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 the Arc repressor of bacteriophage P22 was determined by NMR spectroscopy and compared to that of the wild-type Arc repressor. A backbone rmsd versus the average of 0.37 A was obtained for the well-defined core region. For both Arc-MYL and the wild-type Arc repressor, evidence for a fast equilibrium between a packed ("in") conformation and an extended ("out") conformation of the side chain of Phe 10 was found. In the MYL mutant, the "out" conformation is more highly populated than in the wild-type Arc repressor. The Phe 10 is situated in the DNA-binding beta-sheet of the Arc dimer. While its "in" conformation appears to be the most stable, the "out" conformation is known to be present in the operator-bound form of Arc, 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 temperatures 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 Arc repressor were further characterized by 15N relaxation and hydrogen-deuterium exchange experiments. The increased main chain mobility at the DNA binding site compared to that of the core of the protein as well as the reorientation of the side chain of Phe 10 is suggested to play an important role in specific DNA binding.
D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D013696:Temperature; D014508:Urea / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics; D054334:Viral Regulatory and Accessory Proteins