High resolution crystal structures of the receptor-binding domain of Clostridium botulinum neurotoxin serotypes A and FA

Cloning and constructs

The genes encoding the binding domain of BoNT/A1 (H_C/A1) and BoNT/FA (H_C/FA) were provided by Ipsen Bioinnovation Ltd. Each was cloned into the pJ401 expression vector (DNA 2.0, Menlo Park, CA, USA) with an N-terminal 6× histidine tag using standard molecular biology techniques and confirmed by sequencing (Eurofins Genomics, Germany).

Protein expression

His₆-H_C/A1 and His₆-H_C/FA were expressed in E. coli strain BL21(DE3) (Novagen, Madison, WI, USA) using the following protocol. A glycerol stock was used to inoculate 100 mL TB medium containing 50 µg/mL kanamycin, and grown at 37 °C for 16 h. From this, 10 mL of culture was used to inoculate 1 L TB medium containing 50 µg/mL kanamycin and grown at 37 °C to an OD₆₀₀ of 0.6. The temperature was reduced to 16 °C and the cells grown to an OD₆₀₀ of 1.0 at which point 0.5 mM isopropyl-d-1-thiogalactopyranoside (IPTG) was added to induce expression. Cells were grown for an additional 16 h at 16 °C and then harvested by centrifugation at 4,000× g for 30 min.

Protein purification

Expression cell pastes were resuspended in 0.5 M NaCl, 50 mM Tris–HCl pH 7.4, 20 mM imidazole and lysed using a Constant Systems cell disruptor at 20 kPSI. Cell debris were removed by centrifugation at 80,000× g for 30 min and the supernatant was filtered through a 0.22 µm membrane syringe filter. The clarified lysate was loaded onto a 5 mL HisTrap column (GE Healthcare, Little Chalfont, UK), washed, and the target protein eluted with 0.5 M imidazole. His₆-H_C/A1 and His₆-H_C/FA were further purified by SEC using a Superdex 200 16/60 column (GE Healthcare, Little Chalfont, UK) and 0.5 M NaCl, 50 mM Tris-HCl pH 7.4. Purified samples were concentrated to 10 mg/mL using a 10 kDa MWCO centrifugal filter (Millipore, Billerica, MA, USA).

Crystallography

Crystals of His₆-H_C/FA and His₆-H_C/A1 were grown at 16 °C using a 1:1 ratio of protein solution (10 mg/mL) to well solution using the sitting-drop vapour-diffusion method—4 M sodium formate, 0.1 M sodium acetate pH 5.5 for the former, and 0.1 M MIB pH 4.0, 25% w/v PEG 1500 for the latter. Crystals were soaked in cryoprotectant (equal volume of reservoir solution and 50% glycerol) before vitrification in liquid nitrogen. Complete datasets were collected on beamline I03 and I04, respectively, at the Diamond Light Source (Didcot, UK). Diffraction images were processed using DIALS (Gildea et al., 2014) and scaled using AIMLESS (Evans & Murshudov, 2013) from the CCP4 suite (Winn et al., 2011). Data collection statistics are summarised in Table 1. A combination of R_pim and CC_1/2 value were used to determine the resolution cut-off of 1.95 Å and 1.45 Å, respectively. Phase information was determined by molecular replacement using PHASER (McCoy et al., 2007) and a previous structure of H_C/A1 (PDB: 2VUA; Stenmark et al., 2008) as the initial search model. Multiple rounds of structure refinement were performed by manual correction in COOT (Emsley et al., 2010) followed by restrained refinement with REFMAC5 (Murshudov et al., 2011). Final validation was performed with MolProbity (Chen et al., 2010). Secondary structures were annotated using Stride (Frishman & Argos, 1995) and figures were prepared using PyMol (Schrödinger, LLC, New York, NY, USA) and CCP4mg (McNicholas et al., 2011).

