Enzymatic autocatalysis of botulinum A neurotoxin light chain

Highly purified recombinant zinc-endopeptidase light chain of the botulinum neurotoxin serotype A underwent autocatalytic proteolytic processing and fragmentation. In the absence of added zinc, initially 10-28 residues were cleaved from the C-terminal end of the 448-residue protein followed by the appearance of an SDS-stable dimer and finally fragmentation near the middle of the molecule. In the presence of added zinc, the rate of fragmentation was accelerated but the specificity of the cleavable bond changed, suggesting a structural role for zinc in the light chain. The C-terminal proteolytic processing was reduced, and fragmentation near the middle of the molecule was prevented by adding the metal chelator TPEN to the light chain. Similarly, adding a competitive peptide inhibitor (CRATKML) of the light-chain catalytic activity also greatly reduced the proteolysis. With these results, for the first time, we provide clear evidence that the loss of C-terminal peptides and fragmentation of the light chain are enzymatic and autocatalytic. By isolating both the large and small peptides, we sequenced them by Edman degradation and ESIMS-MS, and mapped the sites of proteolysis. We also found that proteolysis occurred at F266-G267, F419-T420, F423-E424, R432-G433, and C430-V431 bonds in addition to the previously reported Y250-Y251 and K438-T439 bonds.     

Localization of the structural gene for botulinum neurotoxin type A using synthetic DNA probe for neurotoxin light chain

To obtain the information on the genetic control of toxin production in the botulism causative agents, the oligonucleotides were synthesized as the molecular probes by translation of the amino acid sequence of the botulinic type A neurotoxin. The optimal conditions for hybridization of botulinic DNA with the synthetic DNA probes were determined and the probes specificity was demonstrated. The DNA fragments homologous to the probes used were shown to belong to bacterial genome, but not to bacteriophage one.     

Site-directed mutagenesis identifies active-site residues of the light chain of botulinum neurotoxin type A.

Botulinum neurotoxins (BoNTs) are metalloproteases which block neuroexocytosis via specific cleavage and inactivation of SNARE proteins. Such proteolysis accounts for the extreme toxicity of these neurotoxins and of their prolonged effect. The recently determined structures of BoNT/A and/B allows one to design active-site mutants to probe the role of specific residues in the proteolysis of SNARE proteins. Here we present the results of mutations of the second glutamyl residue involved in zinc coordination and of a tyrosine and a phenylalanine residues that occupy critical positions within the active site of BoNT/A. The spectroscopic properties of the purified mutants are closely similar to those of the wild-type molecule indicating the acquisition of a correct tertiary structure. Mutation of the Glu-262* nearly abolishes SNAP-25 hydrolysis as expected for a residue involved in zinc coordination. The Phe-266 and Tyr-366 mutants have reduced proteolytic activity indicating a direct participation in the proteolytic reaction, and their possible role in catalysis is discussed.     

High-level expression, purification, and characterization of recombinant type A botulinum neurotoxin light chain

Botulinum neurotoxin light chain (BoNT LC, 50 kDa) is responsible for the zinc endopeptidase activity specific for proteins of neuroexocytosis apparatus. We describe the expression of recombinant type A BoNT LC in Escherichia coli as well as the purification and characterization of the recombinant protein. A high level of expression of BoNT/A LC was obtained by an extended postinduction time of 15 h at 30 degrees C. Recombinant BoNT/A LC was isolated from an Ni(2+) column. Due to its high pI ( approximately 8.7), purification was achieved by a single step of passing the protein through anion-exchange chromatography at pH 8.0 without the need of elution. The purified recombinant BoNT/A LC retained proteolytic activity and had a secondary structure similar to that of native LC determined by CD measurement.     

Translocation of botulinum neurotoxin light chain protease through the heavy chain channel

Clostridial botulinum neurotoxins (BoNTs) abort the process of neurotransmitter release at presynaptic motor nerve terminals, causing muscle paralysis. An enigmatic step in the intoxication process is the mechanism by which the neurotoxin heavy chain (HC) forms the conduit for the translocation of the light chain (LC) protease across the endosomal membrane into the cytosol, its site of action. Here we investigate the mechanism of LC translocation by using the combined detection of channel currents and substrate proteolysis, the two hallmark activities of BoNT. Our data are consistent with the translocation of the LC through the HC channel and show that the LC protease activity is retrieved in the trans compartment after translocation. We propose that the BoNT HC-LC complex embedded in the membrane is a transmembrane chaperone, a dynamic structural device that prevents aggregation and achieves translocation of the LC. In this regard, the complex is similar to the protein conducting/translocating channels of the endoplasmic reticulum, mitochondria and chloroplasts.     

