title: Substrate specificity of MarP, a periplasmic protease required for resistance to acid and oxidative stress in Mycobacterium tuberculosis.
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.
authors.affiliation: Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York 10065, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Small
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: O'Donoghue
authors.forename: Eva C
authors.initials: EC
authors.lastname: Boritsch
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Tsodikov
authors.forename: Giselle M
authors.initials: GM
authors.lastname: Knudsen
authors.forename: Omar
authors.initials: O
authors.lastname: Vandal
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
authors.forename: Sabine
authors.initials: S
authors.lastname: Ehrt
mesh_terms: D006863:Hydrogen-Ion Concentration; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D018384:Oxidative Stress / Q000502:physiology*; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D033903:Periplasmic Proteins / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D033903:Periplasmic Proteins; D010447:Peptide Hydrolases
keywords: Multiplex Substrate Profiling; Mycobacterium tuberculosis; Peptide Biosynthesis; Peptides; Positional-scanning Synthetic Combinatorial Library; Serine Protease; Stress Response; pH Regulation