Crystal Structure Of Broad Regulator Ms6564and The Molecular Mechanism For Its Interaction With DNA

Protein-DNA interactions play critically important roles in many biological processes and this is particularly true with transcriptional regulation, for a regulator can function only when it successfully recognizes its target DNA.In recent years, master regulators, which broadly affect expression of diverse genes, have thought to play critical roles in bacterial growth and environmental adaptation. However, the structural basis for such a broad regulation remains largely unclear. More recently, Ms6564is characterized as a master regulator that regulates the expression of339potential target genes in Mycobacterium smegmatis. Therefore, the study of the DNA-binding mechanism of Ms6564will help us to fully understand the interaction between proteins and DNA.In this study, we solved the crystal structure of Ms6564in complex with its operator and undered the DNA binding mechanism of Ms6564from molecular level. Firstly, we designed several truncated Ms6564and the most stable truncated protein was used for crystallization. After selection and optimization of crystallization conditions, the crystal of native Ms6564and SeMet-Ms6564were obtained. Then we solved the crystal structure of Ms6564. The overall structure of Ms6564is similar to that of other TFRs. Strikingly, the a4helix of Ms6564, which is shorter than that of other TFRs, makes Ms6564more flexible. The variability of relative location between DNA binding domain (DBD) and Ligand binding domain (LBD) allows Ms6564broader choice of target DNA.Further more, we obtained the crystals of Ms6564-DNA, SeMet-Ms6564and brominated DNA-Ms6564complex and then we solved the cryatal structure of Ms6564-DNA complex. The structure indicated that two dimeric Ms6564docked on the opposite side of DNA and the symmetry axes of Ms6564dimers lie in the same plane and antiparallel to each other. In addition, the DNA maintained the B-form conformation, even if it was deformed and unwinded upon Ms6564binding. This suggests that the energy required for Ms6564to bind DNA is lower. In the interaction interface, the recognition helix of Ms6564inserts slightly into the DNA major and this implies that the protein can slide on the DNA more easily. Unexpectedly,11disordered water molecules bridged the interface of Ms6564-operator. Within the DNA-binding domain of Ms6564, Lys47and Gln48directly interacted with the bases of cognate DNA. Lys47was found to be essential for the specific DNA binding ability of Ms6564.Our study indicated that Ms6564can bind DNA with strong affinity but makes flexible contacts with DNA, which implis that Ms6564might slide more easily along the genomic DNA and specifically recognize a variety of target DNA. The DNA-binding approach of Ms6564that combines broadness and specificity together is the key to its ability to regulate expression of a large number of genes, and solving the crystal structure of Ms6564-DNA furthers our understanding of the mechanism of interaction between proteins and DNA.