Structural framework for covalent inhibition of Clostridium botulinum neurotoxin A by targeting Cys165

Abstract

Clostridium botulinum neurotoxin type A (BoNT/A) is one of the most potent toxins for humans and a major biothreat agent. Despite intense chemical efforts over the past 10 years to develop inhibitors of its catalytic domain (catBoNT/A), highly potent and selective inhibitors are still lacking. Recently, small inhibitors were reported to covalently modify catBoNT/A by targeting Cys(165), a residue located in the enzyme active site just above the catalytic zinc ion. However, no direct proof of Cys(165) modification was reported, and the poor accessibility of this residue in the x-ray structure of catBoNT/A raises concerns about this proposal. To clarify this issue, the functional role of Cys(165) was first assessed through a combination of site-directed mutagenesis and structural studies. These data suggested that Cys(165) is more involved in enzyme catalysis rather than in structural property. Then by peptide mass fingerprinting and x-ray crystallography, we demonstrated that a small compound containing a sulfonyl group acts as inhibitor of catBoNT/A through covalent modification of Cys(165). The crystal structure of this covalent complex offers a structural framework for developing more potent covalent inhibitors catBoNT/A. Other zinc metalloproteases can be founded in the protein database with a cysteine at a similar location, some expressed by major human pathogens; thus this work should find broader applications for developing covalent inhibitors.

FIGURE 1.

Details of the active site of the catalytic domain of BoNT/A. Residues chelating the zinc ion are displayed in cyan, residues reported to play a role in enzyme catalytic efficiency are displayed in green, cysteine residues are in yellow, and the catalytic zinc ion is shown as a purple sphere.

FIGURE 2.

Superimposition of crystal structure between wild type catBoNT/A catalytic domain (1. 8 Å) and C134S/C165S double mutant (1.2 Å). A closer view of the peptide segment bearing Cys¹⁶⁵ in wild type (yellow) or C134S/C165S in double mutant (cyan) is shown in stick representation.

SCHEME 1.

Chemical structure of MTSEA and MTSPA compounds and chemical reaction with cysteine.

FIGURE 3.

Kitz-Wilson plot for the inactivation of catBoNT/A by MTSEA (cross) and by MTSPA (closed circle). 40 mm HEPES buffer at pH 7 at 37 °C was used.

FIGURE 4.

Crystal structure of catBoNT/covalently modified by MTSEA. This structure reveals the NH₃ group of MTSEA is surrounded by Glu²²⁴, the zinc ion, and a malate molecule from the crystallization buffer, interacting itself with the zinc ion.

FIGURE 5.

Superimposition of the crystal structure of C. botulinum neurotoxin type A (Protein Data Bank code 1XTG) with that of C. botulinum neurotoxin type F (Protein Data Bank code 2A97), thimet oligopeptidase (Protein Data Bank code 1S4B), neurolysin (Protein Data Bank code 1I1I), oligopeptidase F (Protein Data Bank code 2H1J), and T. cruzi metallocarboxypeptidase (Protein Data Bank code 3DWC). The structure overlay was based on the best fit between the zinc ligand (His, His, and Glu) in these proteases.