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
1
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
2
Dept. Chemistry, National Taiwan University, Taipei, Taiwan
3
Institute of Biochemical Sciences, National Taiwan University Taipei, Taiwan
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. 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, M29F and V26F/M29F) and GCGATCGC complexes have
been determined and refined well at 2.25 Å, 2.2 Å, 1.45 Å, 1.9 Å and 1.5 Å, respectively. DNA
bending has long been recognized as an important component of biological activity. The Sac7d
mutants display two kinking modes of DNA conformations that are “smooth bending” and “kinked
bending” into minor groove of DNA duplex. The DNA binding patterns and unit cells of the V26A,
M29A single mutants are similar to those of wild type. Without stacking with one of the base pairs,
the Phe29 side chain of M29F mutant penetrates into the C2pG3 step vertically, together with the
Vla26, induces the largest single-step sharp kink (~70o) and disrupts the stacking of two adjacent
base pairs. For the 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
C2pG3 step as observed in the wild type Sac7d, the V26A/M29A protein binds at the G3pA4 step,
bends DNA helix smoothly and has smaller kink angle (~50o). 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 Phe29 is packed against the G15 ribose
with van der Waals contact. 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 and vary the size of these two side chains seem be able to modulate the extent of
the DNA curvature.