Structure And Function Study Of Human Enteropeptidase

Objective Enteropeptidase(EP)initiates intestinal digestion through the proteolysis of trypsinogen to produce trypsin with catalytic activity.EP dysfunction can cause a series of pancreatic diseases,the most serious of which is acute severe pancreatitis.However,due to the lack of complete structural information,the molecular mechanism of EP activation and substrate recognition remains to be investigated.This study intends to reveal the molecular mechanism of EP involved in the occurrence and development of diseases by deeply exploring the spatial conformation and biological characteristics of EP,as well as its binding with substrates.Methods High-purity wild-type and mutant inactive single chain EP proteins were obtained by gene recombination,protein expression and purification.The two forms of EP proteins were activated by trypsin,and transformed them into an activated double chain mode.Different states of EP were detected by enzyme activity assay,western blotting,SDS-PAGE and native PAGE electrophoresis.Nafamostat was incubated with active EP.Trypsinogen was cross-linked with mutant EP.Subsequently,the interacting complexes of high-purity EP combined with inhibitor or substrate were obtained after the purification of size exclusion chromatography.The three-dimensional structures of inactive,activated,mutant,inhibitor bound and substrate bound EP were analyzed by single particle cryo-electron microscope.Results High-purity glycosylated wild-type and mutant single chain EP proteins were obtained by expression and purification in vitro.SDS/Native-PAGE electrophoresis and western blotting showed that in near physiological state,the molecular weight of the EP was about 150 k D,which was in the form of single chain.After being activated by trypsin,they transformed into the states of double chain containing light chain and heavy chain.The mutation of catalytic triad in light chain(Asp-His-Ser)could lead to the inactivation and the change of surface charge of EP.The cryo-EM structures of human EP in multiple states,covering the functional cycle spanning from inactive to active state and eventually to the inhibitor or substrate binding state were investigated,and with the inactive core region reached an atomic 2.7-?-resolution.The overall morphology showed that the SRCR,LDLR2 and CUB2 domains were surrounded the light chain.Compared to the inactive state,the conformation of the catalytic center,the loop of L1,L2 and LD of EP in active state were changed,and the IVGG sequence turned over about 180°.After binding with the inhibitor,the region of EP-core turned about 120° around the LCM domain,and the LDLR2 domain in the core region relocated and contacted with the LCM domain.The three-dimensional conformation of the substrate showed that the CUB2 domain of EP heavy chain binding with the substrate trypsinogen.In sum,the N-terminus of light chain induced the surface loop remodeling from inactive to active conformation,resulting in a highly dynamic and active EP.Then the heavy chain performed as a hinge for ensuring the flexibility of light chain for substrate recruitment and subsequent cleavage.Conclusion The molecular mechanism model provided by a series of EP structures,including the different states of its functional cycle,and its interaction with inhibitor or substrate,established a foundation for the study of EP structure-function relationships.These discoveries provided clues for the treatment of EP-related diseases,drug research and development,having profound pathobiological and therapeutic targeting implications.