2qn0

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Structure of Botulinum neurotoxin serotype C1 light chain proteaseStructure of Botulinum neurotoxin serotype C1 light chain protease

Structural highlights

2qn0 is a 1 chain structure with sequence from Clostridium botulinum. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:X-ray diffraction, Resolution 1.75Å
Ligands:
Resources:FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

BXC_CBCP Botulinum toxin causes flaccid paralysis by inhibiting neurotransmitter (acetylcholine) release from the presynaptic membranes of nerve terminals of the eukaryotic host skeletal and autonomic nervous system, with frequent heart or respiratory failure (PubMed:16252491, PubMed:7901002, PubMed:8611567). Is unique among characterized BoNTs in having 2 substrates, syntaxin (STX) and SNAP25 (PubMed:7901002, PubMed:7737992, PubMed:8611567, PubMed:9886085, PubMed:17718519). Precursor of botulinum neurotoxin C which unlike most BoNTs seems not to have a proteinaceous coreceptor, and instead recognizes 2 different complex polysialylated gangliosides found on neural tissue probably found in synaptic vesicles (PubMed:21483489, PubMed:23027864). 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 the heavy chain (HC) forms pores that allows the light chain (LC) to translocate into the cytosol (By similarity). 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). In vitro the whole toxin only has protease activity after reduction (PubMed:8611567). Electrical stimulation increases uptake of toxin, presumably by transiently exposing a receptor usually found in eukaryotic target synaptic vesicles (PubMed:19650874). Forms ion-conducting channels at around pH 6.1 (PubMed:2424493). Requires complex eukaryotic host polysialogangliosides for full neurotoxicity (PubMed:19650874, PubMed:21483489). Synaptic vesicle glycoproteins (SV2) do not seem to act as its receptor (PubMed:21483489).[UniProtKB:P0DPI0][1] [2] [3] [4] [5] [6] [7] [8] [9] [10] Has proteolytic activity. After translocation into the eukaryotic host cytosol, inhibits neurotransmitter release by acting as a zinc endopeptidase that cleaves syntaxin-1A/STX1A and syntaxin-1B/STX1B (PubMed:7901002, PubMed:7737992, PubMed:8611567). Cleaves the '253-Arg-|-Ala-254' bond of STX1 and the '252-Arg-|-Ala-253' bond of STX2; also acts on syntaxin 3 (STX3) but not 4 (STX4) (PubMed:7737992). Cleaves the '198-Arg-|-Ala-199' bond of SNAP25 (PubMed:8611567, PubMed:9886085, PubMed:17718519). Recognizes the '93-Asn--Met-202' region of SNAP25 (PubMed:9886085).[11] [12] [13] [14] [15] Responsible for host epithelial cell transcytosis, host nerve cell targeting and translocation of light chain (LC) into eukaryotic 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 eukaryotic target cell surface. It simultaneously recognizes 2 polysialated gangliosides coreceptors in close proximity on host synaptic vesicles (PubMed:23027864, PubMed:21542861). The N-terminus of the TD wraps an extended belt around the perimeter of the LC, protecting Zn(2+) in the active site; it may also prevent premature LC dissociation from the translocation channel and protect toxin prior to translocation (By similarity). The TD inserts into synaptic vesicle membrane to allow translocation into the host cytosol (Probable). The C-terminal half of the HC (residues 864-1291) binds neurons in a dose-dependent manner (PubMed:20731382). The C-terminal half of the HC (residues 863-1291) binds eukaryotic host gangliosides in the order GD1b > GT1b > GD1a > GM1a (PubMed:16115873, PubMed:20731382, PubMed:23027864, PubMed:19650874). Has 2 ganglioside binding sites; Sia-1 prefers a sia7 sialic acid and sugars within the ganglioside (GD1b > GT1b), whereas GBP2 recognizes a sia5 sialic acid (GT1b and GD1a) (PubMed:23027864, PubMed:21542861). Both sites are required for HC to enter neurons, acting via different gangliosides (PubMed:23027864). This suggests that 2 gangliosides serve as toxin receptors (PubMed:16115873, PubMed:20731382, PubMed:21542861, PubMed:23027864). Synaptic activity (depolarization with K(+)) increases uptake by neurons (PubMed:23027864). Treatment of synaptosomes with proteinase K does not reduce HC binding, suggesting there is no protein receptor or it is protected from extracellular proteases (PubMed:16115873). Decreases uptake and toxicity of whole BoNT/A, but also interferes with uptake of BoNT/E and BoNT/F (PubMed:19650874). HC also binds phosphoinositides, which might play a role in membrane-binding (PubMed:22120109).[UniProtKB:P0DPI0][16] [17] [18] [19] [20] [21] [22]

Evolutionary Conservation

Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf.

