Phage-Related Ribosomal Protease (Prp) of Staphylococcus aureus: In Vitro Michaelis–Menten Kinetics, Screening for Inhibitors, and Crystal Structure of a Covalent Inhibition Product Complex

Abstract

Phage-related ribosomal proteases (Prps) are essential for the assembly and maturation of the ribosome in Firmicutes, including the human pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Clostridium difficile. These bacterial proteases cleave off an N-terminal extension of a precursor of ribosomal protein L27, a processing step that is essential for the formation of functional ribosomes. This essential role of Prp in these pathogens has identified this protease as a potential antibiotic target. In this work, we determine the X-ray crystal structure of a covalent inhibition complex at 2.35 Å resolution, giving the first complete picture of the active site of a functional Prp. We also characterize the kinetic activity and screen for potential inhibitors of Prp. This work gives the most complete characterization of the structure and specificity of this novel class of proteases to date.

Phage-Related Ribosomal Proteases (Prps): Discovery, Bioinformatics, and Structural Analysis

“…This grouping of bacteria was initially thought to be limited to Gram-positive low GC species, but has since been expanded to include Firmicutes, Fusobacteria, Synergistetes and some Thermotogae and Tenericutes [ 16 , 26 , 31 ]. In the case of S. aureus , this sequence is 9 AAs (amino acids) long (MLKLNLQFF’ASK…), but sequences as long as 15 AAs have been observed ( Table 2 ) [ 11 , 32 ]. Prp is the protease responsible for this essential cleavage [ 11 ].…”

“…Most species’ long L27 cleavage sequences follow the following motif: MLxx(D/N)LQ(F/L)F’A(S/H)KK, with xx being a variable domain ( Table 2 ) [ 16 ]. However, each species encodes for an L27 with a cleavage sequence that is highly specific to that species’ Prp [ 12 , 32 ]. Purified Sa Prp has been seen to cleave a fluorescently labeled Sa L27-based peptide (FITC-KLNLQFFASKK-Dnp) with a catalytic efficiency (K cat /K M ) of 74,000 ± 38,000 M −1 s −1 [ 32 ].…”

“…However, each species encodes for an L27 with a cleavage sequence that is highly specific to that species’ Prp [ 12 , 32 ]. Purified Sa Prp has been seen to cleave a fluorescently labeled Sa L27-based peptide (FITC-KLNLQFFASKK-Dnp) with a catalytic efficiency (K cat /K M ) of 74,000 ± 38,000 M −1 s −1 [ 32 ]. This cleavage was not inhibited by a library of common protease inhibitors, including many broad-spectrum covalent cystine protease inhibitors, indicating Prp’s resistance to inhibition.…”

“…This cleavage was not inhibited by a library of common protease inhibitors, including many broad-spectrum covalent cystine protease inhibitors, indicating Prp’s resistance to inhibition. Although the thiophilic compound mersalyl acid was used as a positive inhibition control in this assay, its general toxicity and non-selective nature preclude it from clinical consideration [ 32 , 33 ]. Additionally, the Prp of C. difficile was unable to cleave the Sa L27-based peptide, further indicating the specificity of Prp to its endogenous substrate [ 32 ].…”

“…Although the thiophilic compound mersalyl acid was used as a positive inhibition control in this assay, its general toxicity and non-selective nature preclude it from clinical consideration [ 32 , 33 ]. Additionally, the Prp of C. difficile was unable to cleave the Sa L27-based peptide, further indicating the specificity of Prp to its endogenous substrate [ 32 ].…”