The first broad-spectrum antibiotic chloramphenicol and one of the newest clinically important antibacterials, linezolid, inhibit protein synthesis by targeting the peptidyl transferase center of the bacterial ribosome. Because antibiotic binding should prevent the placement of aminoacyl-tRNA in the catalytic site, it is commonly assumed that these drugs are universal inhibitors of peptidyl transfer and should readily block the formation of every peptide bond. However, our in vitro experiments showed that chloramphenicol and linezolid stall ribosomes at specific mRNA locations. Treatment of bacterial cells with high concentrations of these antibiotics leads to preferential arrest of translation at defined sites, resulting in redistribution of the ribosomes on mRNA. Antibiotic-mediated inhibition of protein synthesis is most efficient when the nascent peptide in the ribosome carries an alanine residue and, to a lesser extent, serine or threonine in its penultimate position. In contrast, the inhibitory action of the drugs is counteracted by glycine when it is either at the nascent-chain C terminus or at the incoming aminoacyl-tRNA. The context-specific action of chloramphenicol illuminates the operation of the mechanism of inducible resistance that relies on programmed drug-induced translation arrest. In addition, our findings expose the functional interplay between the nascent chain and the peptidyl transferase center.ribosome | antibiotics | protein synthesis | nascent peptide | oxazolidinones

Cysteine proteases are among the most abundant hydrolytic enzymes produced by bacteria, and this diverse family of proteins have significant biological roles in bacterial viability and environmental interactions. Members of the clostripain-like (C11) family of cysteine proteases from distal gut commensal strains have recently been shown to mediate immune responses by inducing neutrophil phagocytosis and activating bacterial pathogenic toxins. Development of substrates, inhibitors, and probes that target C11 proteases from enteric bacteria will help to establish the role of these proteins at the interface of the host and microbiome in health and disease. We employed mass spectrometry-based substrate profiling method to identify an optimal peptide substrate of PmC11, a C11 protease secreted by the commensal bacterium Parabacteroides merdae. Using this substrate sequence information, a panel of fluorogenic substrates were synthesized to calculate kcat and KM and to evaluate the importance of the P2 amino acid for substrate turnover. A potent and irreversible tetrapeptide inhibitor with an C-terminal acyloxymethyl ketone warhead, Ac-VLTK-AOMK, was then synthesized. We determined the crystal structure of PmC11 in complex with this inhibitor and uncovered key active-site interactions that govern PmC11 substrate recognition and specificity. This is the first C11 protease structure in complex with a substrate mimetic and is also the highest resolution crystal structure of a C11 protease to date at 1.12 Å resolution. Importantly, subjecting human epithelial cell lysates to PmC11 hydrolysis in combination with subtiligase-based N-terminal labeling and tandem mass spectrometry proteomics complemented the stringent substrate specificity observed in the in vitro substrate profiling experiment. The combination of chemical biological, biophysical, and biochemical techniques presented here to elucidate and characterize PmC11 substrate selectivity can be expanded to other proteases and the development of chemical tools to study these essential proteins in biologically relevant samples, such as the highly complex distal gut microbiome.

Supporting information Figure S1. ESI-MS mass traces of WT thetapain light and heavy chains. Mass spectrometric analysis of WT thetapain lacking the N-terminal His6-tag yields exact masses of 14.0 and 28.3 kDa for the small Nterminal and large C-terminal chains, respectively. These weights correspond to self-activation from a single cleavage event after Arg147.

The clostripain-like (C11) family of cysteine proteases are ubiquitously produced by the vast majority of the bacterial strains that make up the human distal gut microbiome. Recent reports show that some C11 proteases promote host immune responses and bacterial pathogenesis, including the induction of neutrophil phagocytosis and the activation of bacterial pathogenic toxins, respectively. The crystal structure of distapain, the only C11 protease predicted within the genome of the commensal bacterium Parabacteroides distasonis, was determined in the inactive zymogen state to 1.65 Å resolution. This is the first C11 protease structure of a zymogen, and the structure helped to uncover key unique conformations among critical active-site residues that are likely to assist in preserving the inactive protease. His135, a member of the catalytic dyad, is repositioned approximately 5.5 Å from the orientation found in active C11 structures and forms a hydrogen bond to Asp180 and a -stacking interaction with Trp133. The structure sheds light on the potential importance of Asp180 and Trp133, as these residues are highly conserved across C11 proteases. Structure elucidation of C11 proteases will ultimately help to identify new ways to chemically and/or biologically regulate this family of enzymes, which represent potential drug-discovery targets in microbiome-related gastrointestinal diseases.

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