Engineering trypsin for inhibitor resistance

Abstract

The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity. A key step in developing inhibitor resistance is the identification of key residues in protease-inhibitor interaction. Given that majority of the protease therapeutics currently in use are trypsin-fold, trypsin itself serves as an ideal model for studying protease-inhibitor interaction. To test the importance of several trypsin-inhibitor interactions on the prime-side binding interface, we created four trypsin single variants Y39A, Y39F, K60A, and K60V and report biochemical sensitivity against bovine pancreatic trypsin inhibitor (BPTI) and M84R ecotin. All variants retained catalytic activity against small, commercially available peptide substrates [k cat /K M 5 (1.2 6 0.3) 3 10 7 M 21 s 21 . Compared with wild-type, the K60A and K60V variants showed increased sensitivity to BPTI but less sensitivity to ecotin. The Y39A variant was less sensitive to BPTI and ecotin while the Y39F variant was more sensitive to both. The relative binding free energies between BPTI complexes with WT, Y39F, and Y39A were calculated based on 3.5 ms combined explicit solvent molecular dynamics simulations. The BPTI:Y39F complex resulted in the lowest binding energy, while BPTI:Y39A resulted in the highest. Simulations of Y39F revealed increased conformational rearrangement of F39, which allowed formation of a new hydrogen bond between BPTI R17 and H40 of the variant. All together, these data suggest that positions 39 and 60 are key for inhibitor binding to trypsin, and likely more trypsin-fold proteases.

Dimerization misalignment in human glutamate-oxaloacetate transaminase variants is the primary factor for PLP release

“…The monomer energy EM represents the combined energy of the two monomers in the dimer (EM1 + EM2). The dimerization energy (EE) represents the interaction energies between the two monomers in the dimer, and the PLP binding free energy (EL) calculations were based on procedures used by us and others [ 29 – 33 ]. The EL energies were calculated as the sum ofboth PLP binding sites.…”

Use of Legumes and Yeast as Novel Dietary Protein Sources in Extruded Canine Diets

“…Anti-nutritional factors are protective metabolites in plants that deter consumption of the plant by birds or pests (43). Trypsin inhibitors, classified into either Kunitz or Bowman-Birk families, reduce digestion and absorption of protein in legumes through modulation of trypsin, an important protease (44,45). However, heat processing methods have been proven to be effective in reducing or eliminating trypsin inhibitors (45,46) increasing digestion and improving palatability of legumes (47).…”

Mechanical couplings of protein backbone and side chains exhibit scale-free network properties and specific hotspots for function

“…The trypsin fold of RT containing NT and CT barrels ( Fig. 5 A) is a useful structural template for therapeutic design [54] , [55] . The high-strength eigenvectors indeed have high values and Fig.…”