Probing the mechanistic role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain

Type A botulinum neurotoxin (BoNT/A) is a zinc endopeptidase that contains the consensus sequence HEXXH (residues 223-227) in the toxic light chain (LC). The X-ray structure of the toxin has predicted that the two histidines of this motif are two of the three zinc-coordinating ligands and that the glutamate is a crucial amino acid involved in catalysis. The functional implication of E224 in the motif of LC was investigated by replacing the residue with glutamine and aspartate using site-directed mutagenesis. Substitution of Glu-224 with Gln (E224Q) resulted in a total loss of the endopeptidase activity, whereas substitution with Asp (E224D) retained about 1.4% of the enzymatic activity (k(cat) 140 vs 1.9 min(-1), respectively). However, K(m) values for wild-type and E224D BoNT/A LC were similar, 42 and 50 microM, respectively. Global structure, in terms of secondary structure content and topography of aromatic amino residues, Zn(2+) content, and substrate binding ability are retained in the enzymatically inactive mutants. Titration of Zn(2+) to EDTA-treated wild-type and mutant proteins indicated identical enthalpy for Zn(2+) binding. These results suggest an essential and direct role of the carboxyl group of Glu-224 in the hydrolysis of the substrate. The location of the carboxyl group at a precise position is critical for the enzymatic activity, as replacement of Glu-224 with Asp resulted in almost total loss of the activity.     

Spectroscopic analysis of low pH and lipid-induced structural changes in type A botulinum neurotoxin relevant to membrane channel formation and translocation

Botulinum neurotoxin (BoNT) is an extremely toxic protein to animals and humans. In its mode of action, one of its subunits mediates its translocation by integrating itself into the membrane bilayer. We have examined the membrane channel activity of type A BoNT (BoNT/A) and its heavy (H) chain in planar lipid membrane under various pH conditions to understand the possible role of the channel activity in the translocation of the BoNT/A light (L) chain under physiological conditions. Only BoNT/A H chain, and not the BoNT/A, exhibited membrane channel activity for translocation of ions. The H chain-induced increase in conductance did not require a pH gradient across the lipid membrane, although it was enhanced by a pH gradient. To understand the molecular basis of the membrane channel activity and the translocation of the L chain, the secondary structure of BoNT/A and its H and L chains were analyzed using circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectroscopy at different pH values. BoNT/A showed no structural alternation upon acidifying the buffer pH. However, an increase in beta-sheet content of BoNT/A H chain at low pH was noted when examined by FT-IR. The L chain structure significantly changed with decrease in pH, and the change was mostly reversible. In addition, the neurotoxin and its subunit chains induced a partially reversible aggregation of liposomes at low pH, which indicated their integration into the lipid bilayer. Temperature-induced denaturation studies of BoNT/A H chain indicated major structural reorganization upon its interaction with membrane, especially at low pH.     

Catalytic features of the botulinum neurotoxin A light chain revealed by high resolution structure of an inhibitory peptide complex

The Clostridium botulinum neurotoxin serotype A light chain (BoNT/A-LC) is a Zn(II)-dependent metalloprotease that blocks the release of acetylcholine at the neuromuscular junction by cleaving SNAP-25, one of the SNARE proteins required for exocytosis. Because of the potential for use of the toxin in bioterrorism and the increasingly widespread application of the toxin in the medical field, there is significant interest in the development of small-molecule inhibitors of the metalloprotease. Efforts to design such inhibitors have not benefited from knowledge of how peptides bind to the active site since the enzyme-peptide structures available previously either were not occupied in the vicinity of the catalytic Zn(II) ion or did not represent the product of SNAP-25 substrate cleavage. Herein we report the 1.4 A-resolution X-ray crystal structure of a complex between the BoNT/A-LC and the inhibitory peptide N-Ac-CRATKML, the first structure of the light chain with an inhibitory peptide bound at the catalytic Zn(II) ion. The peptide is bound with the Cys S gamma atom coordinating the metal ion. Surprisingly, the cysteine sulfur is oxidized to the sulfenic acid form. Given the unstable nature of this species in solution, is it likely that oxidation occurs on the enzyme. In addition to the peptide-bound structure, we report two structures of the unliganded light chain with and without the Zn(II) cofactor bound at 1.25 and 1.20 A resolution, respectively. The two structures are nearly identical, confirming that the Zn(II) ion plays a purely catalytic role. Additionally, the structure of the Zn(II)-bound uncomplexed enzyme allows identification of the catalytic water molecule and a second water molecule that occupies the same position as the peptidic oxygen in the tetrahedral intermediate. This observation suggests that the enzyme active site is prearranged to stabilize the tetrahedral intermediate of the protease reaction.     

