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

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.