host presynaptic cell membrane during neurotransmitter release, when the toxin heavy chain (HC) binds to them. Upon synaptic vesicle recycling the toxin is taken up via the endocytic pathway. When the pH of the toxin-containing endosome drops a structural rearrangement occurs so that the N-terminus of HC forms pores that allows the light chain (LC) to translocate into the cytosol. Once in the cytosol the disulfide bond linking the 2 subunits is reduced and LC cleaves its target protein on synaptic vesicles, preventing their fusion with the cytoplasmic membrane and thus neurotransmitter release (By similarity). Artificially assembled BoNT/X cleaves synaptobrevin-2/VAMP2 and VAMP4 in cultured rat neurons and causes flaccid paralysis in mice (PubMed:28770820).[UniProtKB:P0DPI0][1] Has proteolytic activity. After translocation into the eukaryotic host cytosol, LC hydrolyzes the '66-Arg-|-Ala-67' bond in synaptobrevin-2/VAMP2, and the equivalent bonds in 'Arg-|-Ala' bonds in VAMP1 and VAMP3, thus blocking neurotransmitter release (PubMed:28770820). Has a wider target range than most BoNTs, as it also cleaves the '87-Arg-|-Ser-89' bond in VAMP4, the '40-Arg-|-Ser-41' bond in VAMP5 and the '173-Lys-|-Ser-174' bond in YKT6; whether these are physiologically relevant substrates is unknown (PubMed:28770820). BoNT/X is 10-fold more efficient than BoNT/B and 40-fold more efficient than TeTX on an artificial human VAMP1 substrate (PubMed:29540745).[2] [3] Responsible for epithelial cell transcytosis, host nerve cell targeting and translocation of light chain (LC) into host cytosol. Composed of 3 subdomains; the translocation domain (TD), and N-terminus and C-terminus of the receptor-binding domain (RBD). The RBD is responsible for the adherence of the toxin to the cell surface. It simultaneously recognizes 2 coreceptors; polysialated gangliosides and an unknown receptor protein in close proximity on host synaptic vesicles. The N-terminus of the TD wraps an extended belt around the perimeter of the LC, protecting Zn(2+) in the active site (By similarity). The TD inserts into synaptic vesicle membrane to allow translocation into the host cytosol (By similarity). Protein ligation of the RBD to the rest of the toxin (creates an artificial whole toxin) greatly increases VAMP2 degradation, and thus neuron uptake (PubMed:28770820).[UniProtKB:P10844][4] Publication Abstract from PubMedAlthough bespoke, sequence-specific proteases have the potential to advance biotechnology and medicine, generation of proteases with tailor-made cleavage specificities remains a major challenge. We developed a phage-assisted protease evolution system with simultaneous positive and negative selection and applied it to three botulinum neurotoxin (BoNT) light-chain proteases. We evolved BoNT/X protease into separate variants that preferentially cleave vesicle-associated membrane protein 4 (VAMP4) and Ykt6, evolved BoNT/F protease to selectively cleave the non-native substrate VAMP7, and evolved BoNT/E protease to cleave phosphatase and tensin homolog (PTEN) but not any natural BoNT protease substrate in neurons. The evolved proteases display large changes in specificity (218- to >11,000,000-fold) and can retain their ability to form holotoxins that self-deliver into primary neurons. These findings establish a versatile platform for reprogramming proteases to selectively cleave new targets of therapeutic interest. Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity.,Blum TR, Liu H, Packer MS, Xiong X, Lee PG, Zhang S, Richter M, Minasov G, Satchell KJF, Dong M, Liu DR Science. 2021 Feb 19;371(6531):803-810. doi: 10.1126/science.abf5972. PMID:33602850[5] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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Categories: Clostridium botulinum | Large Structures | Blum TR | Dong M | Lee PG | Liu DR | Liu H | Minasov G | Packer MS | Richter M | Satchell KJF | Xiong X | Zhang S