Accelerated trypsin autolysis by affinity polymer templates

Abstract

Self-cleavage of proteins is an important natural process that is difficult to control externally. Recently a new mechanism for the accelerated autolysis of trypsin was discovered involving polyanionic template polymers; however it relies on unspecific interactions and is inactive at elevated salt loads. We have now developed affinity copolymers that bind to the surface of proteases by specific recognition of selected amino acid residues. These are highly efficient trypsin inhibitors with low nanomolar IC₅₀ levels and operate at physiological conditions. In this manuscript we show how these affinity copolymers employ the new mechanism of polymer-assisted self-digest (PAS) and act as a template for multiple protease molecules. Their elevated local concentration leads to accelerated autolysis on the accessible surface area and shields complexed areas. The resulting extremely efficient trypsin inhibition was studied by SDS-PAGE, gel filtration, CD, CZE and ESI-MS. We also present a simple theoretical model that simulates most experimental findings and confirms them as a result of multivalency and efficient reversible templating. For the first time, mass spectrometric kinetic analysis of the released peptide fragments gives deeper insight into the underlying mechanism and reveals that polymer-bound trypsin cleaves much more rapidly with low specificity at predominantly uncomplexed surface areas.

Fig. 1. Concept of multivalent protein surface recognition by linear affinity copolymers from amino acid-selective binding monomers. (A) Schematic of the affinity copolymer concept – each color symbol represents an amino acid/binding monomer pair. (B) Molecular recognition pattern for the specific targeting of arginine (left) and serine residues (right) on the protein surface by designed binding sites in affinity copolymers. Note the noncovalent combination of ionic hydrogen bonds and π-cation attraction between bisphosphonate dianion and alkylguanidinium side chain (blue), and reversible covalent formation of a cyclic boronate ester between the aminomethylphenylboronic acid moiety and the primary alcohol (red). (C) Lewis structures of homopolymer P1 and copolymer P2.

Fig. 2. Affinity polymers P1/P2 accelerate trypsin self-digestion. (A) SDS-PAGE. Autolysis of 60 μM trypsin over time (75 mM TRIS buffer, pH 8.0, 37 °C). Left: Trypsin alone; right: trypsin with 6.7 μM P1/P2. (B) CD spectroscopy. Time-dependent CD spectra monitoring the kinetics of polymer-accelerated trypsin autolysis (60 μM trypsin, 75 mM borate buffer). Left: Trypsin alone; right: trypsin with 6.7 μM P1/P2. (C) Gel filtration. Gel filtration chromatograms depicting the molecular weight distribution of trypsin alone and in the presence of P1 during the self-digest process (100 mM borate buffer, denaturated with 6 M urea and 30 mM DTT). Left: Trypsin alone, right: trypsin with P1/P2. P1/P2 concentrations refer to the full polymer molecular weight.

Fig. 3. The new polymer-accelerated self-digest can be simulated by a simple computational model. (A) Proposed mechanistic model starting with intact protease and polymer (A), followed by polymer collapse (B), cannibalistic autolysis (C) and released peptide fragments (D and E). (B) Polymer (red) stabilizes a trypsin dimer (blue, green), which favors cannibalistic autolysis (active center in red sticks). (C) Red isosurfaces of increased affinity level (−1k_BT) between trypsin and the bisphosphonate monomer, which binds to K/R without shielding the active site (yellow). (D) Lattice model simulation results. DP = degree of polymerization. Polymer contact −2kT/−4kT. (A) Mean monomer–monomer distance R_m; (B) average number n_ee of enzyme–enzyme contacts; (C) average number n_ep of enzyme–polymer contacts; (D) average number n_b of enzymes bound to polymer (total enzymes = 10). (E) Cubic lattice box with 10 enzymes and polymer of DP. (F) Simulation snapshots. (A) 30-mer polymer (red) interacts with trypsin molecules (blue) at contact energies E_cont = −2k_BT (A) and E_cont = −4k_BT (B) at the same trypsin concentration. Only in (B) enzymes contact each other, illustrating the importance of strong single interactions.

Fig. 4. Overlay of epitopsy calculation with cleavage sites identified by MS. The active site of the enzyme is depicted in orange and represents the front of the enzyme. (A) Black cleavage sites found in peptides produced with increasing concentration upon treatment with P1. (B) Yellow cleavage sites from peptides with decreased concentration upon treatment with P1. (C) Blue peptides with no significant abundance change upon P1 treatment. (D) Accelerated autolysis leads to shorter peptide fragments. Each point corresponds to one specific digest peptide. Presence of P1 decreases abundance of longer peptides and increases abundance of shorter peptides.