Understanding the structure and function of HPI, a rubber tree serine protease inhibitor, and its interaction with subtilisin

Abstract

Protease inhibitors are crucial in regulating enzymatic activity and have extensive applications in medicine, biotechnology, and agriculture. This study characterizes a recombinant protease inhibitor from Hevea brasiliensis (rHPI), highlighting its unique structural features and inhibitory potential. Using Matrix-Assisted Laser Desorption/Ionization (MALDI) analysis, the inhibitor exhibits one distinct peak around 7.54 kDa. Enzymatic assays using N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate confirmed the inhibitor's activity against subtilisin Carlsberg, a widely utilized serine protease in industry and biotechnology. The crystal structure of rHPI, resolved at 1.73 Å, reveals a topology closely resembling eglin c, including a single alpha-helix, two parallel beta-strands, and a distinctive binding loop spanning residues 40–51. Disordered regions at the N- and C-termini contribute to its structural uniqueness. Despite lacking disulfide bonds and featuring an Arg residue instead of Trp at the P′₈ position, rHPI maintains a high affinity for subtilisin. Isothermal titration calorimetry (ITC) showed that this interaction is entropically driven. Molecular docking and dynamics simulations of the rHPI-subtilisin complex revealed the formation of antiparallel β-sheets, hydrogen bonding involving the protein backbone, and a salt bridge between His64 of subtilisin and Asp47 of rHPI. These findings provide valuable insights into the molecular basis of rHPI's inhibitory activity and offer a framework for the rational design of novel subtilisin inhibitors with potential applications in agricultural and industrial settings.