The essential transfer protein TraM binds to DNA as a tetramer

Abstract

The TraM proteins encoded by F-like plasmids are sequence specific DNA binding proteins that are essential for conjugative DNA transfer. We investigated the quarternary structure and the DNA binding properties of the TraM wild-type protein of the resistance plasmid R1 and two mutant forms thereof. Size-exclusion chromatography and differential scanning calorimetry showed that purified Tram protein (amino acids 2-127) forms stable tetramers in solution. A truncated version of the protein termed TraMM26 (amino acids 2-56) forms dimers. Thus, the dimerization and tetramerization domains can be assigned to the N-terminal and C-terminal domains of TraM, respectively. Further analyses using chemical cross-linking and light scattering corroborated the preferentially tetrameric nature of the protein but also suggest that Tram has a tendency to form higher aggregates. Band-shift and fluorescence spectroscopy investigations of TraM-DNA complexes revealed that the Tram protein is also tetrameric when bound to its minimal DNA binding site. The deduced binding constant in the range of 108 M-1 demonstrated a very strong binding of Tram to its preferred DNA sequence. Secondary structure analysis based on CD measurements showed that Tram is mainly α- helical with a significant increase in α-helicity (48 to 58%) upon DNA- binding, indicating an induced fit mechanism.