Structural Basis For CD97 And EMR2 Recognition Of Complement Regulatory Factors CD55 And FHR1

Among GPCR superfamily,adhesion GPCRs(a GPCRs)used their extracellular adhesion domains to participate in cell-cell or cell-matrix interactions,thus to regulate cell adhesion.Meanwhile,a GPCRs,studies revealed that the binding signal of adhesion domains are transmitted to the followed GAIN domain and induced release of the Stachel peptide,which in turns docked to the transmembrane domain and triggered the intracellular G protein signaling.CD97 and EMR2 belongs to the ADGRE/EGF subfamily of a GPCRs,here we conducted structural and mechanism studies of the two pairs of inflammation related complex,CD97-CD55 and EMR2-FHR1.CD97 and its ligand complement decay accelerating factor CD55 are important binding partners in the human immune system.Dysfunction in this binding has been linked to immune disorders such as multiple sclerosis and rheumatoid arthritis,as well as various cancers.Previous literatures have indicated that these EGF domains at N-terminus of CD97 can bind to the N-terminal SCR domains of CD55;however,the details of this interaction remain elusive,especially why the CD55 binds with the highest affinity to the shortest isoform of CD97(EGF_1,2,5).Herein,we designed a chimeric expression construct with the EGF_1,2,5 domains of CD97 and the SCR_1-4domains of CD55 connected by a flexible linker,and determined the complex structure to 3.19?.The SAXS analysis together with SPR binding experiments confirmed the physiological relevant of the solved crystal lattice.We found the V137 of EGF₅involved in a hydrophobic interaction network with the SCR₁ residues,thus explained the binding specificity between CD55 and the CD97 isoforms.Our data reveal that CD97-CD55 adopts an overall anti-parallel binding mode involving the SCR_1-3domains of CD55 and all three EGF domains of CD97,this binding mode together with the binding kinetics revealed by SPR suggest the complex can withstand the tensile force in the circulating environment and transmit the signal to the downstream GAIN domain and produce the release of the Stachel peptide.Meanwhile,we are using single molecular methods to study the folding kinetic of the GAIN domain to further uncover the mechanism for the release of Stachel sequence.FHR1 and CD55 are both among regulatory proteins of complement.Studies have revealed that the interaction between FHR1 on necrotic cells and EMR2 on monocytes activates NLRP3 to release IL-1?,TNF?,IL-18 and IL-6.To uncover the molecular mechanism for the unconventional NLRP3 pathway regulated by the FHR1 we tried to solve the complex structure of FHR1-EMR2.We identified the binding site of FHR1 to the N-terminal SCR₁₂ domains by SPR measurements,and then made a similar chimera EMR2-linker-FHR1 construct.Although we can crystallize the chimera protein but can only solve density for the EMR2 fragment but the FHR1 fragment is missing,potential because of proteolysis during crystallization.We will optimize the linker between the two proteins and then continue the crystallization and try to solve the EMR2-FHR1complex which involved in the activation of NLRP3 inflammatory bodies,which not only deepens our understanding of this important immune response,but also will be used as a potential treatment for inflammatory body-related diseases.