期刊
PROTEIN AND PEPTIDE LETTERS
卷 22, 期 3, 页码 239-247出版社
BENTHAM SCIENCE PUBL LTD
DOI: 10.2174/0929866522666141126162839
关键词
Cysteine proteinase; enzyme-inhibitor interactions; fluorometric titration; molecular dynamics simulation; pH dependence; pK(a) shift; protein-protein docking
资金
- National Council of Science and Technology (CONACyT, Mexico) [181049, 168692]
- [28595]
Cysteine proteinases are involved in many aspects of physiological regulation. In humans, some cathepsins have shown another function in addition to their role as lysosomal proteases in intracellular protein degradation; they have been implicated in the pathogenesis of several heart and blood vessel diseases and in cancer development. In this work, we present a fluorometric and computational study of the binding of one representative plant cysteine proteinase, chymopapain, to one of the most studied inhibitors of these proteinases: chicken cystatin. The binding equilibrium constant, K-b, was determined in the pH range between 3.5 and 10.0, revealing a maximum in the affinity at pH 9.0. We constructed an atomic model for the chymopapain-cystatin dimer by docking the individual 3D protein structures; subsequently, the model was refined using a 100 ns NPT molecular dynamics simulation in explicit water. Upon scrutiny of this model, we identified 14 ionizing residues at the interface of the complex using a cutoff distance of 5.0 angstrom. Using the pK(a) values predicted with PROPKA and a modified proton-linkage model, we performed a regression analysis on our data to obtain the composite pK(a) values for three isoacidic residues. We also calculated the electrostatic component of the binding energy (Delta G(b,elec)) at different pH values using an implicit solvent model and APBS software. The pH profile of this calculated energy compares well with the experimentally obtained binding energy, Delta G(b). We propose that the residues that form an interchain ionic pair, Lys139A from chymopapain and Glu19B from cystatin, as well as Tyr61A and Tyr67A from chymopapain are the main residues responsible for the observed pH dependence in the chymopapain-cystatin affinity.
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