Highly Sensitive Quenched Fluorescent Substrate of Legionella Major Secretory Protein (Msp) Based on Its Structural Analysis

• Published in: The Journal of biological chemistry 2012-06, Vol.287 (24), p.20221-20230
• Main Authors: Poras, Hervé, Duquesnoy, Sophie, Dange, Emilie, Pinon, Anthony, Vialette, Michèle, Fournié-Zaluski, Marie-Claude, Ouimet, Tanja
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Enzyme Structure; Enzymes; Enzymology; Fluorescence; Fluorescent Substrate; Legionella pneumophila; Legionella pneumophila - enzymology; Metalloprotease; Metalloproteases - chemistry; Models, Molecular; Peptide Chemical Synthesis; Structural Homology, Protein; Three-dimensional Model Structure
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.334334

Legionella pneumophila has been shown to secrete a protease termed major secretory protein (Msp). This protease belongs to the M4 family of metalloproteases and shares 62.9% sequence similarity with pseudolysin (EC 3.4.24.26). With the aim of developing a specific enzymatic assay for the detection and quantification of Msp, the Fluofast substrate library was screened using both enzymes in parallel. Moreover, based on the crystal structure of pseudolysin, a model of the Msp structure was built. Screening of the peptide library identified a lead substrate specifically cleaved by Msp that was subsequently optimized by rational design. The proposed model for Msp is consistent with the enzymatic characteristics of the studied peptide substrates and provides new structural information useful for the characterization of the protease. This study leads to the identification of the first selective and high affinity substrate for Msp that is able to detect picomolar concentrations of the purified enzyme. The identified substrate could be useful for the development of a novel method for the rapid detection of Legionella. Legionella pneumophila secretes a protease without known specific substrate and three-dimensional structure. Analysis of a quenched peptide library identified a lead substrate, ameliorated by rational design, using an Msp structural model obtained by the x-ray structure of pseudolysin. The study identifies the first selective substrate for Msp. This substrate could be useful for Legionella detection.