Crystal structure of BoNT/FA H_C domain

We identified crystallisation conditions which yielded crystals of H_C/FA in space group P422, with two molecules related by non-crystallographic symmetry in the asymmetric unit. This is different to a recently reported structure, PDB: 5V38 (Yao et al., 2017) and reveals an important loop that is involved in receptor binding (average temperature factor (B-factor) 74 Ų). A high-multiplicity dataset was collected containing 360 degrees of data over 3,600 images. No significant radiation damage was observed over the course of data collection and thus all data were used. The CC_1/2 value for the outer shell was 0.832, indicating there was still very usable data at this resolution (Evans & Murshudov, 2013). The overall structure of H_C/FA is shown in Fig. 1C and the crystallographic statistics are listed in Table 1. As with all reported structures of the BoNT receptor binding domain, present is a characteristic β-jellyroll fold at the N-terminal half and a predominantly β-trefoil fold at the C-terminal half of the protein (Figs. 1A–1D). Both molecules in the asymmetric unit overlay well with a root mean square deviation (RMSD) value of 0.35 Å between all atoms. The B-factors for each chain are low overall (24.74 Ų and 26.84 Ų respectively) with a corresponding overall Wilson B-factor of 24.55 Ų. As expected, our structure is highly similar to PDB: 5V38 with an RMSD value for combinations of chains between structures ranges from 0.54 Å to 0.36 Å (Fig. 1D). However, it further reveals the presence of a loop (R1261–R1268) that has been shown in other subtypes to be involved in ganglioside receptor binding (Fig. 2A). Crystal packing has enabled neighbouring chains to interact directly with this loop, provide sufficient stabilisation to produce good electron density. Yao et al. (2017) suggested that the lack of density was due to high flexibility, which is consistent with the high B-factors we observed in this region relative to the rest of the protein.

The specific ganglioside receptor for BoNT/FA is not yet known; however, considering that GT1b binds with high affinity to BoNT/A1 (Fig. 2B) and that H_C/FA and H_C/A1 are structurally very similar, we propose that BoNT/FA possesses a similar binding specificity. The overall conformation of this region is also similar to that of BoNT/A1 in complex with a ganglioside receptor (Fig. 2). The detailed conformation of this loop is important for understanding receptor binding and our structure confirms that BoNT/FA could bind to gangliosides in a similar manner to BoNT/A. Proximate to the ganglioside binding region, we observe the presence of a disulphide bond between Cys1227 and Cys1272 (Fig. 3C) which is also not present in the structure 5V38. The equivalent bond has been observed previously in some, but not all crystal structures of BoNT H_C Domains; therefore, it is uncertain what role, if any, it may have towards BoNT function.

Crystal structures of the BoNT/A1 binding domain

We have identified a single crystallisation condition that produced two crystal forms of H_C/A1—one possessed the equivalent disulphide bond whereas the other did not. Using 25% w/v PEG 1500 and 0.1 M MIB pH 4.0, H_C/A1 crystallised into orthorhombic crystals with the space group P2₁2₁2₁ (crystal form 1) that diffracted to a resolution of 1.45 Å. No disulphide bond was observed in this structure (Fig. 1A). Instead, Cys1235 rotated away from the junction due to a backbone rotation, bringing the neighbouring Lys1236 toward Cys1280 (Fig. 3A). This is consistent with previous H_C/A1 structures either in complex with GT1b (PDB: 2VU9; Stenmark et al., 2008), SV2-LD (PDB: 4JRA, 5JLV; Benoit et al., 2014; Yao et al., 2016) or in the apo form (PDB: 2VUA; Stenmark et al., 2008). Using the same conditions, monoclinic crystals were obtained six months later with the space group P2₁ (crystal form 2) that diffracted to 1.8 Å resolution. Inspection of this structure (Fig. 1B) revealed the presence of a disulphide bond between C1235 and C1280, the equivalent of which was also observed in our H_C/FA crystal structure (Figs. 3A and 3B) and in a full-length BoNT/A1 crystal structure (PDB: 3BTA; Lacy & Stevens, 1999). Our findings suggest that the crystallisation condition is not the only determinant as to whether the bond is present or not. The conservation of these cysteine residues across the BoNT sub-serotypes suggests they are very important (Fig. 4); however, their precise function is unknown. Almost all BoNT subtypes that cause human botulism contain the two residues. They may be required for disulphide bond formation for structural stability or for efficient function of a nearby ganglioside-binding pocket. It must be noted that the expression system does not appear to select for reduced or non-reduced forms of these cysteine residues—each have been observed in different structures of BoNT/A purified from the native Clostridium botulinum (Garcia-Rodriguez et al., 2007; Stenmark et al., 2008; Fu et al., 2009; Gu et al., 2012; Przedpelski et al., 2013; Benoit et al., 2014; Yao et al., 2017; Davies et al., 2017).