Probing the DNA kink structure induced by the hyperthermophilic chromosomal protein Sac7d

Abstract

Sac7d, a small, abundant, sequence-general DNA-binding protein from the hyperthermophilic archaeon Sulfolobus acidocaldarius, causes a single-step sharp kink in DNA (approximately 60 degrees) via the intercalation of both Val26 and Met29. These two amino acids were systematically changed in size to probe their effects on DNA kinking. Eight crystal structures of five Sac7d mutant-DNA complexes have been analyzed. The DNA-binding pattern of the V26A and M29A single mutants is similar to that of the wild-type, whereas the V26A/M29A protein binds DNA without side chain intercalation, resulting in a smaller overall bending (approximately 50 degrees). The M29F mutant inserts the Phe29 side chain orthogonally to the C2pG3 step without stacking with base pairs, inducing a sharp kink (approximately 80 degrees). In the V26F/M29F-GCGATCGC complex, Phe26 intercalates deeply into DNA bases by stacking with the G3 base, whereas Phe29 is stacked on the G15 deoxyribose, in a way similar to those used by the TATA box-binding proteins. All mutants have reduced DNA-stabilizing ability, as indicated by their lower T m values. The DNA kink patterns caused by different combinations of hydrophobic side chains may be relevant in understanding the manner by which other minor groove-binding proteins interact with DNA.

Figure 1

Sac7d-DNA interface. (a) Ribbon diagram of Sac7d V26F/M29F-GCGATCGC complex with two intercalating phenylalanine residues depicted as ball and stick. The aromatic ring of Phe26 residue stacks with the G3 base, whereas the phenyl ring of Phe29 is stacked on the deoxyribose of G15. (b) The (2F_o − F_c) Fourier electron density maps of the V26AM29A–GCGATCGC complex (contoured at 1.5σ level) in the regions at the protein–DNA interface (upper panel). The indole NH group of Trp24 forms hydrogen bond (3.0 Å) to the base N3 of A4. Adjacent to the intercalating site, Ser 31 (OG) forms a water-mediated hydrogen bond with C14 O2 (lower panel).

Figure 2

Two modes of DNA bending. (a) The structure of V26A/M29A–DNA complex (right) and schematic diagram (left) stress gradual curvature by smooth bending. (b) The structure of M29F–DNA complex shows localization of curvature by kinked bending from minor groove of DNA. The contributions of each base-pair-step curvature to overall helix bending are listed with corresponding base-pair numbers. (c) Local base-pair step parameter, roll, for all eight Sac7d mutant–DNA binary structures, calculated using the X3DNA program. Note the largest roll at certain kinked step (G3pA4 in V26A/M29A, C2pG3 or A3pA4 in the others).

Figure 3

Variations in Sac7d-DNA interactions. (a) The local structures of the M29A– (cyan) and wt-Sac7d– (red) in complex with GTAATTAC are superimposed at the intercalation site. In the M29A structure the isopropyl group of Val 26 penetrates horizontally into DNA base step, but in the wt-Sac7d structure (and other mutants), it intercalates vertically. The electron density map (contoured at 1.5σ level) of the side chain Val26 in M29A is shown in yellow. (b) V26A/M29A–GCGATCGC and (c) V26F/M29F–GCGATCGC complexes, colored in cyan, were superimposed on the wild-type Sac7d–DNA structures, colored in red. The side chain of Arg42 is flipped to opposite side and forms new hydrogen bonds (dashed lines), and the side chain of Ser31 is also rotated in both double mutant complexes.

Figure 4

Local structures of the minor groove-binding proteins. (a) Sac7d V26F/M29F, (b) Sac7d M29F, (c) yTBP, (d) HMG1, (e) LEF-1 and (f) SRY in complex with DNA are displayed near the intercalation sites. The polypeptide backbones are shown in purple, the side chains in coral and the DNA in gold.