Identifi cation 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 10-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 (twofold) substrate cleavage rates. The eCLiPS method provides a new tool to profile the ligand specificity of protease exosites and to develop improved substrates.

Non-invasive imaging and cellular tracking of pulmonary emboli by near-infrared fluorescence and positron-emission tomography

“…3h ). The kinetic advantages gained on exosite inclusion are similar to those reported in other substrate designs suggesting the membrane-binding module does not negatively influence binding characteristics 25 . Catalytic efficiency of thrombin-mediated PAR1–RIP activation, k cat /K M of 8.4±0.3 × 10 6 M −1 s −1 , is 100- to 900-fold greater than previous thrombin-targeted probes that can localize to sites of its activity for in vivo imaging entirely due to its lowered K M 6 26 27 .…”

Incorporating Bacteria as a Living Component in Supramolecular Self-Assembled Monolayers through Dynamic Nanoscale Interactions

“…We could clearly see that the strain displaying the β-trypsin inhibitor knottin had a large population that was fluorescently labeled. 43 In a prior work, the number of displayed peptides has been estimated to be in the order 10 3 À10 4 on the bacterial surface. 43 Supramolecular Assembly of Bacteria in Solution.…”

“…43 In a prior work, the number of displayed peptides has been estimated to be in the order 10 3 À10 4 on the bacterial surface. 43 Supramolecular Assembly of Bacteria in Solution. In the case of the strain engineered to display our GGWGG peptide sequence-containing miniprotein 1, we expected that CB [8] could cause aggregation of a bacterial culture if the miniproteins were sufficiently accessible.…”

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

“…However, the C-terminal region of MarP may also be involved in protein-protein interactions or form an exosite (20) regulating protease activity. Exosites that enhance enzymatic activity upon binding to a preferred ligand have been found in other proteases (51). In addition, it is possible that the transmembrane domain or fulllength linker region, which is absent from the recombinant enzyme, are important for substrate recognition.…”