SOLUTION STRUCTURE OF THE DNA-BINDING PROTEIN SAC7D FROM THE HYPERTHERMOPHILE SULFOLOBUS-ACIDOCALDARIUS

The Sac7 proteins from the hyperthermophile Sulfolobus acidocaldarius are a heterogeneous mixture of small, thermostable, nonspecific DNA-bi nding proteins. One of these proteins, Sac7d, has been overexpressed i n Escherichia coli to provide a homogeneous preparation for structure, stability, and function studies. We present here essentially complete sequence-specific H-1 NMR assignments for Sac7d, a delineation of sec ondary structural elements, and the high-resolution solution structure obtained from a full relaxation matrix refinement. The final structur e provides an excellent fit to the NMR data with an NOE R-factor of 0. 27 for backbone NOEs. The structure has a compact globular fold with 8 2% of the sequence involved in regular secondary structure: an antipar allel two-stranded beta-ribbon with a tight turn, followed by a short 3(10) helix, an antiparallel three-stranded beta-sheet, another short 3(10) helix, and finally four turns of alpha-helix. The amphipathic cr -helix packs across the hydrophobic face of the three-stranded beta-sh eet in an open-faced sandwich arrangement with at least one turn of th e helix exposed beyond the sheet. The hydrophobic face of the beta-rib bon packs against a corner of the twisted beta-sheet. The single trypt ophan responsible for the 88% fluorescence quenching upon DNA binding is exposed on the surface of the three-stranded beta-sheet. Lysines 5 and 7, whose monomethylation may be associated with enhanced thermosta bility, are highly solvent exposed along the inner edge of the two-str anded ribbon. The structure of Sac7d differs in many respects from tha t reported for the homologous native Sso7d [Baumann et al. (1994) Natu re Struct. Biol. 1, 808] with a backbone RMSD greater than 3.0 Angstro m, largely due to the packing and length of the C-terminal alpha-helix which may be important in Sac7d DNA binding.