Effects Of Non-inhibitory Sites Trp10 And Ile25 On Structure And Activity Of Buckwheat Trypsin Inhibitor

Protease Inhibitor(PI)mostly adopts a substrate like approach to insert the binding loop(Inhibitory active site)deep into the substrate binding pocket of the protease.Due to a specific binding mechanism(Clogged gutter mechanism)between the inhibitor and the enzyme,the protease inhibitor will not be hydrolyzed as fast as the substrate by the enzyme.The amino acid composition of the binding loop determines the type of inhibitor,while the amino acids in the non-binding ring portion distinguish the inhibitor from the substrate.However,whether and how the conserved structure of an inhibitor effect the activity of inhibitors has not been reported.Recombinant buckwheat trypsin inhibitor(rBTI)is a kind of trypsin inhibitor of Potato type I derived from buckwheat.Trp10 is far from the inhibitory activity center in rBTI,but forms hydrogen bonds with both the helical region and the Cterminus,which stabilizes the overall structure of rBTI.Considering the site-specific and highly conserved nature of Trp10,it may have an impact on its overall structure.Therefore,it is important to explore the effects of Trp10 and Ile25 interacting with Trp10 on the structure and activity of inhibitors.Mutants rBTI-W10 A and rBTI-I25 A were successfully obtained by site-directed mutagenesis of rBTI,and single bands was purified by ion exchange chromatography and gel chromatography purification.The properties of rBTI and mutants were determined by SDS-PAGE(Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis),Native-PAGE and CD(Circular Dichroism).Meanwhile,the degradation of rBTI,rBTI-W10 A and rBTII25 A by bovine trypsin was analyzed by both SDS-PAGE and HPLC(High Performance Liquid Chromatography).The tertiary structures of the mutants were predicted by Alpha Fold2 and the predicted structures were justified by PROCHECK and SAVES server.The predicted structures were visualized by pymol software.The structural differences between the mutants and rBTI were compared,and then analyzed the causes of the altered inhibitory activity of rBTI.The results showed that the rBTI-W10 A chromatographic behavior changed compared to rBTI.rBTI-W10 A was no longer bound to the ion column during ion exchange chromatography and showed a forward shift of the peak position in gel chromatography,while there was no significant difference between rBTI-I25 A and rBTI during purification.SDS-PAGE and Native-PAGE analysis revealed that the position of the rBTI-W10 A band was shifted upward,indicating that the inhibitor structure might become relaxed after the Trp10 mutation.However,the secondary structures of rBTI-W10 A and rBTI-I25 A did not change significantly by CD spectroscopy analysis.Determination of the inhibitory activity of the three against trypsin revealed that rBTI-W10 A activity was largely lost while rBTII25 A retained about 50% of the original inhibitory activity.SDS-PAGE and HPLC results showed that both mutants rBTI-W10 A and rBTI-I25 A were more readily hydrolyzed by bovine trypsin compared with rBTI,especially rBTI-W10 A was completely converted into a substrate for trypsin.Alpha Fold2 was used to predict the mutant structure.In comparison with the rBTI crystal structure,the RMSD of rBTI-W10 A differs the most from that of rBTI,and the mutation of Trp10 causes a more pronounced structural change.Hydrogen bonding analysis revealed that the number of hydrogen bonds in the inhibitor was significantly reduced after the Trp10 mutation.The binding network formed by these hydrogen bonds is a key factor in maintaining the structural stability of the inhibitor.It was also found that the Trp10 mutation not only deflected the Arg45 conformation at the P1 site of the inhibitor but also disrupted the hydrogen bond between Arg45 and bovine trypsin binding.This caused the loss of rBTI-W10 A inhibitory activity and even turned it into a substrate for the enzyme.This finding provides a theoretical basis for further understanding of the functional relationship between inhibitors and enzymes.