The Enfolding Arms of EcoRI Endonuclease as Probed by ESR Experiments

Abstract

Our research focuses on adducing general principles applicable to site-specific protein-DNA interactions by linking function to structural, thermodynamic and dynamic properties. We use as a model the interaction of EcoRI endonuclease with specific, miscognate (EcoRI∗), and nonspecific DNA sequences. The crystal structure of the specific complex shows that the EcoRI “arms”, invisible (disordered) in the structure of the apoenzyme, enfold cognate DNA upon binding. We are using four pulse Double Electron-Electron Resonance (DEER) FT-ESR experiments to map distances and distance distributions between nitroxide spin labels placed on cysteine-substituted residues in the two “arms” of the EcoRI homodimer, between Cu2+ ions bound near the active sites, and between nitroxide to Cu2+ positions. Our data show that the mean point-to-point distances between the “outer arms”, between the “inner arms” and from the “outer arm” to the main domain are the same in specific, EcoRI∗, and nonspecific complexes. This implies that the EcoRI arms must enfold the DNA in all three classes of complexes. However, an increase in the breadth of distance distributions is observed for noncognate complexes relative to that observed for the tightly complementary specific complex. These results are consistent with inferences from our thermodynamic analyses that the equilibrium ensemble of conformational microstates is larger for noncognate than specific complexes. Our continuous wave (CW) ESR experiments probing the dynamics of the arms support this hypothesis. Nonspecific complexes have been shown to have an important function in accelerating the location of correct recognition sites. It is striking that the EcoRI arms also embrace the DNA in the sliding nonspecific complex.