Structure- and Substrate-based Inhibitor Design for Clostridium botulinum Neurotoxin Serotype A

Abstract

The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins cleave specific soluble N-ethylmaleimidesensitive factor attachment protein receptor complex proteins and block the release of neurotransmitters that cause flaccid paralysis and are considered potential bioweapons. Botulinum neurotoxin type A is the most potent among the clostridial neurotoxins, and to date there is no post-exposure therapeutic intervention available. To develop inhibitors leading to drug design, it is imperative that critical interactions between the enzyme and the substrate near the active site are known. Although enzyme-substrate interactions at exosites away from the active site are mapped in detail for botulinum neurotoxin type A, information about the active site interactions is lacking. Here, we present the crystal structures of botulinum neurotoxin type A catalytic domain in complex with four inhibitory substrate analog tetrapeptides, viz. RRGC, RRGL, RRGI, and RRGM at resolutions of 1.6 -1.8 Å . These structures show for the first time the interactions between the substrate and enzyme at the active site and delineate residues important for substrate stabilization and catalytic activity. We show that OH of Tyr 366 and NH 2 of Arg 363 are hydrogen-bonded to carbonyl oxygens of P1 and P1 of the substrate analog and position it for catalytic activity. Most importantly, the nucleophilic water is replaced by the amino group of the N-terminal residue of the tetrapeptide. Furthermore, the S1 site is formed by

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

“…A quick look at the sequences of the products suggested that acetyl-SNKTRIDEANQ with two acidic residues and two acid amides might have a preferential interaction with the highly positively charged LcA C terminus (KNFTGLFEFYKLLCVR-GIITSKTKSLDKGYNK). Lack of electron density of LcA residues 420 -448 in the crystal structure of BoNT/A (9) and failure of full-length LcA to form crystals (12,13,33) suggest that the LcA C terminus is highly mobile. Our results of the thermal denaturation experiment described in Fig.…”

“…However, when the catalytic activity was measured on an intermediate-sized peptide substrate, a 1-425-residue LcA displayed only 25% of the activity, 4 and a 1-424-residue construct displayed 25% of the fulllength LcA activity as well (12). Thus, it is important that this anomaly is more thoroughly investigated.…”

“…The structure was thus short by 17 residues from the full-length BoNT/A LC, by 10 residues from that of a proposed mature 444-residue BoNT/A LC (10), or by seven residues from the mature 438-residue BoNT/A LC (11) based on their isolation from culture filtrates of C. botulinum. After attempts to crystallize the full-length 448-residue light chain of serotype A (LcA) failed, investigators turned to C-terminally truncated LcA to determine its high resolution structures (12)(13)(14)(15). Thus, we gained no knowledge on the structural importance or role of the C-terminal sequence on the function of the proteins.…”

“…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.…”

Fungal BIS-Naphthopyrones as inhibitors of botulinum neurotoxin serotype A

“…5 DOVIS was used to systematically dock the MLSMR compounds to the catalytic binding site of the BoNTA protein target (PDB code 3C8A). 6 The compounds were sorted by AutoDock 4.0 score, 7 and the top 20000 compounds were chosen and minimized within a fixed BoNT/A catalytic site. After minimization, the compounds were rescored with three different scoring functions, AutoDock 4, 7 LigScore 2, 8 and X-Score.…”