SNAP-25 Substrate Peptide (Residues 180–183) Binds to but Bypasses Cleavage by Catalytically Active Clostridium botulinum Neurotoxin E*

Clostridium botulinum neurotoxins are the most potent toxins to humans. The recognition and cleavage of SNAREs are prime evente in exhibiting their toxicity. We report here the crystal structure of the catalytically active full-length botulinum serotype E catalytic domain (BoNT E) in complex with SNAP-25 (a SNARE protein) substrate peptide Arg180-Ile181-Met182-Glu183 (P1–P3′). It is remarkable that the peptide spanning the scissile bond binds to but bypasses cleavage by the enzyme and inhibits the catalysis fairly with Ki ∼69 μm. The inhibitory peptide occupies the active site of BoNT E and shows well defined electron density. The catalytic zinc and the conserved key residue Tyr350 of the enzyme facilitate the docking of Arg180 (P1) by interacting with its carbonyl oxygen that displaces the nucleophilic water. The general base Glu212 side chain interacts with the main chain amino group of P1 and P1′. Conserved Arg347 of BoNT E stabilizes the proper docking of the Ile181 (P1′) main chain, whereas the hydrophobic pockets stabilize the side chains of Ile181 (P1′) and Met182 (P2′), and the 250 loop stabilizes Glu183 (P3′). Structural and functional analysis revealed an important role for the P1′ residue and S1′ pocket in driving substrate recognition and docking at the active site. This study is the first of its kind and rationalizes the substrate cleavage strategy of BoNT E. Also, our complex structure opens up an excellent opportunity of structure-based drug design for this fast acting and extremely toxic high priority BoNT E.

binding of the toxin to specific neuronal receptors, and the N-terminal domain enables the catalytically active light chain translocation to the cytosol (5). The light chain is a zinc-dependent endopeptidase, which recognizes and cleaves one of the three SNARE proteins (SNAP-25 (synaptosome-associated protein of 25,000 daltons), VAMP-2 (vesicle-associated membrane protein 2), or syntaxin) (5). SNARE proteins associate to form a low energy ternary complex required for fusion and docking of neurotransmitter carrying vesicles to target membranes of peripheral neurons enabling neurotransmitter release (6). Cleavage of any one of them prevents complex formation, resulting in flaccid paralysis. BoNTs A and E cleave SNAP-25, whereas BoNTs B, D, F, and G cleave VAMP-2. BoNT C is unique and cleaves both SNAP-25 and syntaxin (7).
BoNTs are listed as category A bioterrorism agents by the Centers for Disease Control and Prevention. Accordingly, developing inhibitors to counter their activity is a priority. BoNTs A, B, and E are more potent than the other serotypes. Although BoNTs E and A cleave the same substrate SNAP-25, E blocks neurotransmission faster (8). Crystal structures of catalytic domains of all serotypes are available (9 -15). However, insight into the substrate recognition and binding in each serotype, crucial for developing common and/or serotype specific inhibitors, has yet to be established.
As of now, the structure of enzyme-substrate peptide (residues 146 -206) complex has been reported only for BoNT A (Protein Data Bank code 1XTG) (16). However, to prevent cleavage during co-crystallization, an inactive double mutant enzyme was used. Although this structure maps the exosite interactions, the region flanking the scissile bond (about 6 or 7 residues) is disordered and does not make contact with the active site residues of the enzyme (16). Recently, crystal structures of BoNT A in complex with peptide inhibitors Arg-Arg-Gly-Cys (RRGC) and its variants at the Cys position and N-Ac-Cys-Arg-Ala-Thr-Lys-Met-Leu (N-Ac-CRATKML) have been reported (17)(18)(19). However, variation of substrate residues flanking the scissile bond and their subsites in BoNT E and other serotypes warrants determination of structures of substrate-enzyme complex. Structural information of substrate recognition and binding at the active site is crucial in better understanding the substrate specificity among various serotypes and for developing potent serotype-specific inhibitors.
