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Publication Abstract from PubMed

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 A, 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 beta-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.

Understanding the structure and function of HPI, a rubber tree serine protease inhibitor, and its interaction with subtilisin.,Terron-Hernandez J, Gomez-Velasco H, Pinzon-Yaya L, Hernandez-Santoyo A, Garcia-Ramirez B, Rodriguez-Romero A Biochem Biophys Res Commun. 2025 May 12;763:151801. doi: , 10.1016/j.bbrc.2025.151801. Epub 2025 Apr 11. PMID:40233429^[2]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.