Study On The Interaction Between Protein And Ligand Through NMR Spectroscopy

Protein as the executor of cell biological functions mainly depends on their three-dimensional structures,which include both native folded proteins and intrisically disordered proteins(IDPs).In living cells,different proteins can carry out diverse physiological functions.Protein three-dimensional structure determination contributes to elucidate the molecular mechanism by which protein functions.Therefore,the study of protein structure has always been an important part in the field of life science.The interaction of protein and ligand molecules is the basis of the cells to achieve specific functions.Protein-ligand interaction is one of the most important biological events involved in all biological processes of cell cycle,such as signal transduction,energy metabolism,gene regulation,enzyme catalysis,immune reaction and other processes associated with binding of related protein to substrate,ligand or small molecule.The study of protein-ligand interaction and its network are not only helpful for us to understand the molecular mechanism of life activities,but also of great significance for the diagnosis and treatment of clinical diseases.Nuclear Magnetic Resonance(NMR)spectroscopy is a powerful tool used for studying protein structure and its interaction with other biological biomolecules,especially for proteins in solution.In this thesis,human protein GAS7-SH3 and nanobody Nb26 from Vicugna pacos with folded structure and TGIF1-RD2(256-401)predicted to be an intrinsically disordered protein(IDP)are picked up as study targets.Intensive studies focusing on their structures and functions were carried out using NMR spectroscopy and other biophysical/biochemical techniques,the results are briefly presented as follows:1.Structure determination and screening of specific ligand peptides of human protein GAS7-SH3.In human,three GAS7 isoforms can be expressed from a single GAS7 gene,while only the longest isoform,GAS7c,possesses an SH3 domain at the N-terminus.To date,the structure and function of GAS7-SH3 domain are still unclear.Here,we reported the solution NMR structure of GAS7-SH3 domain,which displays a typical SH3 ?-barrel fold comprising five ?-strands and one 310-helix.Structural and sequence comparison showed that GAS7-SH3 shares high similarity with ABL-SH3 domain,which binds to a high-affinity proline-rich peptide P41 in a canonical SH3-ligand binding mode through two hydrophobic pockets and a specificity site in the RT-loop.However,unlike ABL-SH3,only six residues in the RT-loop and two residues adjacent to but not in the two hydrophobic pockets of GAS7-SH3 showed significant chemical shift perturbations in NMR titrations,suggesting a low affinity and a noncanonical binding mode of GAS7-SH3 for P41.Furthermore,four peptides selected from phage display libraries also bound weakly to GAS7-SH3,and the binding region of GAS7-SH3 was mainly located in the RT-loop as well.The study implied that although GAS7-SH3 adopts a typical SH3 fold,it probably possesses distinctive ligand-binding specificity.2.Homologous oligomerization of human protein TGIF1-RD2(256-401)and molecular mechanism of its recognition of corepressor SIN3A-PAH2.The human transforming growth-interacting factor TGIF1 is an essential regulatory protein for cell differentiation.An in-depth study on the structure-function relationship of TGIF-HD domain,which functions on specific DNA recognition,had been carried out by our group.Here,we focused on the C-terminal domain of TGIF1,termed TGIF1-RD2,which participates in multiple cell signal pathways.The protein sample of TGIF1-RD2(256-401)was successfully obtained by recombinant protein technique.Structural studies showed that TGIF1-RD2(256-401)was an IDP in solution and contain a certain proportion of ?-helical structure at its C-terminal between residue 376 and 401.The Y2H experiments verified that TGIF1-RD2(256-401)could undergo homologous oligomerization in cell,and the homologous oligomerization was mediated primarily by the region between residue 376 and 401,wherein the residues of F379,L381,L382,V385 and L387 were essential for its homologous oligomerization.In addition,further Y2H experiments suggested that the region between 376 and 401 residues within TGIF1-RD2(256-401)was required for the interaction with SIN3A-PAH2.NMR titration and mutations indicated that the hydrophobic residues F379,L382 and V385 were the key sites for the interaction of TGIF1-RD2(256-401)with SIN3A-PAH2.This study indicates that the C-terminal between residue 376 and 401 of TGIF1-RD2(256-401)is not only essential for the homologous oligomerization but also the interaction with SIN3A-PAH2.The crucial residues for the homologous oligomerization of TGIF1-RD2(256-401)and its interaction with SIN3A-PAH2 are highly consistent.Whether there is any competition between the homologous oligomerzation of TGIF1-RD2(256-401)and its interaction with SIN3A-PAH2,as well as the deep connection between the two biological functions remain to be further studied.3.Structural insights into the mechanism of nanobody 26(Nb26)binding to AFB1.Nanobody is the smallest antigen-binding entity and has gained increasing attentions due to its convenience in biotechnological applications.Recently,numerous structural models of Nbs binding to protein antigens have been well characterized,but there are few studies on the structure of Nbs that recognize small haptens.We reported here the solution structure of Nb26 against the hapten AFB1 determined by NMR spectroscopy.The overall structure of Nb26 displays a typical folding pattern of the heavy chain variable region(VH).Fluorescence and NMR titration showed that Nb26 had a high affinity with AFB1,and the FR2 and CDR3 of Nb26 made an important contribution to the AFB1 recognition,suggesting a novel binding pattern.Our studies provide useful structural information for further development and modification of hapten-specific NbsIn summary,the structure and the interaction with the specific ligands of GAS7-SH3,TGIF1-RD2(256-401)and nanobody Nb26 were investigated in this thesis through liquid NMR.Structural study and ligand identification of GAS7-SH3 domain revealed a typical SH3 fold but a non-canonical ligand-binding mode and deepened the understanding of the functional mechanism of GAS7-SH3.Studies on homologous oligomerization of TGIF1-RD2(256-401)and the interaction with corepressor SIN3A-PAH2 deepen the understanding of the molecular mechanism of TGIF1 as a "hub" protein in the cellular signal transduction network,and provide the structural basis for the diagnosis and drug discovery of related diseases.The molecular mechanism of Nb26 to recognize AFB1 revealed a novel small molecular hapten binding pattern,which subsequently provides guidance for the functional modification of Nb26 at the molecular level.