The solution structure and dynamics of an arc repressor mutant reveal premelting conformational changes related to DNA binding

The solution structure of the hyperstable MYL mutant (R31M/E36Y/R40L) of th e Are repressor of bacteriophage P22 was determined by NMR spectroscopy and compared to that of the wild-type Are repressor. A backbone rmsd versus th e average of 0.37 Angstrom was obtained for the well-defined core region. F or both Arc-MYL and the wild-type Are repressor, evidence for a fast equili brium between a packed ("in") conformation and an extended ("out") conforma tion of the side chain of Phe 10 was found. In the Mn mutant, the "out" con formation is more highly populated than in the wild-type Are repressor. The Phe 10 is situated in the DNA-binding beta-sheet of the Are dimer. While i ts "in" conformation appears to be the most stable, the "out" conformation is known to be present in the operator-bound form of Are, where the Phe 10 ring contacts the phosphate backbone [Raumann, B. E., et al. (1994) Nature 367, 754-757]. As well as DNA binding, denaturation by urea and high temper atures induces the functionally active "out" conformation. With a repacking of the hydrophobic core, this characterizes a premelting transition of the Arc repressor. The dynamical properties of the Arc-MYL and the wild-type A re repressor were further characterized by N-15 relaxation and hydrogen-deu terium exchange experiments. The increased main chain mobility at the DNA b inding site compared to that of the core of the protein as well as the reor ientation of the side chain of Phe 10 is suggested to play an important rol e in specific DNA binding.