第八屆生物物理新知研討會
The 8th Symposium on Recent Advances in Biophysics
P44
/Probing the DNA kink structure induced by the hyperthermophilic chromosomal protein Sac7d using site-directed mutagenesis and x-ray crystallography
Chin-Yu Chen (陳青諭),1,2 Ting-Wan Lin (林婷婉),1 Chia-Cheng Chou (周家丞),1 Tzu-Ping Ko (柯子平)1 and Andrew H.-J. Wang (王惠鈞)1,3.1Institute of Biological Chemistry, Academia Sinica, 2Dept. Chemistry, National Taiwan University, 3Institute of Biochemical Sciences, National Taiwan University.
The protein Sac7d belongs to a class of small chromosomal proteins from the hyperthermophilic archaeon Sulfolobus acidocaldarius. The protein has high thermal, acid and chemical stability. It binds DNA without marked sequence preference and increases the Tm of DNA by ~40 o C. The crystal structures of Sac7d complexed with two DNA sequences (GCGATCGC and GTAATTAC) have been solved to high resolution as described previously (Nature,392, 202-205, 1998). This early study shows that Sac7d causes a single-step sharp kink in DNA (~60o) via the intercalation of both Val26 and Met29. In this paper, site-directed mutagenesis techniques were applied to change the side chains of Val26 and Met29 systematically to either smaller or larger sizes. The crystal structures of Sac7d mutants (V26A, M29A, V26A/M29A, and V26F/M29F) and GCGATCGC complexes have been determined and refined at 2.25 Å, 2.2 Å, 1.9 Å and 2.15 Å, respectively. The DNA binding patterns and unit cells of the single mutants are similar to those of wild type. For the two double mutants, the unit cells of crystals are both smaller than that of wild type and the crystal structures reveal some surprising unprecedented change. Instead of binding at the C2G step as observed in the wild type Sac7d, the V26A/M29A protein binds at the G3A step. The hydrophobic side chain of the residue Phe26 in V26F/M29F-GCGATCGC complex intercalates deeply into the gap of DNA bases by - stacking interactions with G3 base, whereas the side chain of Phe29 can not insert itself into C2pG3 most likely due of the steric hindrance effect. The crystal data of Sac7d mutants and DNA complexes (M29F-GCGATCGC/GTAATTAC, V26F/M29F-GTAATTAC) have been collected and shown to have different unit cells. Thermodynamic studies show that all mutants still bind to DNA, but with weaker affinity. The determinations of the crystal structure of these Sac7d mutant and DNA complexes will help elucidate the molecular basis of protein-induced DNA kinks, which is poorly understood so far.