Interaction And Molecular Recongnition Between Human Plasminogen Kringle 5 And VWA1 Domain In Cochlin

The growth,invasion,and metastasis of tumors mainly depend on the formation of new blood vessels.Human plasminogen Kringle 5 is a potential angiogenesis inhibitor,and it has broad application prospects in the treatment of vascular diseases and tumors.However,the current research on Kringle 5 mainly focuses on its mechanism of inhibiting angiogenesis,and there are few studies on its binding target and mechanism in vivo.The short peptides(IGNSNTL)with high affinity to Kringle 5 had been screened through the Ph.D.-7 phage display peptide library during the previous experiment,combined with BLAST tool to search for binding proteins of Kringle 5,sorted and screened by matching amino acids number,E value and protein function.It further investigated the binding mechanism of Kringle 5 with possible targets in vivo by using blot overlay,magnetic bead assay,enzyme-linked immunosorbent assay,and frontal affinity chromatography for experimental validation,with the help of molecular dynamics simulation in order to lay some foundation for the development of novel drugs for Kringle 5.The full text is as follows:(1)It was preliminarily determined that the vWA1 domain in the cochlin protein may be one of the binding targets of Kringle 5 by screening.The vWA1 domain was cloned and expressed as inclusion bodies in E.coli(BL21).After refolding by dilution and dialysis of inclusion bodies,the concentration of vWA1 was 0.46 mg/m L.(2)The interaction between Kringle 5 and vWA1 protein was proved through the blot overlay and magnetic bead assay.The enzyme-linked immunosorbent assay combined with biotin-avidin system further revealed the specificity and saturation of both,the value of the dissociation constant(K_d 0.16μM)indicated that there was a strong interaction between Kringle 5 and vWA1.The analysis of frontal chromatographic experiments showed that the binding constant(K_a 4.18×10~4 L/mol),and only one type of binding site existed between Kringle 5 and vWA1 protein.(3)Using homology modeling technology combined with dynamic optimization and evaluation to obtain the three-dimensional structure of vWA1.The final structure consists of 5β-sheets in the center surrounded by 6α-helices,which conforms to the Rossmann type of fold.It was found that the amino acids involved in the interaction of Kringle 5 and vWA1were Arg32,Arg69,Lys70,Leu71,Tyr72,and Tyr74,which were speculated to be binding through the lysine binding site.Meantime,Tyr72 in Kringle 5 contributed the most by the energy decomposition of the MM/GBSA method.After site-directed mutation,the van der Waals force contributed by Tyr72 increased by 57.2%,indicating that Tyr72 was indeed a critical amino acid residue.After vWA1 interacted with Kringle 5,loop1(47-50)and loop2(110-118)on the binding surface had obvious fluctuations,there were also large fluctuations in loop3(154-165),and part of theα6-helix(149-153)became random coil,indicating that the effect of inducing fit effect during the interaction.