Abstract
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 Arg(180)-Ile(181)-Met(182)-Glu(183) (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 K(i) approximately 69 microm. The inhibitory peptide occupies the active site of BoNT E and shows well defined electron density. The catalytic zinc and the conserved key residue Tyr(350) of the enzyme facilitate the docking of Arg(180) (P1) by interacting with its carbonyl oxygen that displaces the nucleophilic water. The general base Glu(212) side chain interacts with the main chain amino group of P1 and P1'. Conserved Arg(347) of BoNT E stabilizes the proper docking of the Ile(181) (P1') main chain, whereas the hydrophobic pockets stabilize the side chains of Ile(181) (P1') and Met(182) (P2'), and the 250 loop stabilizes Glu(183) (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.
Highlights
Clostridium botulinum neurotoxins (BoNTs)2 are produced as an inactive single chain (ϳ150 kDa) and released as active dichains, heavy (ϳ100 kDa) and light chain (ϳ50 kDa) [1,2,3,4]
We report here the crystal structure of the catalytically active full-length botulinum serotype E catalytic domain (BoNT E) in complex with SNAP-25 substrate peptide Arg180-Ile181-Met182Glu183 (P1–P3)
We present for the first time the crystal structure of the full-length BoNT E catalytic domain
Summary
Data collection statistics Wavelength (Å) Resolution (Å) Space group Cell dimensions Molecules/asymmetric unit Redundancy (overall/outermost shell) I/(I) Rmerge (overall/outmost shell) Completeness (%) (overall/outmost shell) No of Reflections. The S1, S1Ј, S2Ј, and S3Ј pockets of BoNT E and their crucial interactions with P1–P3Ј residues are identified. 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
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