Substrate Specificity of MarP, a Periplasmic Protease Required for Resistance to Acid and Oxidative Stress in Mycobacterium tuberculosis

Jennifer L Small,Anthony J O'Donoghue,Eva C Boritsch,Oleg V Tsodikov,Giselle M Knudsen,Omar Vandal,Charles S Craik,Sabine Ehrt

doi:10.1074/jbc.m113.456541

Abstract

The transmembrane serine protease MarP is important for pH homeostasis in Mycobacterium tuberculosis (Mtb). Previous structural studies revealed that MarP contains a chymotrypsin fold and a disulfide bond that stabilizes the protease active site in the substrate-bound conformation. Here, we determined that MarP is located in the Mtb periplasm and showed that this localization is essential for function. Using the recombinant protease domain of MarP, we identified its substrate specificity using two independent assays: positional-scanning synthetic combinatorial library profiling and multiplex substrate profiling by mass spectrometry. These methods revealed that MarP prefers bulky residues at P4, tryptophan or leucine at P2, arginine or hydrophobic residues at P1, and alanine or asparagine at P1'. Guided by these data, we designed fluorogenic peptide substrates and characterized the kinetic properties of MarP. Finally, we tested the impact of mutating MarP cysteine residues on the peptidolytic activity of recombinant MarP and its ability to complement phenotypes of Mtb ΔMarP. Taken together, our studies provide insight into the enzymatic properties of MarP, its substrate preference, and the importance of its transmembrane helices and disulfide bond.

Highlights

MarP is a serine protease critical for pH homeostasis in Mycobacterium tuberculosis
The transmembrane serine protease MarP is important for pH homeostasis in Mycobacterium tuberculosis (Mtb)
An internally quenched octapeptide, methoxycoumarin-4-acetic acid (MCA)-PSLIAKWV-K(DNP), was synthesized that corresponded to the P4-P4Ј amino acids of a substrate observed in the Multiplex Substrate Profiling (MSP)-MS assay at the earliest time point

Summary

Introduction

MarP is a serine protease critical for pH homeostasis in Mycobacterium tuberculosis. Results: MarP is localized in the periplasm, and its substrate specificity was uncovered using synthetic peptides. The transmembrane serine protease MarP is important for pH homeostasis in Mycobacterium tuberculosis (Mtb). We determined that MarP is located in the Mtb periplasm and showed that this localization is essential for function. Using the recombinant protease domain of MarP, we identified its substrate specificity using two independent assays: positional-scanning synthetic combinatorial library profiling and multiplex substrate profiling by mass spectrometry. These methods revealed that MarP prefers bulky residues at P4, tryptophan or leucine at P2, arginine or hydrophobic residues at P1, and alanine or asparagine at P1؅. Our studies provide insight into the enzymatic properties of MarP, its substrate preference, and the importance of its transmembrane helices and disulfide bond

Methods

Results

Conclusion