Effects of pH on the Association between the Inhibitor Cystatin and the Proteinase Chymopapain

Title:Effects of pH on the Association between the Inhibitor Cystatin and the Proteinase Chymopapain

Volume: 22 Issue: 3

Author(s): Francisco Reyes-Espinosa, Alfonso Arroyo-Reyna, Ponciano Garcia-Gutierrez, Iris N. Serratos and Rafael A. Zubillaga

Affiliation:

Keywords: Cysteine proteinase, enzyme–inhibitor interactions, fluorometric titration, molecular dynamics simulation, pH dependence, pK_a shift, protein–protein docking.

Abstract: 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 Å. 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_avalues for three isoacidic residues. We also calculated the electrostatic component of the binding energy (ΔG_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, ΔG_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.

Cite this article as:

Reyes-Espinosa Francisco, Arroyo-Reyna Alfonso, Garcia-Gutierrez Ponciano, Serratos N. Iris and Zubillaga A. Rafael, Effects of pH on the Association between the Inhibitor Cystatin and the Proteinase Chymopapain, Protein & Peptide Letters 2015; 22 (3) . https://dx.doi.org/10.2174/0929866522666141126162839

DOI https://dx.doi.org/10.2174/0929866522666141126162839	Print ISSN 0929-8665
Publisher Name Bentham Science Publisher	Online ISSN 1875-5305