Structural And Functional Investigation Of Transcriptional Regulatory Related Protein TGIF1 And SP 0782

As a performer of biological functions,proteins have a strict transcriptional regulation of their spatial structure and biological functions.Transcriptional regulation can make genetic information change according to a certain time program,and adjust with the change of internal and external environmental conditions,so as to maintain the normal growth and development process of organisms,or enable specific tissues and organs to play their corresponding functions.The regulation process realized with the participation of various regulatory elements such as promoters,enhancers,transcription factors,activators,and regulatory proteins such as RNA polymerase ??Pol??.In eukaryotes,this process requires additional transcriptional cofactors to facilitate the assembly of transcription initiation complexes and thus improve the transcription efficiency.Organism usually forms a complex network of gene expression regulation through the interaction of some specific DNA and a variety of transcription factors and transcriptional cofactors.If an important transcription factor or a cofactor is abnormal in their structure or function,it often leads to a wide range of gene transcriptional disorders,which in turn induces developmental defects and various diseases.The study of the molecular mechanism of structure-based protein transcription factors or transcriptional cofactors to bind with DNA will enable people to further understand some life phenomena as well as the cellular behavior and the occurrence mechanism of diseases.In this paper,we selected the human protein TGIF1 and Streptococcus pneumoniae protein SP₀782 as two transcriptional regulatory proteins.By using NMR,X-ray and some other analytical methods,The structure research of atomic resolution level was carried out,and their DNA identification mechanism was further explored by combining biochemical methods.The concrete study content as follows:1.Mechanism of HPE disease induced by TGIF-HD mutants P192A and R219C Studies have shown that TGIF-HD mutants P192A and R219C can induce HPE disease,but the teratogenic mechanism is not clear due to the lack of structural studies of TGIF1-HD.In functional studies,ITC found that the DNA-specific ability of the mutant P192A and R219C binding sequences decreased by 23 and 10 folds,respectively,relative to the wild-type TGIF 1-HD.In structural studies,CD have initially found that the a-helixs in the mutant is reduced.The structure of TGIF1-HD indicates that the domain contains three a-helices,similar to the reported HD homologous proteins.Based on the NMR structure,R219 is located in the a3,P192 is located in the loop region between al and a2,but the spatial distance is very close to the middle of the a3.The 1H-15N HSQC spectrum further confirm that the P192A mutation leads to the amino acid conformations in the al,a2,a3 region change,and thus the mutation may reduce the ability to bind to DNA by affecting the structure of TGIF1-HD.This part of the study explains the molecular mechanism of the decreased binding ability of two mutants of HPE to DNA from the structural view.2.The molecular mechanism of TGIF1-HD binding to DNA.NMR titration is a common method for studying protein-DNA complexes.However,when DNA titration of TGIF1-HD,it was found that the exchange rate between the free state and the bound TGIF1-HD molecule was mainly at a moderate rate?NMR time scale,?s level?,resulting in broadening of the NMR signal.The attenuation hinders the study of the nuclear magnetic structure of the composite.Here,on the one hand,we used HDX-MS to study the HD exchange process between free and bound TGIF1-HD,determined that the DNA binding interface is mainly concentrated on the a3 helix,but the conformation of ?1 and a2 There were also small changes before and after DNA binding.On the other hand,from the kinetic study,in the system,although the medium-speed exchange is dominant,the CEST experiment reveals that there is a slow exchange of the bound TGIF1-HD with an exchange rate of 130 s-1.This type of binding state is often of higher energy level and has important biological functions.3.The molecular mechanism of SP 0782 binding to ssDNA.The 3D structure of SP 0782 was analyzed by NMR.The structural comparison showed that SP₀782 is a homologous protein of transcriptional helper factor PC4,which may have the function of binding ssDNA.NMR titration and various biochemical experiments demonstrated this function and showed that SP 0782 binds different lengths ssDNA and adopts different binding modes.When SP₀782 is relatively high in molar ratio with ssDNA,it tends to form a large complex with ssDNA.X-ray was used to analyze the complex structure of SP 0782 and ssDNA-dT6 and dT12,and compared with the existing complex structure of SP 0782 and dT19G1,and clarified that the two major DNA binding regions in SP 0782 are combined.The role of ssDNA of different lengths is different.At the same time,by comparing with the structure of other proteins in the PC4 family,it was found that the structure of the two DNA binding regions is significantly different between prokaryotic and eukaryotic PC4 proteins,and these differences may be specific to prokaryotic and eukaryotic PC4 proteins.Biological function related.In summary,based on NMR and X-ray,combined with HDX-MS,CEST and other methods,the structure and function of two transcriptional regulatory proteins TGIF1 and SP 0782 were studied.We have successfully obtained the 3D structure of the above two proteins and explained their molecular mechanisms of interaction with DNA from the atomic level,enriching people's understanding of the transcriptional regulation process and the mechanism of related diseases,and helping people to screen for related diseases.The therapeutic drug target provides a theoretical basis for drug development..