Crystal Structure of Botulinum Neurotoxin A2 in Complex with the Human Protein Receptor SV2C Reveals Plasticity in Receptor Binding.

Robert Gustafsson,Geoffrey Masuyer,Min Dong,Pål Stenmark,Sicai Zhang

doi:10.3390/toxins10040153

Robert Gustafsson, Geoffrey Masuyer + Show 3 more

Open Access

https://doi.org/10.3390/toxins10040153

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Abstract

Botulinum neurotoxins (BoNTs) are a family of highly dangerous bacterial toxins, with seven major serotypes (BoNT/A-G). Members of BoNTs, BoNT/A1 and BoNT/B1, have been utilized to treat an increasing number of medical conditions. The clinical trials are ongoing for BoNT/A2, another subtype of BoNT/A, which showed promising therapeutic properties. Both BoNT/A1 and BoNT/A2 utilize three isoforms of synaptic vesicle protein SV2 (SV2A, B, and C) as their protein receptors. We here present a high resolution (2.0 Å) co-crystal structure of the BoNT/A2 receptor-binding domain in complex with the human SV2C luminal domain. The structure is similar to previously reported BoNT/A-SV2C complexes, but a shift of the receptor-binding segment in BoNT/A2 rotates SV2C in two dimensions giving insight into the dynamic behavior of the interaction. Small differences in key residues at the binding interface may influence the binding to different SV2 isoforms, which may contribute to the differences between BoNT/A1 and BoNT/A2 observed in the clinic.

Highlights

Botulinum neurotoxins (BoNTs) are among the most toxic substances known
BoNTs are produced as a 150 kDa polypeptide chain that is post-translationally cleaved into two fragments, the N-terminal light chain (LC) of 50 kDa and the C-terminal heavy chain (HC) of 100 kDa
Our structure is largely the same as the recently reported crystal structure of Hc of BoNT/A2 (HcA2) + SV2C-L4 complex [26], but our structure offers a higher resolution (2.0 Å compared to 2.3 Å in the previous report), and revealed a different position of SV2C-L4, resulting from a significant rotation and shift of SV2C-L4

Summary

Introduction

Botulinum neurotoxins (BoNTs) are among the most toxic substances known. They are produced by diverse anaerobic bacterium mainly classified as Clostridium botulinum. The structure of the receptor binding domain of BoNT/A1, a subtype of BoNT/A, in complex with the fourth luminal domain of human SV2C (SV2C-L4) [7] as well as its glycosylated form [20] has been reported [7,20]. The Hc of BoNT/A2 (HcA2) displays 13% of amino acid sequence differences from HcA1 These variations are not likely to alter the receptors, but may have a significant impact on receptor binding properties, which in turn may impact the therapeutic efficacy [23]. To understand how residue differences within the receptor binding domain of BoNT/A1 versus BoNT/A2 may impact their receptor recognition properties, we solved a high resolution crystal structure of HcA2 in complex with human SV2C-L4. Our structure is largely the same as the recently reported crystal structure of HcA2 + SV2C-L4 complex [26], but our structure offers a higher resolution (2.0 Å compared to 2.3 Å in the previous report), and revealed a different position of SV2C-L4, resulting from a significant rotation and shift of SV2C-L4

Overall Structure of HcA2 and SV2C

Comparison to Reported HcA2-SV2C-L4 Structure

Comparison to Reported HcA1-SV2C-L4 Structure

SV2 Sequence Comparison

In Vitro Binding Assay

Materials and Methods