Structural modeling and functional analysis of the essential ribosomal processing protease Prp from Staphylococcus aureus

Mol Microbiol. 2017 May;104(3):520-532. doi: 10.1111/mmi.13644. Epub 2017 Mar 2.

Abstract

In Firmicutes and related bacteria, ribosomal large subunit protein L27 is encoded with a conserved N-terminal extension that is removed to expose residues critical for ribosome function. Bacteria encoding L27 with this N-terminal extension also encode a sequence-specific cysteine protease, Prp, which carries out this cleavage. In this work, we demonstrate that L27 variants with an un-cleavable N-terminal extension, or lacking the extension (pre-cleaved), are unable to complement an L27 deletion in Staphylococcus aureus. This indicates that N-terminal processing of L27 is not only essential but possibly has a regulatory role. Prp represents a new clade of previously uncharacterized cysteine proteases, and the dependence of S. aureus on L27 cleavage by Prp validates the enzyme as a target for potential antibiotic development. To better understand the mechanism of Prp activity, we analyzed Prp enzyme kinetics and substrate preference using a fluorogenic peptide cleavage assay. Molecular modeling and site-directed mutagenesis implicate several residues around the active site in catalysis and substrate binding, and support a structural model in which rearrangement of a flexible loop upon binding of the correct peptide substrate is required for the active site to assume the proper conformation. These findings lay the foundation for the development of antimicrobials that target this novel, essential pathway.

MeSH terms

  • Amino Acid Sequence
  • Cysteine Proteases / chemistry*
  • Cysteine Proteases / genetics
  • Cysteine Proteases / metabolism*
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Ribosomal Proteins / chemistry
  • Ribosomal Proteins / genetics
  • Ribosomal Proteins / metabolism*
  • Ribosomes / metabolism
  • Staphylococcus aureus / enzymology*
  • Staphylococcus aureus / genetics
  • Staphylococcus aureus / metabolism

Substances

  • Ribosomal Proteins
  • ribosomal proteins L27
  • Cysteine Proteases