A capillary electrophoresis technique for evaluating botulinum neurotoxin B light chain activity

Botulinum neurotoxin B (BoNT/B) produces muscle paralysis by cleaving synaptobrevin/vesicle-associated membrane protein (VAMP), an 18-kDa membrane-associated protein located on the surface of small synaptic vesicles. A capillary electrophoresis (CE) assay was developed to evaluate inhibitors of the proteolytic activity of BoNT/B with the objective of identifying suitable candidates for treatment of botulism. The assay was based on monitoring the cleavage of a peptide that corresponds to residues 44-94 of human VAMP-2 (V51) following reaction with the catalytic light chain (LC) of BoNT/B. Cleavage of V51 generated peptide fragments of 18 and 33 amino acids by scission of the bond between Q76 and F77. The fragments and parent peptide were clearly resolved by CE, allowing accurate quantification of the BoNT/B LC-mediated reaction rates. The results indicate that CE is suitable for assessing the enzymatic activity of BoNT/B LC.     

Changes in the molecular topography of the light and heavy chains of type A botulinum neurotoxin following their separation

Botulinum neurotoxin serotype A, an approx. 150 kDa protein, is composed of two subunits, the light and heavy chains (approximately 50 and approximately 100 kDa, respectively). The neurotoxin's mode of action is believed to depend on coordinated but independent actions of the two subunit chains. The molecular environments of the aromatic amino acid residues of the dichain neurotoxin and the two isolated subunit chains were analyzed using near-ultraviolet circular dichroism (CD) (between 250 and 320 nm) and second-derivative ultraviolet absorption spectroscopy (between 240 and 320 nm) to investigate the conformational variations of the subunit chains in separated and conjugated forms. The mean residue weight ellipticities showed virtually no change (i.e., 1.7%) in the vicinities of Phe (268 nm), and only a small change (11%) around Tyr (279 nm) residues following dissociation of the subunit chains. However, significant changes (23-26%) at 286 nm as well as at 292 nm were noted, suggesting considerable alteration in the conformation of the subunits. Second-derivative ultraviolet absorption spectra indicated the degree of Tyr exposure in the dichain neurotoxin, isolated heavy and light chains at 70.7, 81.5 and 46.4%, respectively. A weighted mean of the degree of exposed Tyr residues in the separated heavy and light chains was 69.6%, virtually same as the 70.7% exposed Tyr residues observed in the intact dichain neurotoxin, indicating no difference in their Tyr exposure upon separation of the two chains. This was corroborated by the CD data which revealed only small changes in the CD signals of Tyr residues, and no alteration in those of the Phe residues following separation of the subunit chains. However, a change in the CD signal at 292 nm suggested that the conformations of Trp-containing segments of the two chains were significantly influenced upon their separation. The heavy and light chains of the neurotoxin therefore appear to exist as two semi-independent domains, in spite of being linked by disulfide and noncovalent bonds, and at least part of their conformations depends on interactions between them.     