To our knowledge, structural information for BoNT-substrate/analog peptide complexes is not available for any serotype except BoNT A. Here, we present for the first time the crystal structure of the full-length BoNT E catalytic domain with its substrate SNAP-25-(180 -183) Arg-Ile-Met-Glu-amide (RIME; P1:Arg 180 , P1Ј:Ile 181 , P2Ј:Met 182 , P3Ј:Glu 183 ) peptide spanning the scissile bond. The S1, S1Ј, S2Ј, and S3Ј pockets of BoNT E and their crucial interactions with P1-P3Ј residues are identified. We also modeled the P2:Asp 179 residue of the substrate and identified its possible interactions in the S2 pocket. Interestingly, we found that the substrate peptide RIME itself acts as a moderate inhibitor of catalysis. This study rationalizes the substrate cleavage strategy of BoNT E, which could be extended to other serotypes too. The present complex structure is the first report of substrate binding to the active site of BoNT E and helps to better understand the mechanism of catalysis and substrate recognition and binding at and near the active site. This will serve as an important starting point for designing specific structure-based potent inhibitors/drugs for highly toxic BoNT E.

Cloning, Expression, and Purification of BoNT E Catalytic
Domain-The clone for BoNT E catalytic domain in pET9c vector was used as described (20). Protein was expressed using an autoinduction protocol (21) and was purified (20). This yielded Ͼ20 mg protein/500 ml of culture, and the protein was concentrated to 10 mg/ml in Hepes buffer (20 mM, pH 7.8, 200 mM NaCl).
Kinetic Parameters and K i Determination-Kinetic values k m and k cat were determined for SNAP-25-(146 -186) as substrate with BoNT E. Enzyme concentrations were adjusted (0.5 nM) to cleave Ͻ20% substrate at various concentrations of substrate ranging from 35 to 500 M. The reactions were carried out in 20 l of reaction buffer containing 20 mM Hepes, pH 7.4, 2 mM DTT, and 10 M zinc acetate. The reaction mixtures were incubated at 37°C for 15 min and stopped by adding 2 l of 1% trifluoroacetic acid. The samples were run in HPLC (Beckman) with a Discovery Bio-Wide pore C18 column with acetonitrile and aqueous phase gradient (Buffer A: 0.1% trifluoroacetic acid in water; Buffer B: 0.1% trifluoroacetic acid, 90% acetonitrile). The amount of cleaved product was calculated using software provided by Beckman. Kinetic constants were calculated from Lineweaver-Burk plot using Enzyme Kinetic Module and Sigmaplot (Systat Software, Inc.). IC 50 and K i determination for RIME (P1-P3Ј) (50 -950 M concentrations) was performed in similar assay conditions, and data were analyzed using Graph-Pad software and the Cheng-Prusoff equation, respectively. Peptide 2.0 Inc. synthesized this tetrapeptide.
Crystallization and Structure Determination-The catalytic domain was cocrystallized with RIME in 0.5 M ammonium sulfate, 0.1 M sodium citrate buffer, pH 5.6, 2 M Li 2 SO 4 in sitting drop in 24-well plates. Protein at 7 mg/ml and peptide at ϳ10 mM concentration was used for cocrystallization. Lower concentrations of the peptide during cocrystallization led to poor occupancy of the substrate at the active site. Diamond-shaped crystals appeared in 2-7 days at 22°C. Crystals were immersed in mother liquor containing 15% (v/v) glycerol and immediately flash frozen in liquid nitrogen. X-ray data were collected at beamline X29 of the National Synchotron Light Source, Brookhaven National Laboratory, at 1.08 Å wavelength, and crystals diffracted to better than 2.25 Å resolution. Crystals belong to space group P2 1 2 1 2 with 2 molecules/asymmetric unit. A rigid body refinement was performed using a BoNT E catalytic domain model (Protein Data Bank code 1T3A), followed by simulated annealing in CNS (22). The refinement statistics are given in Table 1. Molecules A and B are complete for amino acids 1-411 and 1-409, respectively, but molecule A has weak electron density for amino acids 235-238. Residues 59 -60 are not modeled in both molecules due to poorly defined electron density. The SNAP-25 substrate RIME (P1-P3Ј) bound to molecule B could be modeled unambiguously; however, the C-terminal residue P3Ј and P1 side chain could not be modeled for peptide bound to molecule A because of weak electron density. P1:Arg 180 -P2Ј:Met 182 (RIME) aligns well in both molecules of enzymes (root mean square deviation ϭ 0.051Å) with minor variations in interacting distances of P1:Arg 180 (peptide RIME) main chain amino group with the zinc and Glu 212 of the enzyme. All of our discussion in this paper is based on the substrate peptide RIME bound to the B molecule. Overall Structure and Crucial Interactions of BoNT E with Inhibitory Peptide (RIME)-BoNT E recognizes and cleaves SNAP-25 substrate specifically between P1:Arg 180 -P1Ј:Ile 181 . In our complex structure, RIME binds to the active site cavity of BoNT E without being cleaved (Fig. 2, A and B), interacts with several residues (Fig. 2C), and shows well defined electron density in the omit map in molecule B (Fig. 3). The crucial interactions of various peptide residues (P1-P3Ј) and P2:Asp 179 modeling results are discussed below.

