Characterization of Sac10a, a hyperthermophile DNA-binding protein from Sulfolobus acidocaldarius.
Sac10a is a member of a group of basic DNA-binding proteins thought to be important in chromatin structure and regulation in the archaeon Sulfolobus. We describe here the isolation, gene identification, and biophysical characterization of native Sac10a. The protein exists as a 23.8 kDa homodimer at pH 7 and unfolds with a T degrees of 122 degrees C. Dissociation of the dimer into folded globular subunits is promoted by decreased pH and salt concentration. Thermal unfolding of the monomeric subunits occurred with two transitions, indicating two independent domains. The dimer demonstrated a high affinity for duplex poly(dAdT) with a K(D) of 5 x 10(-)(10) M and a site size of 17 bp (in 0.15 M KCl, pH 7), with only weak binding (K(D) > 5 x 10(-)(6) M) to poly(dA)-poly(dT), poly(dGdC), poly(dG)-poly(dC), and Escherichia coli DNA under similar conditions. Binding to poly(dAdT) resulted in distortions in the DNA duplex that were consistent with overwinding as indicated by inversion of the CD spectrum of the DNA. The monomeric subunits are predicted to adopt a winged helix DNA-binding motif which dimerizes through formation of a two-stranded coiled coil involving an extended C-terminal helix with more than four heptad repeats (about 45 A in length). This is the first example of the conserved archaeal transcription regulator domain COG3432 to be characterized. Sequences for homologous proteins containing both COG3432 and predicted coiled coil domains occur in the genomes of both crenarchaeota (Sulfolobus, Pyrobaculum, Aeropyrum) and euryarchaeota (Methanosarcina, Methanococcus, Archaeoglobus, Thermoplasma), with multiple genes in some species. Sac10a shows no sequence similarity to the other Sulfolobus chromatin proteins Sac7d, Sac8, Sso10b2, and Alba.
Laboratory for Structural Biology, Department of Chemistry, Graduate Program in Biotechnology and Bioengineering, University of Alabama in Huntsville, Huntsville, Alabama 35899, USA.
Stephen P
SP
Edmondson
Mebrahtu A
MA
Kahsai
Ramesh
R
Gupta
John W
JW
Shriver
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D016945:Sulfolobus acidocaldarius / Q000737:chemistry*; D013499:Surface Properties; D013816:Thermodynamics; D014461:Ultracentrifugation
D019843:Archaeal Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C119661:Sac10b protein, Sulfolobus acidocaldarius