The C-terminus of botulinum neurotoxin type A light chain contributes to solubility, catalysis, and stability

Botulinum neurotoxin type A (BoNT/A) is the etiological agent responsible for botulism, a disease characterized by peripheral neuromuscular blockade. BoNT/A is produced by Clostridium botulinum as a single chain protein that is activated by proteolytic cleavage to form a 50 kDa light chain (LC, 448 amino acids) and a disulfide bond-linked 100 kDa heavy chain (HC, 847 amino acids). Whilst HC comprises the receptor binding and translocation domains, LC is a Zn2+-endopeptidase that cleaves at a single glutaminyl-arginine bond corresponding to residues 197 and 198 at the C-terminus of SNAP25. Cleavage of SNAP25 uncouples the neural exocytosis docking/fusion machinery. LC/A (LC 1-448) and several C-terminal deletion proteins of LC/A were engineered and expressed as His-tagged fusion proteins in Escherichia coli. LC 1-448 was purified, but precipitated upon storage. Approximately 40% of LC 1-448 was a covalent dimer due to the formation of inter-chain disulfide bond formation at Cys430. Conversion of Cys430 to Ser abolished dimer formation of LC 1-448, but did not improve solubility. Three C-terminal deletion peptides were engineered; LC 1-425 and LC 1-418 were expressed and could be purified as soluble and stable proteins, whilst LC 1-398 was soluble, but not stable to storage. Kinetic studies showed that LC 1-448 and LC 1-425 efficiently cleaved GST-SNAP25 and the fluorescent substrate SNAPtide, while LC 1-418 catalyzed the cleavage of both the SNAP25 and the fluorescent substrate SNAPtide with a similar Km, but at a 10-fold slower kcat. Thus, regions within the C-terminus of LC/A contribute to solubility, stability, and catalysis.     

Engineering botulinum neurotoxin domains for activation by toxin light chain

Targeted secretion inhibitors (TSI) are a new class of biopharmaceuticals designed from a botulinum neurotoxin protein scaffold. The backbone consists of the 50-kDa endopeptidase light chain and translocation domain (N-terminal portion of the heavy chain), lacks neuronal toxicity, but retains the ability to target cytoplasmic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. TSI are produced as single-chain proteins and then cleaved post-translationally to generate functional heterodimers. Precise proteolytic cleavage is essential to activate the protein to a dichain form. TSI are themselves highly specific proteases. We have exploited this activity to create self-activating enzymes by replacing the native proteolytic site with a substrate SNARE peptide for the TSI protease. We have also created cross-activating backbones. By replacing the proteolytic activation site in one backbone with the substrate SNARE peptide for another serotype, controlled activation is achieved. SNARE peptides encompassing the whole of the coiled-coil region enabled complete activation and assembly of the dichain backbone. These engineered TSI backbones are capable of translocating their enzymatic domains to target intracellular SNARE proteins. They are also investigative tools with which to further the understanding of endopeptidase activity of light chain in SNARE interactions.     

Association of botulinum neurotoxin serotype A light chain with plasma membrane-bound SNAP-25

The Clostridium botulinum neurotoxins (BoNTs) cleave SNARE proteins, which inhibit binding and thus fusion of neurotransmitter vesicles to the plasma membrane of peripheral neurons. BoNTs comprise an N-terminal light chain (LC) and C-terminal heavy chain, which are linked by a disulfide bond. There are seven serotypes (A-G) of BoNTs based upon immunological neutralization. Although the binding and entry of BoNT/A into neurons has been subjected to considerable investigation, the intracellular events that allow BoNT/A to efficiently cleave SNAP-25 within neurons is less well understood. Earlier studies showed that intracellular LC/A bound to the plasma membrane of neurons. In this study, intracellular LC/A is shown to directly bind SNAP-25 on the plasma membrane. Solid phase binding showed that the N-terminal residues of LC/A bound residues 80-110 of SNAP-25, which was also observed in cultured neurons. Association of the N-terminal 8 amino acids of LC/A and residues 80-110 of SNAP-25 also enhanced substrate cleavage. These findings explain how LC/A associates with SNAP-25 on the plasma membrane and provide a basis for LC/A cleavage of SNAP-25 within the SNARE complex.     

Role of zinc binding in type A botulinum neurotoxin light chain's toxic structure

