Guillaume Collet

Structural Framework for Covalent Inhibition of Clostridium botulinum Neurotoxin A by Targeting Cys165

Background: Development of small potent synthetic inhibitors of Clostridium botulinum neurotoxin A remains an unresolved challenge. Results: Small compounds incorporating electrophile moiety can block enzyme activity by covalent modification of Cys165. Conclusion: A structural framework for developing potent covalent inhibitors of Clostridium botulinum neurotoxin A is provided. Significance: This study uncovers a subfamily of zinc proteases containing a conserved cysteine in their active site. 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 Cys165, a residue located in the enzyme active site just above the catalytic zinc ion. However, no direct proof of Cys165 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 Cys165 was first assessed through a combination of site-directed mutagenesis and structural studies. These data suggested that Cys165 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 Cys165. 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.

Grafting of functional motifs onto protein scaffolds identified by PDB screening – an efficient route to design optimizable protein binders

Artificial miniproteins that are able to target catalytic sites of matrix metalloproteinases (MMPs) were designed using a functional motif‐grafting approach. The motif corresponded to the four N‐terminal residues of TIMP‐2, a broad‐spectrum protein inhibitor of MMPs. Scaffolds that are able to reproduce the functional topology of this motif were obtained by exhaustive screening of the Protein Data Bank (PDB) using stamps software (search for three‐dimensional atom motifs in protein structures). Ten artificial protein binders were produced. The designed proteins bind catalytic sites of MMPs with affinities ranging from 450 nm to 450 μm prior to optimization. The crystal structure of one artificial binder in complex with the catalytic domain of MMP‐12 showed that the inter‐molecular interactions established by the functional motif in the artificial binder corresponded to those found in the MMP‐14–TIMP‐2 complex, albeit with some differences in geometry. Molecular dynamics simulations of the ten binders in complex with MMP‐14 suggested that these scaffolds may allow partial reproduction of native inter‐molecular interactions, but differences in geometry and stability may contribute to the lower affinity of the artificial protein binders compared to the natural protein binder. Nevertheless, these results show that the in silico design method used provides sets of protein binders that target a specific binding site with a good rate of success. This approach may constitute the first step of an efficient hybrid computational/experimental approach to protein binder design.