RESULTS AND DISCUSSION
Docking of P1:Arg 180 at the Active Site-P1:Arg 180 makes interactions required for proper docking of P1 residue to the active site (Figs. 2C and 4). Its carbonyl oxygen interacts with both the catalytic zinc (2.69 Å) and Tyr 350 (3.08 Å) and displaces the nucleophilic water (Figs. 2C and 4). In wild type protein structure (Protein Data Bank code 1T3A), the nucleophilic water is at 2.18 Å from zinc. The oxygen of P1 makes a tetrahedral arrangement similar to that of nucleophilic water. Interactions between zinc, Tyr 350 (conserved in all BoNTs), and nucleophilic water are common to all catalytic domain struc-tures. The Y350A mutant of BoNT E led to complete loss of catalytic inactivity (at Յ500 nM concentration) (23). The P1:Arg 180 main chain amino group interacts with both Oe1 and Oe2 of Glu 212 , displacing a water molecule present in the native structure. However, it interacts with another water present in the current structure. Glu 212 , a general base, plays a crucial role in catalysis, and the E212Q mutation led BoNT E to be catalytically inefficient without alteration in protein conformation (23).
In the crystal structure, P1:Arg 180 side chain NH1 and NH2 form a strong salt bridge with Glu 158 and stabilize P1 at the active site (Fig. 2C). Glu 158 is conserved in BoNTs E, F, and B and is Asp in C1, D, and G and Gln in A. The sequence analysis of SNAP-25 and VAMP2 showed that they have one of Q/R/K at the P1 position (except BoNT G), which can complement well with E/D/Q at the equivalent position of Glu 158 (BoNT E) in all serotypes (24). A triple mutation E158A/T159A/N160A in BoNT E decreased the catalytic efficiency by ϳ8-fold and decreased substrate affinity as compared with the wild type enzyme (23). Interestingly, the R180K (P1) variant in murine SNAP-23 still shows cleavage by BoNT E (25).
P1Ј:Ile 181 Is Important for Specificity and Docking at the Active Site-P1Ј:Ile 181 main chain N interacts with the side chain Oe1 and Oe2 of catalytic base Glu 212 and also with Thr 159 oxygen (Figs. 2C and 4). P1Ј oxygen interacts with the side chain NH1 and NH2 groups of the conserved residue Arg 347 . The R347A mutation reduced the catalytic efficiency ϳ1000-fold relative to the wild type (23). These interactions are presumably conserved among the other serotypes with their specific substrates and help to properly position and stabilize the substrate. However, the ones that are not conserved are side chain interactions of P1Ј and the integrity of the S1Ј pocket. Here, the hydrophobic P1Ј:Ile 181 is stabilized in the S1Ј pocket comprising Thr 159 , Phe 191 , and Thr 208 , confirming our earlier prediction (Figs. 2C and 4) (23). This seems to be typically different for various serotypes defining the specificity. Mutational studies on Thr 159 , Thr 208 , and Phe 191 showed markedly lower k cat values by ϳ20, ϳ30, and ϳ80-fold, respectively, for SNAP25 cleavage, relative to wild type (26). F191N mutation also showed a lower (ϳ100-fold) k cat . Mutagenesis of P1Ј:Ile 181 to Cys in 141-206-amino acid-long SNAP-25 led it to be completely resistant to cleavage by BoNT E. 3 However, change to Ala reduced the activity by ϳ75-fold relative to wild type (27).
