Sequence-specific proton NMR assignment and secondary structure of the Arc repressor of bacteriophage P22, as determined by two-dimensional proton NMR spectroscopy

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Abstract
The Arc repressor of bacteriophage P22 is a DNA binding protein that does not belong to any of the known classes of such proteins. We have undertaken a 1H NMR study of the protein with the aim of elucidating its three-dimensional structure in solution and its mode of binding of operator DNA. Here we present the 1H nuclear magnetic resonance (NMR) assignments of all backbone protons and most of the side-chain protons of Arc repressor. Elements of secondary structure have been identified on the basis of networks of characteristic sequential and medium-range nuclear Overhauser enhancements (NOEs). Two .alpha.-helical regions have been found in the peptide regions 16-29 and 35-45. The ends of the helices could not yet be firmly established and could extend to residue 31 for the first helix and to residue 49 for the second. Immediately before the first helix, between residues 8 and 14, a region is present with .beta.-sheet characteristics dominated by a close proximity of the .alpha.-protons of residues 9 and 13. Because of the dimeric nature of the protein there are still two possible ways in which the NOEs in the .beta.-sheet region can be interpreted. If the NOEs are intramonomer, this requires a tight turn involving residues 10-12. Alternatively, if the NOEs are intermonomer, then an antiparallel .beta.-sheet would be implicated comprising two strands of different Arc monomers. While the data presently do not allow an unambiguous choice between these two possibilities, some evidence is discussed that favors the latter (.beta.-sheet between monomers). Since the N-terminal region of Arc is responsible for the sequence-specific recognition of its operator, our findings suggest the existence of a DNA binding motif in which a .beta.-sheet region is present.

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