The Study On Structure And Function Of BR3 Extracellular Domain In Its Selective Binding

B-cell activating factor(BAFF)and A proliferation induced ligand(APRIL)are important for the development of B cells.By binding to receptors on B cells,they activate intracellular signaling pathways,induce anti-apoptosis factors,and inhibit of the expression of apoptotic factors to significantly increase the number of B cells.Therefore,induced expression of APRIL and BAFF will lead to a rapid increase in the numbers of B cells,elevated the production of immune antibodies,and then trigger a series of autoimmune diseases.At present,the use of antagonists to block the binding of BAFF/APRIL to receptors on B cells and to reduce the number of B cells,has become an effective strategy for autoimmne disease.However,only the Belimumab antibody has been by FDA for marketing.Most other antibodies face the problems that the therapeutic effects cannot be expected,the immune function is too low,the infection rate of patients increases,and the price of drugs is expensive.Therefore,the study on the specificity of receptors must continue.BR3 is a specific receptor for BAFF,and elucidation of its structure and function will provide new ideas for the study of BAFF/APRIL receptor antagonists.Through the overlap of crystal structures,we found that the specific binding of BR3 is due to the steric hindrance effect of APRIL and the sequence outside Dx Lmotif in the cysteine-enriched region(CRDs)of the BR3.In order to confirm other roles of sequences other than Dx Lmotif in the specific binding of BAFF,we distinguished BR3 extracellular binding into two parts,BR3-1 and BR3-2,based on previous experiments.The corresponding Fc fusion protein was expressed through genetic engineering.By analyzing the affinity of the fusion protein and BAFF/APRIL,we found that BR3-2 binds not only to BAFF but also to APRIL.Therefore,the BR3-2 region is not a specific recognition function of the signal peptide of BAFF,and the specific binding performance of BR3 is mainly due to steric hindrance,and BR3-2 also contributes to the high affinity of BR3 to BAFF and improves its ability to combine.However,the affinity of BR3-2-Fc for APRIL is greater than that of BAFF,so it is speculated that the binding site of BR3-2 and APRIL may be more compact than BAFF.And BR3-1-Fc + BR3-2-Fc << BR3-Fc,we speculated that the binding of BR3-2 to BAFF may cause a certain degree of spatial allosteric effect to make BAFF more favorable for BR3-1-Fc binding.Next we simulated the binding sites of BR3-2 possibly in BAFF and APRIL by ZDOCK program and RDOCK scoring program calculations.We calculated the two binding sites with high scores and compared the binding sites to the BAFF-BR3 crystal structure in Chimera software.We found that the binding sites of BR3-2 in BAFF were far from BR3 and there is no steric hindrance effect.The binding site between BR3-2 and APRIL was compared with the crystal structure of APRIL-BCMA.We found that the optimal binding site of BR3-2 is closer to the Dx Lmotif binding pocket.There may be steric hindrance between BR3-1 and BR3-2 binding.Therefore,we concluded that BR3-2 can bind to both BAFF and APRIL,and BR3-2 does not work as a signal to direct the specific binding of BR3 to BAFF.The specific binding of BR3 to BAFF is due to the steric hindrance between BR3-2 amino acids residues and APRIL residues.BR3-2 can increase the binding affinity of BR3-1-Fc to BAFF.The binding of BR3-2 maybe cause an allosteric regulation,and make a contribution to the high binding affinity of BR3 to BAFF.BR3-2 can also bind to APRIL and have a high affinity to APRIL,and the optimized binding site of BR3-2 is the hydrophobic pocket of APRIL.There may be a steric hindrance between BR3-1 and BR3-2 binding.And it may be also a reason for BR3 selective binding.The amino acids sequence is important for both BR3 specific binding and its high affinity to BAFF.Although,the exact binding sites of BR3-2 to APRIL and the mechanism remind to identify.But we can conclude that BR3-2 is important for high affinity of BR3 and the specific binding of BR3 is due to the steric hindrance.We can use the structure of BR3 to design high affinity and selectivity antagonist to cure autoimmune diseases.