In this work, we show that the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus displays a cation-dependent ATPase activity with a pH optimum around neutrality and a temperature optimum of 70°C. Measurements of tryptophan fluorescence and experiments that used 1-anilinonaphthalene-8-sulfonic acid as probe demonstrated that ATP hydrolysis induces a conformational change in the molecule and that the binding of the nucleotide triggers the ATP hydrolysis-induced

more »

... nformation of the protein to return to the native conformation. We found that Sso7d rescues previously aggregated proteins in an ATP hydrolysis-dependent manner; the native conformation of Sso7d forms a complex with the aggregates, while the ATP hydrolysis-induced conformation is incapable of this interaction. Sso7d is believed to be the first protein isolated from an archaeon capable of rescuing aggregates. Archaea are microorganisms that are distinct from bacteria and eukarya in the tree of life and mostly thrive in extreme environments (1). The Euryarchaeota branch of the Archaea kingdom includes methanogens and halophiles, while most thermoacidophilic species belong to the Crenarchaeota branch. Crenarchaea, considered the most ancient living cells, have peculiar metabolic pathways and genetics, many of their vital processes still awaiting a clear understanding. The small, basic, nonspecific DNA-binding proteins of Sulfolobales crenarchaea have high sequence identity among them and lack obvious similarity to any other known protein; their tertiary structure (2-6) is very different from that of histones and was found to be similar to the "chromo domain" (7) and SH3 domains (8) involved in protein-protein interactions. The definition of the biological role(s) played by these novel proteins is hampered by the poor knowledge of many DNA-related events in Sulfolobales and by the lack of molecular tools to obtain targeted mutants in these microorganisms. In in vitro approaches, Sso7d from Sulfolobus solfataricus, the best-studied protein of the family, increases the melting temperature of DNA (2), promotes the annealing of complementary DNA strands (9), and induces negative supercoiling (10) and a kink associated with unwinding in oligonucleotides (4, 5). In this paper, we show that Sso7d has an associated ATPase activity that drives the cycling of the molecule between conformational states. We demonstrate that Sso7d rescues aggregated proteins in the presence of ATP hydrolysis. The native conformation of Sso7d binds to the aggregates, while the ATP hydrolysis-induced conformation is incapable of interacting with the aggregated proteins. Sso7d is the only protein present in a S. solfataricus crude extract that has disaggregating activity, and the possible significance of this finding is discussed. EXPERIMENTAL PROCEDURES Materials-Malic enzyme from chicken liver (29 units/mg), lysozyme from chicken egg white (183 units/mg), NADP, and adenosine and guanosine nucleotides were purchased from Sigma. Recombinant ␤-glycosidase of S. solfataricus (Ss␤gly) 1 (13 units/mg) was obtained as described by Moracci et al. (11). ANS was from Aldrich. [␥-32 P]ATP (3,000 Ci/mmol) was from Amersham Pharmacia Biotech. The other chemicals were of the highest grade available. Miscellaneous Methods-Protein concentration was determined by Bradford assay (12) using bovine serum albumin as the standard. The concentration of Sso7d solutions was estimated spectrophotometrically using the extinction coefficient reported by Baumann et al. (2). The concentration of ANS solutions was determined using a millimolar extinction coefficient of 5 OD at 350 nm. SDS-PAGE (15% acrylamide) was carried out according to Laemmli (13). Nondenaturing PAGE (10% acrylamide) was carried out according to Davis (14). Cell Growth and Purification of Sso7d-Cells of S. solfataricus strain MT-4 were grown aerobically at 87°C to the late exponential phase (about 0.8 OD at 600 nm) in DSM 182 culture medium. Cells harvested by centrifugation at 4000 ϫ g for 15 min (6 g from a 4-liter culture) underwent freeze-thawing twice; were added to 6 g of sand and 2 ml of 50 mM Tris-HCl, pH 8.4, containing 0.2 M NaCl, 5% glycerol; and homogenized in an Omni mixer. The homogenate was centrifuged at 4,000 ϫ g for 20 min at 4°C to remove the sand; the supernatant was ultracentrifuged at 160,000 ϫ g for 90 min at 4°C, and the residue was discarded. The crude extract (about 175 mg) was aliquoted and stored at Ϫ20°C. The crude extract (25 mg) was loaded onto a Superdex 75 High Load column (2.6 ϫ 60 cm; Amersham Pharmacia Biotech), which was eluted with 10 mM Tris-HCl, pH 8.4, containing 0.2 M NaCl (buffer A) at a flow rate of 2 ml/min; the fractions containing Sso7d (6 mg) were pooled, concentrated by polyethylene glycol 6,000, and rechromatographed on a Superdex 75 High Load column (1.6 ϫ 60 cm; Amersham Pharmacia Biotech), which was eluted with buffer A at a flow rate of 0.8 ml/min. The peak containing Sso7d (870 g) was dialyzed against 10 mM Tris-HCl, pH 8.4, and concentrated by a Savant vacuum centrifuge. Freshly prepared Sso7d was used in all of the experiments described in this paper.