Clostridial neurotoxins are zinc endopeptidases, and each contains one Zn(2+)/molecule. To investigate the structural/functional role of Zn(2+) in botulinum neurotoxin light chain (the enzymatic subunit of the neurotoxin), the effect of the removal of zinc on protein folding and enzyme kinetics was investigated. The active site Zn(2+), which was easily displaced from the active site by ethylenediaminetetraacetate, reversibly binds to the BoNT/A light chain (LC) in a stoichiometric manner. Enzymatic activity was completely abolished in the zinc-depleted light chain (apo-LC). However, Zn(2+) replenishment partially restored the activity in the re-Zn(2+)-LC (k(cat) = 72 min(-)(1)) compared to the holo-LC (k(cat) = 140 min(-)(1)). Comparable K(m) values in the holo- and re-Zn(2+)-LC were observed (41 and 55 microM, respectively), indicating a similar substrate binding ability. We investigated the structural basis of a 3-fold difference in the catalytic efficiency of the native holo-LC and re-Zn(2+)-LC by analyzing secondary and tertiary structural parameters. Removal of the zinc causes irreversible tertiary structural change while the secondary structure remains unchanged. Zinc binding leads to enhanced thermal stability of the LC, which is not identical in the native holo-LC and re-Zn(2+)-LC.     

Autocatalytically fragmented light chain of botulinum a neurotoxin is enzymatically active

The zinc-endopeptidase light chain of botulinum A neurotoxin undergoes autocatalytic fragmentation that is accelerated by the presence of the metal cofactor, zinc [Ahmed, S. A. et al. (2001) J. Protein Chem. 20, 221-231]. We show in this paper that >95% fragmented light chain obtained in the absence of added zinc retained 100% of its original catalytic activity against a SNAP-25-derived synthetic peptide substrate. In the presence of zinc chloride, when >95% of the light chain had undergone autocatalytic fragmentation, the preparation retained 35% of its original catalytic activity. On the other hand, in the presence of glycerol, the light chain did not display autocatalysis and retained 100% of the original activity. These results suggest that the activity loss by incubation with zinc was not a direct consequence of autocatalysis and that the environment of the active site was not affected significantly by the fragmentation. The optimum pH 4.2-4.6 for autocatalysis was different than that (pH 7.3) for intrinsic catalytic activity. Inhibition of autocatalysis at low pH by a competitive inhibitor of catalytic activity rules out the presence of a contaminating protease but suggests a rate-limiting step of low pH-induced conformational change suitable for autocatalysis. Our results of LC concentration dependence of the fragmentation reaction indicate that the autocatalysis occurs by both intramolecular and intermolecular mechanisms.     

Conformational sampling of the botulinum neurotoxin serotype A light chain: implications for inhibitor binding

Botulinum neurotoxins (BoNTs) are the most potent of the known biological toxins, and consequently are listed as category A biowarfare agents. Currently, the only treatments against BoNTs include preventative antitoxins and long-term supportive care. Consequently, there is an urgent need for therapeutics to counter these enzymes--post exposure. In a previous study, we identified a number of small, nonpeptidic lead inhibitors of BoNT serotype A light chain (BoNT/A LC) metalloprotease activity, and we identified a common pharmacophore for these molecules. In this study, we have focused on how the dynamic movement of amino acid residues in and surrounding the substrate binding cleft of the BoNT/A LC might affect inhibitor binding modes. The X-ray crystal structures of two BoNT/A LCs (PDB refcodes=3BTA and 1E1H) were examined. Results from these analyses indicate that the core structural features of the examined BoNT/A LCs, including alpha-helices and beta-sheets, remained relatively unchanged during 1 ns dynamics trajectories. However, conformational flexibility was observed in surface loops bordering the substrate binding clefts in both examined structures. Our analyses indicate that these loops may possess the ability to decrease the solvent accessibility of the substrate binding cleft, while at the same time creating new residue contacts for the inhibitors. Loop movements and conformational/positional analyses of residues within the substrate binding cleft are discussed with respect to BoNT/A LC inhibitor binding and our common pharmacophore for inhibition. The results from these studies may aid in the future identification/development of more potent small molecule inhibitors that take advantage of new binding contacts in the BoNT/A LC.     

Nicking of single chain Clostridium botulinum type A neurotoxin by an endogenous protease

Botulinum neurotoxin (NT) serotype A isolated from cells from young cultures (approximately 8 h) of Clostridium botulinum type A is a approximately 150 kDa single chain protein. Supernatant from older cultures (96 h) yields approximately 150 kDa dichain NT composed approximately 50 and approximately 100 kDa subunits, that remain associated by disulfide and noncovalent bonds. This had led to the assumption that an endogenous protease cleaves a peptide bond at 1/3rd the distance from the N- or C-terminals of the single chain protein. An endogenous protease that causes such a cleavage (nicking) has now been purified greater than 1,000-fold from C. botulinum type A (Hall strain) culture; this culture also produces the single chain NT and eventually yields the dichain NT. The purified protease nicked the pure preparation of single chain type A NT, in vitro at pH 5.6, into a dichain form that was indistinguishable from the dichain NT normally isolated from 96 h cultures. The protease appears specific for nicking serotype A NT because it did not nick single chain serotype B and E NT nor did it enhance toxicity of serotype A, B and E NT.     