P1Ј is the crucial residue where mutations are hardly tolerated and any mutation leads to either no or poor cleavage (25,27). The S1Ј pocket residues also play a prime role in delivering substrate specificity by allowing optimal docking of a P1Ј residue of specific size and charge. It is tempting to speculate that probably an improper docking of the P1Ј residue also affects the proper docking of the P1 residue, which abrogates the phenotype. We therefore conclude that the P1Ј residue and its respective S1Ј pocket together contribute primarily to the optimum docking of the substrate at the active site and to scissile bond specificity. Not surprisingly, most of the variations observed are at the P1Ј position in various substrates as compared with the P1 position (24).   (Figs. 2C and 4). The above observation is supported by the fact that F191A mutation showed ϳ75-fold lower k cat than the wild type enzyme (26). All of the above three residues are not conserved in various serotypes except for Phe 191 , corresponding to Phe 194 in BoNT A, but the Phe 194 side chain takes a different rotamer position in both native substrate-free (Protein Data Bank code 1E1H) and complex structure of BoNT A (Protein Data Bank code 1XTG), possibly due to the presence of different surrounding residues unlike BoNT E. Mutation of P2Ј:Met 182 to Val reduced the catalytic activity of BoNT E on SNAP-25 by ϳ25% relative to wild type (27).
The current structure confirms that Arg 347 and Tyr 350 play a crucial role in transition state stabilization by allowing proper docking of the main chain of P1, P1Ј, and P2Ј residues at the active site.
P3Ј:Glu 183 Interactions with the 250 Loop-In the native substratefree protein crystal structure (Protein Data Bank code 1T3A), the electron density for the region 234 -244 residues (250 loop) was disordered and thus was not modeled, but in the present structure, the electron density is well defined, allowing this region to be modeled unambiguously in molecule B ( Fig.  2A). Ordering of the 250 loop is probably due to the interactions of P3Ј:Glu 183 with Ile 240 , Thr 241 , Asn 242 , and Thr 246 of BoNT E (Figs. 2C and 4). In the present structure, the carboxylate side chain of P3Ј: Glu 183 takes two rotamer positions, both involved in multiple interactions with the 250 loop. Two additional water molecules and Gly 352 are also contributing in stabilizing the P3Ј (Fig. 2C). A functional importance assay on the P3Ј:E183A mutant showed the catalytic activity reduced by ϳ25%, suggesting the requirement of Glu for optimal activity (27).
Modeling of P2:Asp 179 in BoNT E Structure-P2:Asp 179 is one of the crucial residues for better positioning of the scissile residue of the substrate. Mutation of P2:Asp 179 to Val and Ala reduced the catalytic efficiency of BoNT E by ϳ290and 60-fold, respectively (27). Therefore, to trace the S2 pocket in . BoNT E is shown in a blue and yellow diagram, RIME in a magenta ball and stick model, and zinc in a green sphere model. Only molecule B is shown for clarity. B, BoNT E is shown in a blue surface model and RIME in a white ball and stick model to show that the substrate peptide binds in a cavity at and near the active site. C, the dashed line in orange shows the interactions of P1-P3Ј residues of RIME with various residues of the S1-S3Ј pocket of BoNT E. The RIME and enzyme residues are shown in green and white ball and stick models, respectively. BoNT E secondary structure is shown in a blue loop diagram. The RIME (P1-P3Ј) and BoNT E residues are labeled in green and black, respectively. Figs. 2, 3, 5, and 6 are made using the PYMOL program (38).
BoNT E, the current and BoNT A-SNAP-25 peptide structures (Protein Data Bank code 1XTG) (16) are compared. P2:Asp 179 of SNAP-25 is modeled similar to P2:Asn 196 (in BoNT A), close to P1:Arg 180 in BoNT E structure with some minor adjustments in rotation and translation. P2:Asn 196 (in BoNT A) side chain interacts with His 227 (BoNT A) ND1 in 1XTG, but here, P2:Asp 179 side chain will be stabilized by interactions with Lys 224 NZ and His 215 ND1 of BoNT E (Fig. 5). These interactions are probably stronger than for Asn 196 in BoNT A, since the K224A BoNT E mutant showed reduced (Ͻ80%) catalytic activity (26).
