Study On The Binding Specificity Of Tudor Domains In HTDRD Proteins And PIWI Proteins With Different Methylated Arginine States

piRNA?PIWI-interacting RNA?is a member of the large non-coding RNA family,which usually combines with PIWI?P-element-induced wimpy testis?proteins specifically to form the piRISC?piRNA-induced silencing complex?involved in the transcriptional regulation of normal gametogenesis and reproduction.It is reported that there are arginine methylation modifications in the N-terminal of PIWIs,which provides binding sites for TDRDs?Tudor domain containing proteins?.And the TDRDs-PIWI is the key prerequisite for PIWI to perform its biological function normally.Therefore,it is of great value to study the interaction between the two proteins.At present,the recognizition mechanism of TDRDs-PIWI has been extensively studied.However,it is still unclear for the interactions of part TDRD proteins.TDRDs contain Tudor domain,which is able to bind methylarginine proteins.Among the 13 members of human TDRDs,specific mechanism of the interactions between TDRD2?TDRD3?SND1?staphylococcal nuclease domain-containing 1?and PIWIs have been identified and analyzed by structural analysis,while other TDRDs are poorly understood both in binding selectivity and binding mechanism,which need to be further studied.Based on the identified binding mechanism of SND1,we speculate that the other TDRD proteins may share the same binding mechanism as SND1.That is their Tudor domains might utilize the conservative aromatic cage consisting of aromatic amino acids to recognize the methylated arginine of PIWI proteins.Therefore,in this thesis,we compared the amino acid sequences of all the Tudor domains in hTDRDs and found that some Tudor domains do not contain completely conservative aromatic cage.According to the previous studies and the sequence alignment analysis,we performed further study with five proteins,including TDRD1,TDRD4,TDRD2,TDRD7 and TDRD9.We first designed the mutants for the conservative amino acid residues of the five proteins and successfully constructed the expression vector of 29 mutants through gene recombination.Then we got 30 soluble proteins of mutants and their widetypes.Subsequently,we used FP assay?Fluorescence Polarization Assay?to detect the binding affinity between these soluble proteins and PIWIL1?PIWI-like protein 1?peptides with different methylated arginine states.Based on the results of FP?Fluorescence Polarization Assay?,we co-crystalized Tudor domain proteins with different binding ligands or made homologous modeling.Based on above results,we analysed the binding mechanism of TDRD proteins and PIWIL1 with different methylated arginine states.The results showed that TDRD1₁,TDRD1₂ and TDRD1₃ prefered to bind to PIWIL1 with SDMA?Symmetrical di-methyl arginine?modification and the binding mechanism was similar to that of SND1.Only TDRD4 5 showed a strong binding affinity to PIWIL1 modified by methylated arginine in TDRD4 with a preference for ADMA?Asymmetrical di-methyl arginine?modification of PIWIL1,just like TDRD9.The binding mechanism of TDRD4₅ may be the same as that of SND1 while the binding mechanism of TDRD9 seems to be different.At the same time,we carried out co-crystallization experiment using Tudor domain protein and PIWI proteins with different modifications,but we failed to obtain the crystals of complex.We further performed a co-crystallization using the antibody of TDRD2.prepared by our collaborators and the Tudor domain of TDRD2,and successfully obtained the complex structure of TDRD2-Fab,which lays the foundation on our futher study on the structure of Tudor domains and other proteins.