Publication Abstract from PubMed

Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote alpha-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the alpha-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.

Structural and biochemical studies of botulinum neurotoxin serotype C1 light chain protease: implications for dual substrate specificity.,Jin R, Sikorra S, Stegmann CM, Pich A, Binz T, Brunger AT Biochemistry. 2007 Sep 18;46(37):10685-93. Epub 2007 Aug 24. PMID:17718519[23]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

See Also

References

  1. ↑ Jin R, Sikorra S, Stegmann CM, Pich A, Binz T, Brunger AT. Structural and biochemical studies of botulinum neurotoxin serotype C1 light chain protease: implications for dual substrate specificity. Biochemistry. 2007 Sep 18;46(37):10685-93. Epub 2007 Aug 24. PMID:17718519 doi:10.1021/bi701162d
  2. ↑ Rummel A, Häfner K, Mahrhold S, Darashchonak N, Holt M, Jahn R, Beermann S, Karnath T, Bigalke H, Binz T. Botulinum neurotoxins C, E and F bind gangliosides via a conserved binding site prior to stimulation-dependent uptake with botulinum neurotoxin F utilising the three isoforms of SV2 as second receptor. J Neurochem. 2009 Sep;110(6):1942-54. PMID:19650874 doi:10.1111/j.1471-4159.2009.06298.x
  3. ↑ Peng L, Tepp WH, Johnson EA, Dong M. Botulinum neurotoxin D uses synaptic vesicle protein SV2 and gangliosides as receptors. PLoS Pathog. 2011 Mar;7(3):e1002008. PMID:21483489 doi:10.1371/journal.ppat.1002008
  4. ↑ Donovan JJ, Middlebrook JL. Ion-conducting channels produced by botulinum toxin in planar lipid membranes. Biochemistry. 1986 May 20;25(10):2872-6. PMID:2424493 doi:10.1021/bi00358a020
  5. ↑ Schiavo G, Shone CC, Bennett MK, Scheller RH, Montecucco C. Botulinum neurotoxin type C cleaves a single Lys-Ala bond within the carboxyl-terminal region of syntaxins. J Biol Chem. 1995 May 5;270(18):10566-70. PMID:7737992 doi:10.1074/jbc.270.18.10566
  6. ↑ Blasi J, Chapman ER, Yamasaki S, Binz T, Niemann H, Jahn R. Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin. EMBO J. 1993 Dec;12(12):4821-8. PMID:7901002 doi:10.1002/j.1460-2075.1993.tb06171.x
  7. ↑ Foran P, Lawrence GW, Shone CC, Foster KA, Dolly JO. Botulinum neurotoxin C1 cleaves both syntaxin and SNAP-25 in intact and permeabilized chromaffin cells: correlation with its blockade of catecholamine release. Biochemistry. 1996 Feb 27;35(8):2630-6. PMID:8611567 doi:10.1021/bi9519009
  8. ↑ Vaidyanathan VV, Yoshino K, Jahnz M, Dörries C, Bade S, Nauenburg S, Niemann H, Binz T. Proteolysis of SNAP-25 isoforms by botulinum neurotoxin types A, C, and E: domains and amino acid residues controlling the formation of enzyme-substrate complexes and cleavage. J Neurochem. 1999 Jan;72(1):327-37. PMID:9886085 doi:10.1046/j.1471-4159.1999.0720327.x
  9. ↑ Takeda M, Tsukamoto K, Kohda T, Matsui M, Mukamoto M, Kozaki S. Characterization of the neurotoxin produced by isolates associated with avian botulism. Avian Dis. 2005 Sep;49(3):376-81. PMID:16252491 doi:10.1637/7347-022305R1.1
  10. ↑ Karalewitz AP, Fu Z, Baldwin MR, Kim JJ, Barbieri JT. Botulinum neurotoxin serotype C associates with dual ganglioside receptors to facilitate cell entry. J Biol Chem. 2012 Nov 23;287(48):40806-16. doi: 10.1074/jbc.M112.404244. Epub, 2012 Oct 1. PMID:23027864 doi:http://dx.doi.org/10.1074/jbc.M112.404244
  11. ↑ Jin R, Sikorra S, Stegmann CM, Pich A, Binz T, Brunger AT. Structural and biochemical studies of botulinum neurotoxin serotype C1 light chain protease: implications for dual substrate specificity. Biochemistry. 2007 Sep 18;46(37):10685-93. Epub 2007 Aug 24. PMID:17718519 doi:10.1021/bi701162d
