Structural Basis of Substrate Recognition and Covalent Inhibition of Cdu1 from Chlamydia trachomatis

Abstract

Based on the similarity between the active sites of the deubiquitylating and deneddylating enzyme ChlaDub1 (Cdu1) and the evolutionarily related protease adenain, a target-hopping screening approach on a focused set of adenain inhibitors was investigated. The cyanopyrimidine-based inhibitors identified represent the first active-site-directed small-molecule inhibitors of Cdu1. High-resolution crystal structures of Cdu1 in complex with two covalently bound cyanopyrimidines, as well as with its substrate ubiquitin, were obtained. These structural data were complemented by enzymatic assays and covalent docking studies to provide insight into the substrate recognition of Cdu1, active-site pocket flexibility and potential hotspots for ligand interaction. Combined, these data provide a strong basis for future structure-guided medicinal chemistry optimization of this cyanopyrimidine scaffold into more potent and selective Cdu1 inhibitors.

The Two Deubiquitinating Enzymes from Chlamydia trachomatis Have Distinct Ubiquitin Recognition Properties

“…Site 3 involves the N-terminal β-hairpin of Ub interacting with helix α5 and loop L2 of ChlaDUB2. This site features interactions similar to those of the VR3 recognition element described in a previous work , on ChlaDUB1 where one face of helix α5 (VR3 insertion) was shown to engage the Leu8-Thr9 hairpin turn of Ub. Leu187 and Met190 in helix α5 of ChlaDUB2 appear to make hydrophobic contacts with Thr9 of Ub, while Pro119 seems to pack against the backbone of Leu8 of Ub, providing additional hydrophobic contact (Figure E).…”

“…Taken together, the interactions in the three sites play important roles in recognizing and holding Ub in the distal binding position in a manner that would promote the placement of the scissile peptide bond at Ub Gly76 adjacent to the catalytic Cys of the DUB. The overall orientation and manner of recognition of Ub are similar to that of the S1 binding interactions of ChlaDUB1, with an rmsd of 1.36 Å over the entire Ub-bound complex (Figure B). ,, …”

“…Nevertheless, the copy of the asymmetric unit showing Ub in a better-defined position allowed us to deduce intermolecular contacts between the DUB and Ub at most places with reasonable confidence (Figure ). Ubiquitin binds to ChlaDUB2 in a fashion similar to that in which it binds to ChlaDUB1 (Figure B). , This binding site is termed the distal binding site (also known as the S1 binding site), so named because this is where the distal Ub group in a diubiquitin substrate would interact when it is bound to the DUB. (In a diubiquitin molecule, the Ub group contributing its Gly76 to the isopeptide link is termed the distal Ub and the Ub donating its Lys side chain as the proximal Ub.…”

Insights Into Mitochondrial Dynamics in Chlamydial Infection

“…Thereby, it seems to be an extremely promising strategy to create ligands that exclusively bind to Cdu1 as a competitive inhibitor of Mcl-1 in order to increase the ubiquitination level of Mcl-1 and thus impact mitochondrial dynamics. Similar investigations are currently ongoing: Caroline Kisker’s team discovered that the particular inhibitors cyanopyrimidine 3 and cyanopyrimidine 5 may form covalent connections with Cdu1 and disrupt the protein’s deubiquitination function ( Ramirez et al., 2018 ). This indicates that the chemical may be capable of combating chlamydial infection.…”

Clear Victory for Chlamydia: The Subversion of Host Innate Immunity

“…The formation of this complex inhibits the ubiquitination and degradation of I𝜅Bα and stabilizes it in the cytosol. The ectopic expression of ChlaDub1 also blocks NF-κB signaling downstream of the IKK complex but does not interfere with the upstream components of the pathway, leading to the suppression of NF-κB activation (Le Negrate et al, 2008; Ramirez et al, 2018). Once in the host cytoplasm, the C. pneumoniae -specific inclusion membrane protein (Inc) CP0236 binds to and alters the distribution of NF-κB activator 1 (Act1) in the cytoplasm.…”