Interestingly, variation at the P2 position observed in human SNAP-23 and murine SNAP-23, which have 184 IKRI 187 and 183 IQKI 186 sequence in place of 178 IDRI 181 of SNAP-25 and show resistance and poor cleavage, respectively, by BoNT E (25). P2:Asp 179 (SNAP-25) complements well with His 215 and Lys 224 , whereas P2:Lys 185 (human SNAP-23) in its place will not and may affect drastically the scissile bond alignment and thus the phenotype. However, P2:Gln 184 (murine SNAP-23) still may form a hydrogen bond with Lys 224 and may not affect the phenotype that severely. S1Ј Pockets in BoNTs Are Unique-The BoNTs E/C/D/G crystal structures (Protein Data Bank entries 1T3A, 2QN0, 2FPQ, and 1ZB7) are compared for the integrity of the S1 and S1Ј pockets, since they have a positively charged side chain for P1 (except BoNT G) and a hydrophobic P1Ј residue ( Table 2). The S1Ј pocket of E, C, and D are hydrophobic, but fine variations in S1Ј in each serotype make them very specific for a particular residue. Pro 168 in place of Thr 159 and also relative narrowing of the S1Ј pocket in BoNT C may not allow any bigger side chain residue at P1Ј. Although the S1Ј pocket is unique in all serotypes, BoNT C and D share some similarity. The presence of Ile instead of Ala at position 226 and the flexibility of the S1Ј pocket (Protein Data Bank code 2FPQ), might facilitate BoNT D to dock the bigger side chain of P1Ј. However, P1Ј mutations to Ala did not completely abrogate but rather drastically reduced the activity of BoNTs, since the presence of Ala at P1Ј may not allow a stable docking and optimal alignment at the active site required for catalysis. The lower catalytic efficiency of BoNT C (scissile bond Arg-Ala) could also be due to similar reasons.
Substrate Binding Subsites Vary between BoNT E and A-In order to evaluate the variation/similarities among serotypes  (green ball and stick model, unlabeled for clarity). BoNT E residues interacting with RIME are shown in a white ball and stick model and labeled in black. about substrate recognition/specificity at the active site, we structurally aligned BoNT E-SNAP-25-(180 -183) with the existing structures of BoNT A with inhibitors Arg-Arg-Gly-Cys (Protein Data Bank code 3C88) and N-Ac-CRATKML (Protein Data Bank code 3BOO) (Fig. 6, A and B) (17)(18)(19). DALILITE structural alignment showed a root mean square deviation of 2.3 Å between RIME and RRGC structures for 386 C␣ atoms. Although both BoNT E and A recognize SNAP-25, they cleave at different scissile bonds. BoNT E cleaves between P1:Arg 180 and P1Ј:Ile 181 , whereas BoNT A cleaves between P1:Gln 197 and P1Ј:Arg 198 . N-Ac-CRATKML has SNAP-25 sequence 198 RAT-KML 203 (P1Ј-P6Ј).
A structural alignment with the above two structures showed that the position of the P1 main chain oxygen of the RIME structure aligns reasonably with the oxygen of Arg of RRGC and the sulfur of cysteine of N-Ac-CRATKML, but the spatial position of the amino group of the RIME structure is at variance with RRGC (Fig. 6C). Also, the side chain rotamer position of P1:Arg 180 is different from the side chain of Arg of RRGC (the residue is underlined for clarity). In our structure, the P1:Arg 180 side chain makes a strong salt bridge with the Glu 158 side chain, whereas the side chain of Arg of RRGC interacts with a sulfate ion and Glu 164 (Asn 160 in BoNT E) in BoNT A. However, in the real substrate, P1 is Gln instead of Arg for BoNT A.
Comparison of P1Ј:Ile 181 with Arg in RRGC reveals variation in spatial positioning as well as docking (Fig. 6D). Ile has a smaller side chain and goes deep inside the cavity and is stabilized by the Phe 191 (Phe 194 in BoNT A), Thr 159 (Phe 163 in BoNT A), and Thr 208 (Thr 220 in BoNT A). A residue corresponding to Phe 191 exists in a similar position in BoNT A but has a different rotamer position in both native substrate-free and complex structures (Protein Data Bank codes 1E1H and 1XTG). Also, Thr 159 is replaced with a relatively bigger residue, Phe 163 (Fig.  6D). The long side chain of Arg of RRGC takes a different rotamer position, making a salt bridge interaction with the Asp 370 (BoNT A). Asp 370 (BoNT A) corresponds to Tyr 354 in BoNT E, which has a different rotamer position and helps in stabilizing the P2Ј:Met 182 side chain (Fig. 6D).