Structure of heavy and light chain subunits of type A botulinum neurotoxin analyzed by circular dichroism and fluorescence measurements

Summary The secondary and tertiary structural features of botulinum neurotoxin (NT) serotype A, a dichain protein (M_r 145 000), and its two subunits, the heavy (H) and light (L) chains (M_r 97 000 and 53 000, respectively) were examined using circular dichroism and fluorescence spectorscopy. Nearly 70% of the amino acid residues in each of the three polypeptide preparations were found in ordered structure (sum of helix, sheet and turns). Also, the helix, sheet, turns and random coil contents of the dichain NT were nearly equal to the weighted mean of each of these secondary structure parameters of the L and H chains; e.g., sum of helix of L chain (22%) and H chain (18.7%), as weighted mean, 19.8% was similar to that of NT (20%). These agreements suggested that the secondary structures of the subunits of the dichain NT do not significantly change when they are separated as isolated L and H chains. Fluorescence emission maximum of L chain, 4 nm less (blue shift) than that of H chain, suggested relatively more hydrophobic environment of fluorescent tryptophan residue(s) of L chain. Tryptophan fluorescence quantum yields of L chain, H chain and the NT, 0.072, 0.174 and 0.197, respectively, suggested that a) an alteration in the micro-environment of the tryptophan residues was possibly caused by interactions of L and H chain subunits of the NT and b) quantum yields for L and H chains were altered when they are together as subunits of the NT. Possible implications of structural features of the L and H chains, their interactions and the molecular mechanism of action of botulinum NT are assessed.     

Cloning, expression, and one-step purification of the minimal essential domain of the light chain of botulinum neurotoxin type A

A truncated but functional form of the botulinum neurotoxin A light chain (Tyr 9-Leu 415) has been cloned into the three bacterial expression vectors, pET 28, pET 30, and PGEX-2T, and produced as fusion proteins. This 406-amino-acid light chain was expressed with 1 six-histidine tag (LC-pET28), 2 six histidine tags and a S-tag (LC-pET30), or a six-histidine tag and a glutathione S-transferase tag (LC-pGEX-2T). The three fusion proteins have been overexpressed in Escherichia coli, purified in a soluble form, and tested for protease activity. All three recombinant proteins were found to have similar enzymatic activity, comparable to the light chain purified from the whole toxin. The LC-pET30 protein was the most soluble and stable of the three fusion proteins, and it could be purified using a one-step affinity chromatography protocol. The purified protein was determined to be 98% pure as assessed by SDS-polyacrylamide gel. This protein has been crystallized and initial X-ray data show that the crystals diffract to 1.8 A.     

Structure of botulinum neurotoxin type D light chain at 1.65 A resolution: repercussions for VAMP-2 substrate specificity

The seven serotypes (A-G) of botulinum neurotoxins (BoNTs) function through their proteolytic cleavage of one of three proteins (SNAP-25, Syntaxin, and VAMP) that form the SNARE complex required for synaptic vesicle fusion. The different BoNTs have very specific protease recognition requirements, between 15 and 50 amino acids in length depending on the serotype. However, the structural details involved in substrate recognition remain largely unknown. Here is reported the 1.65 A resolution crystal structure of the catalytic domain of BoNT serotype D (BoNT/D-LC), providing insight into the protein-protein binding interaction and final proteolysis of VAMP-2. Structural analysis has identified a hydrophobic pocket potentially involved in substrate recognition of the P1' VAMP residue (Leu 60) and a second remote site for recognition of the V1 SNARE motif that is critical for activity. A structural comparison of BoNT/D-LC with BoNT/F-LC that also recognizes VAMP-2 one residue away from the BoNT/D-LC site provides additional molecular details about the unique serotype specific activities. In particular, BoNT/D prefers a hydrophobic interaction for the V1 motif of VAMP-2, while BoNT/F adopts a more hydrophilic strategy for recognition of the same V1 motif.