Application Of Molecular Docking Simulation Strategies To The Study Of Interaction Between Porcine CR1-like And C3b

Veterinary studies have shown that some mammalian erythrocytes have an immune adhesion function based on the molecular basis of the Complement receptor I-like(CR1-like)present on the surface of the erythrocyte membrane.One of the ligands of CR1-like is C3 b formed by cleavage of C3 in serum,which is specifically bound by erythrocytes via CR1 to transport sensitised antigens or immune complexes(Immune Complex,IC)to innate immune cells for clearance and to inhibit over-activation of complement.Erythrocytes specifically bind C3 b via CR1 to transport sensitised antigens or immune complexes(Immune Complex,IC)to innate immune cells for clearance and to inhibit over-activation of complement.The specific molecular mechanisms involved have only been investigated in primates and are less well understood in other mammals.The group has shown that porcine CR1-like can bind C3 b,but information on the structure,binding site and binding/dissociation constants of porcine CR1-like and C3 b is still unclear.These issues have hindered the in-depth study of porcine red blood cell immunity and limited the development and application of red blood cell drug carriers associated with it.Based on the above questions,this experiment was conducted as follows:1.Construction of porcine CR1-like extracellular fragment and C3 b interaction model and simulated mutation analysis:A tertiary structural model of the porcine CR1-like extracellular fragment(SCR 12-14 and SCR 19-21)and porcine C3 b was constructed separately using homology modelling techniques.The three-dimensional structural models of CR1-like and C3 b molecules were constructed using homologous proteins with known structures as reference templates,and the structurally conserved regions and corresponding framework regions of the homologous proteins were identified by multiple sequence comparisons;backbone copying,side chain construction,missing residue complementation and loop region optimization were applied to construct the three-dimensional structural models of CR1-like and C3 b molecules,and the generated structures were optimized using Gromacs kinetic simulation software.The results of the evaluation were combined with the PROCHECK tool to analyse the rationality of the stereochemical structure of the model,Verify 3D to evaluate the sequence-structure compatibility of the model and Prosa to assess the rationality of the energy of the model,and the optimal 3D structures of the CR1-like and C3 b molecules were output.Using CR1-like and C3 b molecules as research objects,the molecular docking simulation strategy was applied to predict the binding mode of porcine CR1-like and C3 b by applying two models of free docking and residue-restricted docking,respectively,and to propose a model for the interaction between porcine CR1-like and C3 b.The proposed CR1-like and C3 b interaction model was predicted using the simulated alanine mutation scanning technique to clarify the interaction information such as the binding mode,key sites and affinity parameters of their interaction.2.In vitro expression and affinity determination of porcine CR1-like extracellular fragments with C3b:The porcine CR1-like extracellular fragment(SCR 12-14 and SCR 19-21)was expressed in vitro with C3 b protein using eukaryotic and prokaryotic expression techniques respectively,and the kinetic assay of recombinant protein porcine CR1-like and C3 b was performed using surface plasmon resonance technique to determine the baseline affinity of their interaction.3.Mutational analysis of porcine CR1-like extracellular fragment interaction with C3 b and validation of model structures:Mutants of porcine CR1-like extracellular fragments(SCR 12-14 and SCR 19-21)were expressed in vitro by eukaryotic expression techniques,and the affinity of porcine CR1-like mutants to C3 b was examined by surface plasmon resonance techniques,and the changes in affinity of the reciprocal proteins before and after the mutations were compared and the rationality of the pre-constructed model was verified to reveal the mechanism of action of porcine CR1-like binding to C3 b at the molecular level.like binding mechanism of C3 b at the molecular level.The experimental results are as follows:1.3D structural models of porcine C3 b,CR1-like SCR 12-14 and CR1-like SCR 19-21 were constructed by homology modelling.The interaction models of C3b-CR1-like SCR 12-14 and C3b-CR1-like SCR 19-21 were constructed and optimized by molecular docking and molecular dynamics simulations with reference to the crystal structure of the human CR1-C3 b complex.The prediction of key residues for the interaction of CR1-like and C3 b was performed by binding interface analysis.CR1-like had a total of12 residues that were considered as potential key residues by the prediction tool,of which amino acid residues Tyr761,Arg763,Phe765,Thr789 and Val873 of SCR12-14 and amino acid residues Tyr1210,Asn1244,Val1249,Val1322 and Val1339 are conserved at the binding interface of the human C3b-CR1 SCR 15-17 complex.Among the above residues,Tyr761-Tyr1210 and Val873-Val1322 are conserved allelic residues,and their equivalents on human CR1(Tyr978,Val1090)are hotspot residues validated by mutagenesis analysis.2.E.coli engineered bacteria capable of stably expressing porcine C3 b α-chain and β-chain proteins were obtained by Prokaryotic expression technology and successfully expressed porcine C3 b protein.In addition,two recombinant proteins of porcine CR1-like SCR 12-14 and SCR 19-21 fragments were successfully obtained by eukaryotic expression technique using 293 F cells.The affinity of porcine C3 b α-chain to CR1-like was measured by SPR at 1.06±0.02 μM and the affinity of C3 b β-chain to CR1-like was38±2 μM,which provided a control for subsequent mutagenesis assays.3.The target proteins of porcine CR1-like mutants 761 Y,873V,1210 Y,1322V and 1185K-K were obtained by eukaryotic expression techniques,and the affinity of the five mutants to the porcine C3 b α-chain was examined by SPR.The results showed that the affinity of mutants 761 Y,873V,1210 Y and1322V to the porcine C3 b α chain was reduced after the mutation occurred compared to the normal porcine CR1-like,indicating that the porcine CR1-like residues Tyr761,Tyr1210,Val873 and Val1322 are at the binding interface between porcine CR1-like and C3 b and have a significant effect on the porcine Mutations in 1185K-K had little effect on the binding of porcine CR1-like SCR 19-21 to C3 b,suggesting that Lys1185 and Lys1187 make little contribution to the binding of CR1-like SCR 19-21 to C3 b.Combined with the above experimental results,this study confirms that both porcine CR1-like SCR12-14 and SCR 19-21 can interact with porcine C3 b.In the interaction between porcine CR1-like and C3 b,the affinity of the α-chain of C3 b for CR1-like was more than 30-fold higher than that of the β-chain,and for C3 b its α-chain was the main source of affinity.A significant decrease in the affinity of porcine CR1-like for C3 b was detected after mutation of the amino acids Tyr761,Tyr1210,Val873 and Val1322 to Ala.This suggests that the interaction of porcine CR1-like with C3 b is a multisite interaction and that Tyr761,Tyr1210,Val873 and Val1322 are key residues involved in the interaction of porcine CR1-like with C3 b.