title: Identification of protease exosite-interacting peptides that enhance substrate cleavage kinetics.
abstract: Many peptidases are thought to require non-active site interaction surfaces, or exosites, to recognize and cleave physiological substrates with high specificity and catalytic efficiency. However, the existence and function of protease exosites remain obscure owing to a lack of effective methods to identify and characterize exosite-interacting substrates. To address this need, we modified the cellular libraries of peptide substrates (CLiPS) methodology to enable the discovery of exosite-interacting peptide ligands. Invariant cleavage motifs recognized by the active sites of thrombin and caspase-7 were displayed on the outer surface of bacteria adjacent to a candidate exosite-interacting peptide. Exosite peptide libraries were then screened for ligands that accelerate cleavage of the active site recognition motif using two-color flow cytometry. Exosite CLiPS (eCLiPS) identified exosite-binding peptides for thrombin that were highly similar to a critical exosite interaction motif in the thrombin substrate, protease-activated receptor 1. Protease activity probes incorporating exosite-binding peptides were cleaved ten-fold faster than substrates without exosite ligands, increasing their sensitivity to thrombin activity in vitro. For comparison, screening with caspase-7 yielded peptides that modestly enhanced (two-fold) substrate cleavage rates. The eCLiPS method provides a new tool to profile the ligand specificity of protease exosites and to develop improved substrates.
authors.affiliation: Department of Chemical Engineering, University of California, Santa Barbara, CA 93106, USA.
authors.forename: Abeer M
authors.initials: AM
authors.lastname: Jabaiah
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Getz
authors.forename: Witold A
authors.initials: WA
authors.lastname: Witkowski
authors.forename: Jeanne A
authors.initials: JA
authors.lastname: Hardy
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Daugherty
mesh_terms: D000595:Amino Acid Sequence; D053179:Caspase 7 / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D044463:Receptor, PAR-1 / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D008024:Ligands; D010455:Peptides; D044463:Receptor, PAR-1; D013917:Thrombin; D053179:Caspase 7