Structural And Functional Studies Of DNA Binding Proteins INSM1 And SP₀782 Through NMR Spectroscopy

DNAis the main genetic material in living organisms,storing and encoding almost all the information about the living activities.The basic biological events of DNA,such as replication,recombination,transcription and repair,all require the participation of a large number of DNA-binding proteins from diverse protein families.Therefore,the function of DNA binding proteins is crucial to the organism.It is very important to study the function of DNA binding proteins and explain the molecular mechanism of their function from the perspective of structure.In this study,in-depth investigations focusing on the structure and function of human INSM1 and SP 0782 from Streptococcus pneumoniae were carried out using NMR spectroscopy and other biophysical/biochemical methods.Human insulinoma-related protein INSM1 is a transcriptional repressor that plays a key regulatory role in the development of the neuroendocrine system.Abnormal function of INSM1 can lead to a variety of neuroendocrine system diseases,such as pancreatic cancer and neuroblastoma.Human INSM1 is composed of 510 amino acids,and contains five tandem C2H2 zinc fingers?ZF1?ZF5?in its carboxyl terminal part.In transcriptional regulation,the five ZFs are responsible for binding cis-regulatory elements in gene promoter,that is,a double-stranded DNA?dsDNA?segment containing the sequence 5'-TG/TC/TC/TT/A GGGGG/TCG/A-3'.However,the mechanism by which the ZFs interact with DNA is still unclear.In this study,three INSM1 truncated proteins including ZF1-2?containing the first two ZFs?,ZF3?containing the third ZF?and ZF1-5?containing all five ZFs?were successfully produced using Escherichia coli expression system.Static light scattering and NMR relaxation experiments showed that the three truncated proteins exist as monomers in solution.Because ZF1-2 and ZF3 displayed excellent stability and signals in NMR spectra,while ZF1-5 was unstable and degraded in one day after purification,ZF1-2 and ZF3 were selected in further structural study using NMR.Through a series of 2D and 3D NMR experiments,the chemical shifts of ZF1-2 and ZF3 were assigned with the overall completion rate of 92.4%and 95.6%,respectively.Based on the chemical shift assignments,the structural models of ZF1-2 and ZF3 were preliminarily calculated using the CS-Rosetta software.The structure models showed that ZF2 and ZF3 have typical C2H2 ZF 3D structures,each containing one ?-helix and two antiparallel ?-strands.Although the structure model of ZF1 was also composed of one?-helix and two antiparallel ?-strands,its structure model was poorly convergent.In the research following this paper,NOE constraint information should be added and the 3D structures of ZF1-2 and ZF3 should be solved using the traditional NMR structure calculation method.Although ZF1-5 protein has poor stability and is not suitable for the study of NMR structure,its function can be preliminarily studied by biochemical methods.Thus,the interaction between ZF1-5 and sequence specific dsDNA were investigated by electrophoresis mobility shift assay.The results showed that ZF1-5 could interact with sequence-specific dsDNA,but the binding affinity was weak.Although the binding affinity of the INSM1 ZFs to selected dsDNA was weak under the conditions used in this study,there may be other factors that can enhance the binding in cells or in vivo,which need to be further explored.SP₀782 from S.pneumoniae belongs to the PC4-like single-stranded DNA?ssDNA?binding protein family,and plays a role in DNA repair and bacterial uptake of exogenous DNA.SP 0782 has a homo-dimer structure,and the two subunits can individually bind to a short ssDNA?e.g.,dT6?,or cooperatively bind to a long ssDNA?e.g.,dT12 or dT19G1?.The DNA-binding interface of single subunit can be divided into two regions,namely DNA binding region 1 and 2?DBR1 and DBR2?.The two DBRs act differently in binding to different lengths of ssDNA.On the other hand,two or more SP₀782 dimers can bind with one dT19G1 molecule to form higher-order complexes.In this study,a new amino acid site,leucine 20?L20?,was found to play an important role in the interaction between SP 0782 and ssDNA.Although L20 is not located in DBRs,when it was mutated to alanine,the affinity between the mutant protein L20A and dT19G1 was significantly reduced,and the formation of high-order SP₀782:dT19G1 complex was also affected.Further studies found that the side chain of L20 interacts with the side chains of several amino acid residues on the backside of DBRs in apo SP₀782 structure,and L20A mutation leaded to significant changes of the line shape in circular dichroism?CD?and the chemical shifts in NMR spectrum.The most significantly affected residues in the NMR spectrum included F39 and R49 in DBR2 and K60 in DBR1.Therefore,it can be concluded that L20A mutation caused local conformational change of SP₀782,indirectly affecting the conformation of the DNA binding interface,and thus weakening the affinity of SP.0782 to ssDNA.The study of INSM1 and SP₀782 in this paper deepened the understanding of the functional mechanism of the two proteins.The exploration on the preparation of INSM1 protein samples and the assignments of NMR chemical shifts laid a foundation for the further study on the interaction mechanism between INSM1 and DNA or other biological macromolecules.The study on the L20 site of SP₀782 revealed the effect of local conformational changes of SP₀782 on the function of the whole protein,and suggested that a new amino acid site might be used to design targeted drugs for inhibiting bacterial growth.