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

Abstract

Background: Legionella pneumophila secretes a protease without known specific substrate and three-dimensional structure.Results: 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. Conclusion:The study identifies the first selective substrate for Msp. Significance: This substrate could be useful for Legionella detection.

Zinc metalloprotease ProA of Legionella pneumophila increases alveolar septal thickness in human lung tissue explants by collagen IV degradation

“…Differences mainly pertain to outer flexible loop regions (Figure S3). Compared to the proposed model of ProA by Poras et al (2012), the actual protein structure differs in the same three loops and additionally contains a second β‐sheet region, which cannot be found in the elastase but similarly in vibriolysin (Figure S3b–d). Discrepancies in substrate targeting of TLPs are mainly defined by divergent amino acid residues in specific substrate binding sites.…”

“…Pseudolysin from P. aeruginosa (sequence identity 47.7%) and vibriolysin from Pseudoalteromonas strains display minor structural C‐alpha RMSDs of approx. 1.3 and 0.7 Å, respectively, to ProA (Poras et al, 2012). Differences mainly pertain to outer flexible loop regions (Figure S3).…”

“…Despite high sequence homologies, ProA and the elastase pseudolysin of P. aeruginosa differ in their main substrate, as ProA is not able to cleave elastin (Thompson et al, 1981). The current structural model of ProA is unsuitable for an adequate elaboration of these differences since it is also based on the elastase pseudolysin (Poras et al, 2012). To certainly identify determinants responsible for the divergent target recognition and completing the overall characterisation of ProA, we resolved the protein crystal structure at 1.48 Å.…”

“…In previous studies, it was already shown that this residue is essential for proteolytic activity (Moffat, Black, & Tompkins, 1994). Compared to the proposed model of the zinc metalloprotease from 2012, we resolved discrepancies concerning flexible loops and β‐sheets, since the model was elastase‐based and shared the same differences to ProA as pseudolysin (Poras et al, 2012). Overall, the crystal structure of ProA resembles those of homologous virulence‐associated TLPs implicating functional comparability.…”

“…ProA is a member of the M4 enzyme family of thermolysin‐like proteases (TLPs) and is especially homologous to the elastase pseudolysin from Pseudomonas aeruginosa . ProA is initially translated together with a signal‐ and a propeptide as a 60 kDa large preproenzyme (Adekoya & Sylte, 2009; Poras et al, 2012). The propeptide exhibits chaperone as well as inhibitory functions in TLPs, therefore truncation of the gene sequence results in wrong folding and non‐functional conformation of the protein.…”