Crystal Structures of Botulinum Neurotoxin Subtypes A4 and A5 Cell Binding Domains in Complex with Receptor Ganglioside

Gregory, Kyle S.; Mojanaga, Otsile O.; Liu, Sai Man; Acharya, K. Ravi

doi:10.3390/toxins14020129

Cited by 6 publications

(20 citation statements)

References 38 publications

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“…The quality of the electron density map is good throughout except for a small loop region (Arg 1269-Phe 1277). This loop (which is conserved across all BoNT/A subtypes) precedes the ganglioside binding site (GBS) and appears to be disordered for other H C /A subtypes [ 28 , 29 ]. This is likely due to the inherent flexibility of this loop so that it can accommodate ganglioside binding.…”

Section: Resultsmentioning

confidence: 99%

Structural Features of Clostridium botulinum Neurotoxin Subtype A2 Cell Binding Domain

Gregory

Mahadeva

Liu

et al. 2022

Toxins

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Botulinum neurotoxins (BoNT) are a group of clostridial toxins that cause the potentially fatal neuroparalytic disease botulism. Although highly toxic, BoNTs are utilized as therapeutics to treat a range of neuromuscular conditions. Several serotypes (BoNT/A-/G, /X) have been identified with vastly differing toxicological profiles. Each serotype can be further sub-categorised into subtypes due to subtle variations in their protein sequence. These minor changes have been attributed to differences in both the duration of action and potency for BoNT/A subtypes. BoNTs are composed of three domains—a cell-binding domain, a translocation domain, and a catalytic domain. In this paper, we present the crystal structures of the botulinum neurotoxin A2 cell binding domain, both alone and in complex with its receptor ganglioside GD1a at 1.63 and 2.10 Å, respectively. The analysis of these structures reveals a potential redox-dependent Lys-O-Cys bridge close to the ganglioside binding site and a hinge motion between the HCN and HCC subdomains. Furthermore, we make a detailed comparison with the previously reported HC/A2:SV2C structure for a comprehensive structural analysis of HC/A2 receptor binding.

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Section: Resultsmentioning

confidence: 99%

Structural Features of Clostridium botulinum Neurotoxin Subtype A2 Cell Binding Domain

Gregory

Mahadeva

Liu

et al. 2022

Toxins

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“…The program Dyndom [ 30 ] revealed a large hinge rotation of ~16.8° in H C /A6 (crystal form II) when compared to H C /A6 (crystal form I) and H C /A6:GD1a ( Figure 4 ). To date, this is the largest “hinge motion” in subdomain orientation observed among BoNT/A subtype structures [ 25 , 27 ], and it suggests a high degree of flexibility existing between the H CN and H CC subdomains. The biological implication of this hinge-rotation has not yet been determined; however, it has been previously suggested that it may aid in orientating the H N towards the membrane in preparation for translocation [ 23 ].…”

Section: Resultsmentioning

confidence: 99%

“…This was observed previously for H C /A2, H C /A3, and H C /A5. Furthermore, H C /A6 forms seven hydrogen bonds with GD1a at Sia 5 , Gal 4 , and GalNAc 3 through six residues that are conserved among all BoNT/A subtypes (Figure 2) [23][24][25][26][27]. The H C /A6:GD1a interface is most similar to H C /A5, forming a total of 7 hydrogen bonds with the three monosaccharides GalNAc 3 , Gal 4 and Sia 5 [27].…”

Section: New Crystal Form Of H C /A6mentioning

confidence: 99%

“…GD1a ( Figure 1 A) constitutes one of the four major gangliosides (GM1, GD1a, GT1b and GD1b [ 22 ]) that make up 80–90% [ 20 ] of all gangliosides. The oligosaccharide moiety contains six monosaccharide units, of which the three terminal units (Sialic acid, Galactose, and N -acetylglucosamine) have been shown to form direct hydrogen bonding interactions with the H CC subdomain of BoNT subtypes (/A1 to/A5) [ 23 , 24 , 25 , 26 , 27 ]. The terminal units are conserved among GD1a and GT1b and both have been identified as binding partners with BoNT/A1 [ 28 ].…”

Section: Introductionmentioning

confidence: 99%

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Crystal Structures of the Clostridium botulinum Neurotoxin A6 Cell Binding Domain Alone and in Complex with GD1a Reveal Significant Conformational Flexibility

