Structural Basis And Molecular Mechanism Of The Recognition Of Ligand ENF Peptides By The Binding Protein Of Silkworm

In the process of facing various extreme environmental pressures and pathogen infection,insects have evolved a unique immune system composed of humoral immunity and cellular immunity,which mainly relies on antimicrobial peptides and cytokines in blood.As a cytokine,insect ENF peptide is widely found in Lepidopteran insects,and has been shown to activate the immune response for self-protection of the body.Besides,there is also a class of ENF peptide binding proteins in insects,which can bind with ENF peptide and inhibit its activity,thus playing a negative role in immune regulation.This immune response co-regulated by ENF peptide and ENF peptide binding protein belongs to a feedback regulation of the body's self-protection.However,the current studies on the immune response involved in ENF peptide mainly focus on its upstream activation pathway,while the process of immune release is little known.Therefore,to elucidate the physiological role of ENF peptide-binding proteins in the immune response is of great significance for understanding the basic immune regulation process of insects,especially the immune suppression mechanism.In this study,we determined the structural basis and molecular mechanism of ENF peptide binding protein(BmENF-BP)bind with ENF peptide(PP)in silkworm,Bombyx mori,and revealed the role of ENF peptide binding protein(BmENF-BP)in the immune response.The main results are as follows:1.Crystal structure of ENF peptide binding protein from silkwormWe overexpressed the BmENF-BP using E.coli and purified the recombinant protein.Crystal of BmENF-BP through crystal screening and optimization,which diffracted by X-ray diffraction at SSRF and its dataset was collected at 3 A.The pahse information and the final three-dimensional structure of BmENF-BP were determined by SAD(Single-wavelength Anomalous Dispersion)and Molrep methods.The whole structure of BmENF-BP composed of three domains:the homologous domain of Pseudomonas odorata(PPD),the full a helical domain and full ? folded domain.The N-terminal domain consists of eight ? folds and four ? helices,and the ? folds and ?helices are connected by loop.The N-terminal domain contains a large number of loop regions which may be the binding region of ENF peptide.The full ? helical domain includes six ? helices and the full ? folded domain is a full ?-folded domain.These two domains showed a high similarity with the full ? helical domain and full ? folded domain of BmLP7,the classic 30K protein of B.mori.Therefore,BmENF-BP may have the same function as BmLP7 to bind to some lipid material.In order to determine the effect of the full ? helical domain and full ? folded domain of ENF peptide-binding protein on the folding and stability of N-terminal domain,we cloned and overexpressed the N-terminal domain of BmENF-BP,and purified and crystalized the recombinant protein.The three-dimensional structure of the N-terminal domain was determined at 2.2 A.Structural comparison of the single N-terminal structure and the N-terminal domain in BmENF-BP suggest that the two structures are highly similar with an RMSD value of 0.265 A,indicating that the N-terminal domain as a single domain is very stable.2?The structural basis of BmENF-BP protein bind with the ENF peptide.We tried co-crystallized the BmENF-BP protein and the N-terminal domain with PP,but all failed.Thus,we fused the coding sequence of PP with the BmENF-BP-N region by linker,and the fused protein of PP-ENF-BP-N were obtained.The diffraction data of the complex were collected by crystal screening,optimization and X-ray diffraction.When analyzing the structure of the complex,it was found that the N-terminal domain element could be successfully constructed,while the PP peptide could only be tracked due to the low electronic density.However,the approximate binding region of PP bound in the N-terminal domain has been identified,which is located at the N-terminal acidic area.Subsequently,molecular docking simulation of BmENF-BP and PP was performed based on the general binding region,and the complex model of BmENF-BP and PP was obtained.In terms of the structure model,the N-terminal surface of the N-terminal domain has acidic groove constituted by Asp and Glu,while the PP peptide has a large alkaline interface,which provides a suitable environment for the binding of BmENF-BP and ligand PP.The PP peptide have two conserved Cys residues,which form an intramolecular disulfide in the active PP(oxidized PP).In this study,we found that the BmENF-BP can only bind with the reduced PP,not the oxidized PP.The structural analyses suggested that the disulfide was broken in the reduced PP that exposed the alkaline residues,which facilitate the recognition and tightly binding of the alkaline interface in PP with corresponding acidic interface in the BmENF-BP.Moreover,we mutated the eight acidic residues in BmENF-BP that presumed to involve in the PP binding,and determined their binding ability towards PP peptide.There are four amino acids E19,E23,D25,and D 27 located at the N-terminal domain participated in the binding of reduced PP.3.The physiological role of the ENF peptide binding protein in the PP-induced immune response.Both PP and pathogenic bacteria were injected into the 3rd day of 5th instar larvae of B.mori.It was found that both could induce the expression of BmENF-BP gene and the downstream antimicrobial peptide genes,indicating that PP could activate humoral immunity.Quantitative results showed that the induced time of BmENF-BP gene is later that of the antimicrobial peptide genes.This suggests that BmENF-BP should play a role after humoral immunity is activated,which may be related to the elimination of immunity.In addition,the binding ability of PP to the receptor(BmE cells)was weaker than that of PP to BmENF-BP,suggesting that BmENF-BP could competitively bind PP from the receptor.Co-localization of BmENF-BP,BmE cells and PP revealed that the receptor on the BmE cell membrane can only bind to oxidized PP,while BmENF-BP can only bind to PP-reduced.BmENF-BP can be co-localized to the BmE cell membrane only when oxidized PP binding on the BmE cell membrane.This indicates that oxidized PP-Oxidized binding with receptors in the cell membrane results in a conformational change that mimes the conformation of PP in its reduced state(or directly into a reduced state),which could bind to BmENF-BP to eliminate immunity.Comprehensive the above results,there is a putative model of PP-controled humoral immune response:oxidized PP bind with receptors on the cell and activate humoral immunity,then the PP undergoes a conformation change induced by receptor that could be easily recognized and bound with BmENF-BP.The BmENF-BP bing the PP peptide from the cell receptor through a competitive combination,and remove the immune stimulation.For the transformation of PP from oxidized to reduced state,there are two hypothesis:(1)the cell receptor causes the transformation of PP from oxidation to reduced state;(2)Exogenous stimulation causes body fluid in a reduced state that induced the change of PP from oxidation to reduced state.To further verify the above conjectures,we identified the receptor proteins of PP by co-immunoprecipitation method combined with mass spectrometry,and finally screened out 10 possible target proteins for further research.