Tailored Peptide Phenyl Esters Block ClpXP Proteolysis by an Unusual Breakdown into a Heptamer-Hexamer Assembly

Angew Chem Int Ed Engl. 2019 May 20;58(21):7127-7132. doi: 10.1002/anie.201901056. Epub 2019 Apr 17.

Abstract

The proteolytic complex ClpXP is fundamental to bacterial homeostasis and pathogenesis. Because of its conformational flexibility, the development of potent ClpXP inhibitors is challenging, and novel tools to decipher its intricate regulation are urgently needed. Herein, we present amino acid based phenyl esters as molecular probes to study the activity and oligomerization of the ClpXP complex of S. aureus. Systematic screening of (R)- and (S)-amino acids led to compounds showing potent inhibition, as well as stimulation of ClpXP-mediated proteolysis. Substoichiometric binding of probes arrested ClpXP in an unprecedented heptamer-hexamer assembly, in which the two heptameric ClpP rings are dissociated from each other. At the same time, the affinity between ClpX and ClpP increased, leading to inhibition of both enzymes. This conformational arrest is beneficial for the consolidated shutdown of ClpXP, as well as for the study of the oligomeric state during its catalytic cycle.

Keywords: activation; caseinolytic proteases; conformational selection; inhibitors; structure-activity relationships.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / antagonists & inhibitors*
  • Bacterial Proteins / metabolism
  • Endopeptidase Clp / antagonists & inhibitors*
  • Endopeptidase Clp / metabolism
  • Esters / chemistry
  • Esters / pharmacology*
  • Peptides / chemistry
  • Peptides / pharmacology
  • Protein Conformation
  • Protein Multimerization / drug effects*
  • Proteolysis / drug effects*
  • Serine Proteinase Inhibitors / chemistry
  • Serine Proteinase Inhibitors / pharmacology*
  • Staphylococcus aureus / enzymology*
  • Stereoisomerism
  • Structure-Activity Relationship

Substances

  • Bacterial Proteins
  • Esters
  • Peptides
  • Serine Proteinase Inhibitors
  • ClpXP protease, Streptococcus
  • Endopeptidase Clp