Gregory

Newell

Mojanaga

et al. 2022

IJMS

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Clostridium botulinum neurotoxin A (BoNT/A) targets the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, by cleaving synaptosomal-associated protein of 25 kDa size (SNAP-25). Cleavage of SNAP-25 results in flaccid paralysis due to repression of synaptic transmission at the neuromuscular junction. This activity has been exploited to treat a range of diseases associated with hypersecretion of neurotransmitters, with formulations of BoNT/A commercially available as therapeutics. Generally, BoNT activity is facilitated by three essential domains within the molecule, the cell binding domain (HC), the translocation domain (HN), and the catalytic domain (LC). The HC, which consists of an N-terminal (HCN) and a C-terminal (HCC) subdomain, is responsible for BoNT’s high target specificity where it forms a dual-receptor complex with synaptic vesicle protein 2 (SV2) and a ganglioside receptor on the surface of motor neurons. In this study, we have determined the crystal structure of botulinum neurotoxin A6 cell binding domain (HC/A6) in complex with GD1a and describe the interactions involved in ganglioside binding. We also present a new crystal form of wild type HC/A6 (crystal form II) where a large ‘hinge motion’ between the HCN and HCC subdomains is observed. These structures, along with a comparison to the previously determined wild type crystal structure of HC/A6 (crystal form I), reveals the degree of conformational flexibility exhibited by HC/A6.

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“…During the simulation, we found that in addition to GBS, gangliosides also interact with BoNT/A1 via a structure that we referred to as the Ganglioside Binding Loop “GBL”, referring to the appellation used in previous studies 41, 42 . The GBL is defined by the amino acid sequence 1253-HQFNNIAK-1260 for which H1253 was identified to interact with ganglioside 43 . After performing a structural alignment of the heavy chain of BoNT/A1, BoNT/E1 and BoNT/F1, we observed that the GBL is not conserved in serotype E1 and F1 ( figure 7 C and D ).…”

Section: Discussionmentioning

confidence: 99%

Structural basis of botulinum neurotoxin serotype A1 binding to human SV2A or SV2C receptors

Azzaz

Hilaire

Fantini

2022

Preprint

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Botulinum neurotoxin A1 (BoNT/A1) is the most potent serotype in humans with the highest clinical duration. BoNT/A1 interacts with synaptic vesicle glycoprotein 2 (SV2) and gangliosides to be taken up by neurons. In this study, we present three molecular dynamics simulations in which BoNT/A1 is in complex with singly or doubly glycosylated SV2C or singly glycosylated SV2A, in a ganglioside rich (lipid raft) context. Our computational data suggest that the N-glycan at position 480 (N480g) in the luminal domain of SV2C (LD-SV2C) indirectly enhanced the contacts of the neurotoxin surface with the second N-glycan at position 559 (N559g) by acting as a shield to prevent N559g to interact with residues of LD-SV2C. The N-glycosylation at the position N573 (N573g) in the luminal domain of SV2A has a slightly lower affinity for the surface of BoNT/A1 compared to 559g because of possible intermolecular contacts between N573g and residues of the luminal domain of SV2A (LD-SV2A). In addition to the ganglioside binding site (GBS) conserved across serotypes B, E, F and G, the lipid-raft associated GT1b interacted with a structure we coined the ganglioside binding loop (GBL) which is homologous to the lipid binding loop (LBL) in serotypes B, C, D, D/C and G. Finally, we proposed a global model in which BoNT/A1 interacts with its glycosylated protein receptor, one molecule of GT1b interacting in the GBS and five molecules of GT1b interacting with the GBL and residue Y1133.

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Crystal Structures of Botulinum Neurotoxin Subtypes A4 and A5 Cell Binding Domains in Complex with Receptor Ganglioside

Cited by 6 publications

References 38 publications

Structural Features of Clostridium botulinum Neurotoxin Subtype A2 Cell Binding Domain

Structural Features of Clostridium botulinum Neurotoxin Subtype A2 Cell Binding Domain

Crystal Structures of the Clostridium botulinum Neurotoxin A6 Cell Binding Domain Alone and in Complex with GD1a Reveal Significant Conformational Flexibility

Structural basis of botulinum neurotoxin serotype A1 binding to human SV2A or SV2C receptors

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