Kunitz‐type trypsin inhibitor from durian (<i>Durio zibethinus</i>) employs a distinct loop for trypsin inhibition

Kunitz-type trypsin inhibitors are ubiquitous in plants. They have been proposed to be a part of a defense mechanism against herbivores. Trypsin inhibitors also have potential applications in the biotechnology industry, such as in mammalian cell culture. We discovered that durian (Durio zibethinus) seed contains Kunitz-type trypsin inhibitors as identified by N-terminal sequencing and mass spectrometry. Eleven new trypsin inhibitors were cloned. The D. zibethinus trypsin inhibitors (DzTIs) that are likely expressed in the seed were produced as recombinant proteins and tested for trypsin inhibitory activity. Their inhibitory activity and crystal structures are similar to the soybean trypsin inhibitor. Surprisingly, a crystal structure of the complex between DzTI-4, the DzTI with the lowest inhibitory constant, and bovine trypsin revealed that DzTI-4 utilized a novel tryptophan-containing β1-β2 loop to bind trypsin. Site-direct mutagenesis confirmed the inhibitory role of this loop. DzTI-4 was not toxic to the HEK293 cells and could be used in place of the soybean trypsin inhibitor for culturing the cells under serum-free conditions. DzTI-4 was not toxic to mealworms. However, a mixture of DzTIs extracted from durian seed prevented weight gain in mealworms, suggesting that multiple trypsin inhibitors are required to achieve the antinutritional effect. This study highlights the biochemical diversity of the inhibitory mechanism of Kunitz-type trypsin inhibitors and provides clues for further application of these inhibitors.