Structural basis of DNA binding mechanism of the hyperthermophilic chromosomal protein Sac7d from Sulfolobus acidocaldarius

• Main Authors: Chin-Yu Chen, 陳青諭
• Other Authors: 王惠鈞
• Format: Others
• Language: en_US
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/90016770588971108378

博士 === 國立臺灣大學 === 化學研究所 === 94 === Sac7d is a small (~7600 Da.), but abundant, chromosomal proteins from the hyperthermophilic archaeon Sulfolobus acidocaldarius. The protein is extremely stable to heat, acid and chemical agents. Sac7d binds to DNA as monomer non-cooperatively with micro-molar affinity, without marked sequence preference and increases the Tm of DNA by ~ 40°C. Previously, two crystal structures of Sac7d-octamer complexes have been solved at high resolution. These structures reveal that Sac7d binds in the minor groove of DNA and causes a single-step sharp kink in DNA (~60°) via the intercalation of both Val26 and Met29. These two amino acids were systematically changed in size to probe their effects on DNA kinking. DNA bending has long been recognized as an important component of biological activity. 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 (~50°). The M29F mutant inserts the Phe29 side chain orthogonally to the C2pG3 step without stacking with base pairs, inducing a sharp kink (~80°). 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 Tm 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. Two new crystal forms of Sac7d in complex with the DNA decamers CCTATATCGG and CCTACGTACC were obtained and their structures were determined by molecular replacement. The protein structures are similar to the previously determined structure of Sac7d-GCGATCGC, but the DNA molecules are more bent overall, by 14-20°. Analysis the interactions of the same protein bound to different DNA sequences showed weak DNA binding sequence preferences of Sac7d, a sequence-general DNA binding protein. The preferred intercalation sites in DNA were found at either the CpG, TpT(=ApA) or TpA steps, likely due to their weak stacking forces. The base at the 3’ end of the intercalating site is always a purine (G or A), with Trp24 NE1 forming a hydrogen bond to its N3 atom. The second or third base at the 3’-side of the intercalation site is a thymine that forms hydrogen bond(s) with its O2 atom to Arg42. The spectroscopic methods such as UV-Vis, fluorescence and Raman spectroscopy have been used to study variety properties of Sac7d in solution. A Raman spectroscopic analysis of Sac7d binding to decamer GAGGCGCCTC reveals that large changes in the DNA backbone and partial B- to A-form DNA transitions in the DNA structure upon complex formation. A hydrophobic cluster on the surface of Sac7d is composed of Trp24, Val26, and Met29 residues which play a key role in defining thermal stability and DNA binding affinity of Sac7d. All of the Sac7d W24/V26/M29 mutations resulted in a decrease in protein thermal stability and DNA binding affinity. In addition, the photochemical study show evidence for the photoinduced specific electron transfer process from Trp24 to bromo-uracil containing DNA in the Sac7d-DNA complex. Recent surveys of high-resolution protein-DNA crystal structures have noted that solvent molecules are commonly present within the protein-DNA interfaces. Putting these results together has revealed that protein-DNA complexes are quite diverse in their use of water. In the non-sequence specific DNA binding proteins such as Sac7d, interfacial water molecules may act as “modulators” for their binding to DNA of varying sequence without adding specificities. When sequence specific DNA binding proteins bound to non-cognate DNA, more waters remained at the interface of the complexes. These waters may behave as a kind of molecular glue allowing the protein to slide along the DNA for their target sites. Some proteins switch their specificity, i.e., transformation of the high affinity complex to a low affinity complex revealed that direct hydrogen bonds at the interface of protein and DNA are often replaced by water-mediated hydrogen bonds. Water molecules could act as major contributions to stability and specificity in some specific protein-DNA complexes. Since DNA hydration patterns are sequence dependent, proteins recognize the DNA hydration structures rather than DNA sequence upon forming the complexes.