Interestingly, in the N-Ac-CRATKML structure, the P1Ј position is occupied by the N-terminal acetate moiety. The methyl group of the acetate occupies the same position as CG1 of P1Ј:Ile 181 in the RIME structure and is stabilized by Phe 163 (Thr 159 ) and Phe 194 (Phe 191 ). However, the carboxylate part of acetate interacts with the main chain amino and carbonyl groups of the enzyme, indicating a facilitated docking.
In BoNT E, P2Ј:Met 182 is stabilized in a hydrophobic pocket formed by Phe 191 , Tyr 354 , and Tyr 356 . BoNT A lacks this pocket due to the presence of Asp 370 (Fig. 6D). Gly in RRGC and Ala in N-Ac-CRATKML do not interact with protein, unlike P2Ј in BoNT E. P3Ј:Glu 183 interacts with 250 loop residues, whereas Cys (RRGC) takes a different turn (Fig. 6B) and is stabilized by the hydrophobic pocket of the 250 loop. But the actual P3Ј residue is Thr, which might not bind in a similar manner as Cys. In BoNT A, the 250 loop is bigger and has a spatial position different from that of the BoNT E 250 loop. Thr in N-Ac-CRATKML was stabilized by a water molecule interacting with Asp 370 in BoNT A. Interestingly, RATKML (P1Ј-P6Ј) of N-Ac-CRATKML represents P2Ј-P7Ј and occupies the S2Ј-S7Ј subsites instead of S1Ј-S6Ј (Fig. 6A). This is because the oxidized cysteine side chain occupies the place of the P1 main chain, and its N-terminal acetate part goes in the S1Ј pocket, and from then on the substrate residues slide by one. Surprisingly, this indicated the very interesting possibility that the docking at the S1 and S1Ј pocket can decide the overall docking of other residues in BoNT A by challenging the substrate specificity. The P1Ј-P6Ј residues dock in the S2Ј-S7Ј pockets, although they do not match with the actual P2Ј-P6Ј residues.
Overall analysis of structures suggested a variation in the S1Ј-S3Ј pocket integrity between BoNT E and A. However, the presence of hydrophobic residues at the S1Ј pocket in BoNT A, Phe 194 (Phe 191 in BoNT E) and Phe 163 (Thr 159 in BoNT E) might allow docking of hydrophobic side chains, but the hydrophobic interactions of both phenylalanines and a different rotamer position of Phe 194 , unlike Phe 191 (BoNT E), cause a shallower S1Ј pocket in BoNT A than in E. The Arg side chain at P1Ј takes a suitable rotamer position and is stabilized differently than Ile in BoNT E. Also, in BoNT E, the Phe 191 side chain adopts different rotamer positions, opening the S1Ј pocket for residues like Ile of suitable size to fit in. The absence of a similar residue at the Asp 370 (BoNT A) position will restrict the binding of residues like Arg or charged residue at P1Ј and P2Ј position in BoNT E.

TABLE 2
The comparison of the S1 and S1 side chain pocket residues of BoNT E with other serotypes (C/D/G) displaying similarity in P1 and P1 residue by charge and hydrophobicity, respectively Substrate Cleavage Strategy for BoNT E-Initially, it was a surprise in our crystal structure to have well defined electron density for the full tetrapeptide. However, combining and comparing the results of the enzyme inhibition and structural studies led us to better understand the molecular events of binding and cleavage. We assume that the reason the peptide did not get cleaved is due to the requirement of a minimal length as mentioned earlier. However, the sequence of 178 IDRIME 183 as an N-terminal GST fusion protein preceded by SNAP-25-(93-146) is also shown as cleavable length (27). The SNAP-25-(166 -181) sequence that has C-terminal Arg-Ile showed inhibition (50%) of the catalytic activity of BoNT E at 400 M concentration (27).
Also, our crystal structure clearly reveals that the mere presence of P1-P1Ј and additional P2Ј and P3Ј are not sufficient for the cleavage of the substrate but are sufficient for recognition and binding at the active site. Thus, both P1 and P1Ј with either N-or C-terminal extensions are not sufficient enough to bring the proper cleavage. Rather, they can serve as potential candidates for substrate inhibitors.
