NMR Structure And Function Study On ZF(4-5)domain Of The Human Protein INSM1

Human insulinoma-associated 1(INSM1)protein is mainly expressed during the development of neuroendocrine system,and plays an essential role in regulating neuroendocrine development.INSM1 is a transcription factor containing zinc-finger domains(ZFs),and ZF domain normally requires the coordination of one or more zinc ions with specific residues to take a finger form.ZFs regulate a variety of basal biological progresses including DNA replication,gene transcription,mRNA splicing,and translation in living organisms.Human INSM1 consists of 510 amino acids,and contains five C2H2-type ZFs at its C-terminus playing a role in recognizing the sequence-specific consensus DNA.At present,the structures of these ZFs in INSM1 and molecular mechanisms of the interaction between these ZFs and the consensus DNA remains unclear.In order to elucidate the structures and the DNA recognition mechanism of the ZFs in human INSM1,different constructs have been made including ZF(1-5),ZF(1-3),ZF(2-3)and ZF(4-5).In this thesis,we focused on the NMR structure and function study of ZF(4-5).First,high-yield,pure and stable ZF(4-5)protein samples with or without the13 C and 15 N isotope labeling were obtained after the expression condition optimization.ZF(4-5)exists predominantly as a monomer under the NMR study conditions.The well-dispersion of 2D 1H-15 N HSQC spectrum of ZF(4-5)and good signal-to-noise ratio indicated that ZF(4-5)was structurally well-folded in solution,suitable for structure determination using NMR spectroscopy.Then,the solution NMR structure of ZF(4-5)was solved.Each ZF in ZF(4-5)consists of one short antiparallel ?-sheet comprising two?-strands and one ?-helix,adopting a canonical ??? fold.Finally,the interaction between ZF(4-5)and the consensus DNA(5'-TG/TC/TC/TT/AGGGGG/TCG/A-3')was further studied by NMR titration.Chemical shift perturbations from five residues were clearly observed that indicated ZF(4-5)could bind this sequence-specific DNA.Further isothermal titration calorimetry(ITC)experiments confirmed this interaction,although in a relatively weak manner(Kd ~ 20 ?mol/L).In summary,our experimental results revealed that INSM1 ZF(4-5)participated in the recognition of sequence-specific DNA,but the weak interaction indicated that ZF(4-5)may not play the main role in DNA recognition.The structure and function study on other ZFs in INSM1 need further exploration.