  12. ↑ Schiavo G, Shone CC, Bennett MK, Scheller RH, Montecucco C. Botulinum neurotoxin type C cleaves a single Lys-Ala bond within the carboxyl-terminal region of syntaxins. J Biol Chem. 1995 May 5;270(18):10566-70. PMID:7737992 doi:10.1074/jbc.270.18.10566
  13. ↑ Blasi J, Chapman ER, Yamasaki S, Binz T, Niemann H, Jahn R. Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin. EMBO J. 1993 Dec;12(12):4821-8. PMID:7901002 doi:10.1002/j.1460-2075.1993.tb06171.x
  14. ↑ Foran P, Lawrence GW, Shone CC, Foster KA, Dolly JO. Botulinum neurotoxin C1 cleaves both syntaxin and SNAP-25 in intact and permeabilized chromaffin cells: correlation with its blockade of catecholamine release. Biochemistry. 1996 Feb 27;35(8):2630-6. PMID:8611567 doi:10.1021/bi9519009
  15. ↑ Vaidyanathan VV, Yoshino K, Jahnz M, Dörries C, Bade S, Nauenburg S, Niemann H, Binz T. Proteolysis of SNAP-25 isoforms by botulinum neurotoxin types A, C, and E: domains and amino acid residues controlling the formation of enzyme-substrate complexes and cleavage. J Neurochem. 1999 Jan;72(1):327-37. PMID:9886085 doi:10.1046/j.1471-4159.1999.0720327.x
  16. ↑ Tsukamoto K, Kohda T, Mukamoto M, Takeuchi K, Ihara H, Saito M, Kozaki S. Binding of Clostridium botulinum type C and D neurotoxins to ganglioside and phospholipid. Novel insights into the receptor for clostridial neurotoxins. J Biol Chem. 2005 Oct 21;280(42):35164-71. PMID:16115873 doi:10.1074/jbc.M507596200
  17. ↑ Rummel A, Häfner K, Mahrhold S, Darashchonak N, Holt M, Jahn R, Beermann S, Karnath T, Bigalke H, Binz T. Botulinum neurotoxins C, E and F bind gangliosides via a conserved binding site prior to stimulation-dependent uptake with botulinum neurotoxin F utilising the three isoforms of SV2 as second receptor. J Neurochem. 2009 Sep;110(6):1942-54. PMID:19650874 doi:10.1111/j.1471-4159.2009.06298.x
  18. ↑ Karalewitz AP, Kroken AR, Fu Z, Baldwin MR, Kim JJ, Barbieri JT. Identification of a Unique Ganglioside Binding Loop within Botulinum Neurotoxins C and D-SA . Biochemistry. 2010 Aug 23. PMID:20731382 doi:10.1021/bi100865f
  19. ↑ Strotmeier J, Gu S, Jutzi S, Mahrhold S, Zhou J, Pich A, Eichner T, Bigalke H, Rummel A, Jin R, Binz T. The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites. Mol Microbiol. 2011 May 4. doi: 10.1111/j.1365-2958.2011.07682.x. PMID:21542861 doi:10.1111/j.1365-2958.2011.07682.x
  20. ↑ Zhang Y, Varnum SM. The receptor binding domain of botulinum neurotoxin serotype C binds phosphoinositides. Biochimie. 2012 Mar;94(3):920-3. PMID:22120109 doi:10.1016/j.biochi.2011.11.004
  21. ↑ Karalewitz AP, Fu Z, Baldwin MR, Kim JJ, Barbieri JT. Botulinum neurotoxin serotype C associates with dual ganglioside receptors to facilitate cell entry. J Biol Chem. 2012 Nov 23;287(48):40806-16. doi: 10.1074/jbc.M112.404244. Epub, 2012 Oct 1. PMID:23027864 doi:http://dx.doi.org/10.1074/jbc.M112.404244
  22. ↑ Donovan JJ, Middlebrook JL. Ion-conducting channels produced by botulinum toxin in planar lipid membranes. Biochemistry. 1986 May 20;25(10):2872-6. PMID:2424493 doi:10.1021/bi00358a020
  23. ↑ Jin R, Sikorra S, Stegmann CM, Pich A, Binz T, Brunger AT. Structural and biochemical studies of botulinum neurotoxin serotype C1 light chain protease: implications for dual substrate specificity. Biochemistry. 2007 Sep 18;46(37):10685-93. Epub 2007 Aug 24. PMID:17718519 doi:10.1021/bi701162d

2qn0, resolution 1.75Å

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