A Stabilizing #/#-Hydrophobic Core Greatly Contributes to Hyperthermostability of Archaeal [P62A]Ssh10b

The hyperthermophilic Ssh10b from Sulfolobus shibatae is a member of the Sac10b family, which has been postulated to play a role in chromosomal organization in Archaea. Ssh10b is capable of significantly constraining negative DNA supercoils at elevated temperatures. In this study, the solution structure of the dimeric P62A mutant Ssh10b ([P62A]Ssh10b) was determined by multidimensional NMR spectroscopy. The backbone 15N dynamics, H/D exchange with and without the denaturant GdmSCN, and chemical and thermal denaturation experiments were performed to investigate the molecular basis of high thermostability of [P62A]Ssh10b. Data analysis has revealed an R/ -hydrophobic core consisting of two R-helices and one -sheet which are stabilized by cooperative hydrophobic and hydrogen-bonding interactions. This stabilizing R/ -hydrophobic core of [P62A]Ssh10b exhibiting highly restricted internal motions is composed of residues having highly protected amide protons which exchange with solvent mostly by means of a global unfolding process. The K40N mutation greatly destabilizes the mutant [P62A]Ssh10b because this mutation disturbs the packing of R-helix against the -sheet reducing the stability of the R/ -hydrophobic core in the mutant protein. In comparison with homologous mesophilic and thermophilic proteins, it can be presumed that the stabilizing R/ -hydrophobic core in the [P62A]Ssh10b structure greatly contributes to the high thermostability of the protein. Proteins having molecular mass of 7, 8, and 10 kDa have been extracted from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius grown at 75-80 °C and pH 4.5-4.8. Sac10b is the second most abundant protein in S. acidocaldarius (1). The members of Sac10b family, postulated as chromosomal DNA binding proteins, are highly conserved among most archaeal and eukaryal proteins having homologous sequences identified from higher plants, protists, and vertebrates (2, 3). Ssh10b and Sso10b are the members of the Sac10b family. Ssh10b has been isolated from Sulfolobus shibatae and Sso10b having identical sequence to Ssh10b with the exception of amino acids 1-3 has been isolated from Sulfolobus solfataricus (2, 4). Sso10b was found to exist predominantly in an acetylated form in vivo and in deacetylated form after incubation with Sir2 in the presenceofNAD+, regulating theDNAbindingaffinity(2,5,6). More recently, the crystal structures of Sso10b (also named Alba) have been published (7), which show structural homology with the C-terminal domain of the Escherichia coli translation factor IF3 and the N-terminal DNA-binding domain of DNase I. Ssh10b, a protein thermally stable up to 402 K (8), binds DNA without apparent sequence specificity, and is capable of significantly constraining negative DNA supercoils at elevated temperatures (4). The previous NMR studies (9, 10) have revealed that two forms of Ssh10b homodimer coexisted in solution, and the slow cis-trans isomerization of the L61-P62 peptide bond is responsible for the conformational heterogeneity of the Ssh10b homodimer. The P62A mutant Ssh10b ([P62A]Ssh10b) adopted only the trans-form homodimer. Similar to Ssh10b, [P62A]Ssh10b is a highly thermostable protein and shows the same features upon interaction with DNA at high temperatures (10). However, what leads to the high thermostability of these proteins has not been deciphered