Identification of the DNA binding surface of H-NS protein from Escherichia coli by heteronuclear NMR spectroscopy

Abstract

The DNA binding domain of H-NS protein was studied with various N-terminal deletion mutant proteins and identified by gel retardation assay and heteronuclear 2D- and 3D-NMR spectroscopies. It was shown from gel retardation assay that DNA binding affinity of the mutant proteins relative to that of native H-NS falls in the range from 1/6 to 1/25 for H-NS60-137, H-NS70-137 and H-NS80-137, whereas it was much weaker for H-NS91-137. Thus, the DNA binding domain was defined to be the region from residue A80 to the C-terminus. Sequential nuclear Overhauser effect (NOE) connectivities and those of medium ranges revealed that the region of residues Q60-R93 in mutant protein H-NS60-137 forms a long stretch of disordered, flexible chain, and also showed that the structure of the C-terminal region (residues A95-Q137) in mutant H-NS60-137 was nearly identical to that of H-NS91-137. 1H and 15N chemical shift perturbations induced by complex formation of H-NS60-137 with an oligonucleotide duplex 14-mer demonstrated that two loop regions, i.e. residues A80-K96 and T110-A117, play an essential role in DNA binding. Copyright (C) 1999 Federation of European Biochemical Societies.