Substrate Binding Mode and Its Implication on Drug Design for Botulinum Neurotoxin A

Abstract

The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins, the causative agents of botulism, block the neurotransmitter release by specifically cleaving one of the three SNARE proteins and induce flaccid paralysis. The Centers for Disease Control and Prevention (CDC) has declared them as Category A biowarfare agents. The most potent among them, botulinum neurotoxin type A (BoNT/A), cleaves its substrate synaptosome-associated protein of 25 kDa (SNAP-25). An efficient drug for botulism can be developed only with the knowledge of interactions between the substrate and enzyme at the active site. Here, we report the crystal structures of the catalytic domain of BoNT/A with its uncleavable SNAP-25 peptide 197QRATKM202 and its variant 197RRATKM202 to 1.5 Å and 1.6 Å, respectively. This is the first time the structure of an uncleavable substrate bound to an active botulinum neurotoxin is reported and it has helped in unequivocally defining S1 to S5′ sites. These substrate peptides make interactions with the enzyme predominantly by the residues from 160, 200, 250 and 370 loops. Most notably, the amino nitrogen and carbonyl oxygen of P1 residue (Gln197) chelate the zinc ion and replace the nucleophilic water. The P1′-Arg198, occupies the S1′ site formed by Arg363, Thr220, Asp370, Thr215, Ile161, Phe163 and Phe194. The S2′ subsite is formed by Arg363, Asn368 and Asp370, while S3′ subsite is formed by Tyr251, Leu256, Val258, Tyr366, Phe369 and Asn388. P4′-Lys201 makes hydrogen bond with Gln162. P5′-Met202 binds in the hydrophobic pocket formed by the residues from the 250 and 200 loop. Knowledge of interactions between the enzyme and substrate peptide from these complex structures should form the basis for design of potent inhibitors for this neurotoxin.

The C Terminus of the Catalytic Domain of Type A Botulinum Neurotoxin May Facilitate Product Release from the Active Site

“…Phe 423 forms the 1) S3 pocket in the RRGC-LcA structure (17), 2) S4 pocket in the CRATKML-LcA structure (13), and 3) S5 pocket in the RRATKM-LcA structure (17). However, K m of the two shorter versions of the LcA used in this study was little affected compared with the effect on k cat by the C-terminal truncations (Table 2).…”

“…Not visible are ␣1, ␣2, ␣3, and ␣4 in the ␣-exosite and 370 loop in the ␣-exosite residues (15) that are located on the other face of the molecule. Because the first BoNT/A structure (Protein Data Bank code 3BTA (9)) identified the maximum number of C-terminal residues in any reported LcA crystal structure, its coordinates for residues 415-431 are included in this figure along with the high resolution 1.5-Å substrate-bound structure of LcA showing residues 1-423 (Protein Data Bank code 3DDA (17)) that clearly identified the active site and catalytic and exosite residues. The figure was generated by C␣ alignment of the first 1-423 residues (root mean square deviation of 1.3 Å except in the 200 and 250 loops) from both structures using the program VMD 1.9 beta 1 (47).…”

“…Although the active site pocket of LcA has been described as highly flexible (33), one of the reasons for very low catalytic activity of LcA420 may be the fact that it lacks Phe 423 , and as a result, it may not form a substrate-binding pocket (13,17). Phe 423 forms the 1) S3 pocket in the RRGC-LcA structure (17), 2) S4 pocket in the CRATKML-LcA structure (13), and 3) S5 pocket in the RRATKM-LcA structure (17).…”

“…However, K m of the two shorter versions of the LcA used in this study was little affected compared with the effect on k cat by the C-terminal truncations (Table 2). Thus, Phe 423 might play only an auxiliary role in forming the S3, S4, and S5 pockets (13,17), or flexibility of the active site (33) could compensate for the missing Phe 423 . Fig.…”

“…Thus, we gained no knowledge on the structural importance or role of the C-terminal sequence on the function of the proteins. Moreover, although mutagenesis and x-ray crystallographic studies have unequivocally defined the S1-S5Ј sites of the catalytic domain (12,(15)(16)(17), none of residues involved in either substrate interaction or catalysis, all of which are located in a 20 -24-Å-deep pit, were identified beyond Phe 423 of the 448-residue protein. In addition, the two exosites involved in the large substrate recognition (15), although located at the surface, are well removed from the C terminus of LcA.…”