To understand BoNT E specificity for P1:Arg 181 -P1Ј:Ile 181 , we analyzed the SNAP-25 sequence (amino acids . It has at least three regions containing a similar scissile bond (RI), 57 LERIEE 62 , 178 IDRIME 183 , and 189 KTRIDE 194 , but BoNT E selects only 178 IDRIME 183 for cleavage. This is probably due to the prime requirement of exosite interactions and/or cleavage region in larger peptides. Also, the size and type of P2 and P2Ј may play significant roles.
Therefore, we assume that at least three residues on either side of the scissile bond are needed for precise alignment of the scissile bond required for the cleavage. However, all of them may not contribute to substrate affinity or specificity, although longer substrates serve better for high affinity binding and faster scissile bond cleavage. Also, simultaneous substrate recognition both at the exosites and the active site facilitates the high affinity binding and improved rate of cleavage in longer substrates. Together, the unique exosites and subsites at and near the active site determine the substrate selectivity and scissile bond specificity, which are unique in all BoNTs.
Implications in Inhibitor Design for BoNT E-The extreme toxicity and fast action of BoNT E in blocking neurotransmission requires advancement in developing countermeasures against it. Although several inhibitors are being developed for BoNT A and B and a few for BoNT F (18, 19, 28 -36), a knowledge gap exists for BoNT E. The unavailability of structural information regarding substrate-enzyme interactions is the major reason for the existing gap. We for the first time report the substrate binding at the active site of full-length catalytically active enzyme on any BoNTs. Also, the identification of S1-S3Ј subsites, hydrophobic nature of S1Ј and S2Ј pockets, and the positive charge nature of S2 can be exploited in designing of better specific inhibitors for BoNT E. Interestingly, the substrate RIME itself acts as an inhibitor and specifically binds at the active site. Overall, the BoNT E-substrate peptide complex crystal structure delivers the specific required information essential for structure-based design of potent inhibitors.
Catalytic Mechanism of BoNT Serotype E-Based on our complex structure analysis and previous mutational analysis of active site residues of BoNT E, we propose a mechanism of catalysis for botulinum neurotoxins (Fig. 7). Here in our structure, Arg 181 carbonyl oxygen displaces the nucleophilic water, and the main chain nitrogen displaces the water coordinated to Glu 212 . Glu 212 , being a general base, activates a water molecule, which donates OH to the carbonyl oxygen of Arg 180 and hydrogen to the leaving amino of Ile 181 . In our structure, we do not see any water close to Glu 212 . Perhaps in the presence of larger substrates with P2/P3 residues and/or the S4 SNARE motif Here the P1, P1Ј, P2Ј, and P3Ј positions in the RIME structure are Arg-Ile-Met-Glu; in RRGC they are Arg-Arg-Gly-Cys; and in N-Ac-CRATKML they are oxidized Cys-acetate-Arg-Ala. BoNT E, RIME, BoNT A-RRGC, and BoNT A-N-Ac-CRAT-KML structures are shown in white, green, magenta, and cyan ball and stick models, respectively. Zinc is in a deep cyan color for RIME (zinc of other structures is not shown for clarity). A, the position of oxidized cysteine (CSO), acetate (ACE), Arg, and Ala in the N-Ac-CRATKML structure aligns well with the P1:Arg-, P1Ј:Ile-, P2:Met-, and P3Ј:Glu-of the BoNT E-RIME structure but for BoNT A in actual RATKML represents P1Ј-P6Ј. B, the P1-P1Ј-P2Ј of RRGC aligns with RIME with few differences. Cys of RRGC goes in a very different spatial position, which is different from the equivalent P3Ј:Glu for BoNT E and Ala in N-Ac-CRATKML. C, superposition of P1 residues and S1 pockets in all three structures. Only BoNT E residues are labeled. D, superposition of P1Ј residues and S1Ј pockets in all three structures. Black and magenta labels represent BoNT E and A, respectively.
(37), there is a possibility of subtle rearrangements at the active site to achieve the transition state. The transition state is often the result of strain or distortion of the reactants to form the particular electronic structure needed for the proper collision and product formation. However, due to smaller substrate length, a transition state is not achieved, which leads RIME to act as a substrate inhibitor.