Dimer–dimer stacking interactions are important for nucleic acid binding by the archaeal chromatin protein Alba

Abstract

Archaea use a variety of small basic proteins to package their DNA. One of the most widespread and highly conserved is the Alba (Sso10b) protein. Alba interacts with both DNA and RNA in vitro, and we show in the present study that it binds more tightly to dsDNA (double-stranded DNA) than to either ssDNA (single-stranded DNA) or RNA. The Alba protein is dimeric in solution, and forms distinct ordered complexes with DNA that have been visualized by electron microscopy studies; these studies suggest that, on binding dsDNA, the protein forms extended helical protein fibres. An end-to-end association of consecutive Alba dimers is suggested by the presence of a dimer–dimer interface in crystal structures of Alba from several species, and by the strong conservation of the interface residues, centred on Arg59 and Phe60. In the present study we map perturbation of the polypeptide backbone of Alba upon binding to DNA and RNA by NMR, and demonstrate the central role of Phe60 in forming the dimer–dimer interface. Site-directed spin labelling and pulsed ESR are used to confirm that an end-to-end, dimer–dimer interaction forms in the presence of dsDNA.

Biochemical and Structural Insights into RNA Binding by Ssh10b, a Member of the Highly Conserved Sac10b Protein Family in Archaea

“…The patchmediated dimer-dimer interaction appears to play a negligible role in the binding of RNA by Ssh10b because substitution of either Phe-60 or all four key residues of the patch by Glu resulted in little change in the affinity of the protein for the RNA. This is in contrast with the report that an F60A mutant reduced the affinity of Sso10b for DNA by 10-fold (41). It is speculated that the patch-mediated Ssh10b interaction may help bring RNA strands together.…”

The Archaeal Sac10b Protein Family: Conserved Proteins with Divergent Functions

“…In vivo, the association of Sso10b and other Sac10b protein dimers via dimer-dimer interfaces may facilitate the packaging of dsDNA by bending [30] and/or bridging [1]. The NMR experiments of Jelinska et al [46] suggested also that, contrary to the model of the Sso10b-dsDNA complex proposed by Wardleworth et al [27] (Fig. (1b)), the two 3-4 hairpins of the Sso10b dimer do not bind in minor groove segments of dsDNA because in the complex the chemical shifts in the loop regions do not change and the loops remain flexible.…”

“…The dimer-dimer interfaces observed in most of the crystal structures of Sac10b family proteins [27][28][29] are formed between the strongly conserved hydrophobic residues of the 1 helix and the C-terminal tip of the 2 helix of one subunit each of the dimers. NMR studies of Sso10b by Jelinska et al [46] demonstrated the importance of the two phenylalanine residues of the interface (drawn as sticks in (Fig. (1c)) for dimer-dimer formation and DNA binding; replacement of the phenylalanine by site-directed mutagenesis resulted in weaker DNA binding.…”

“…To date, only structures of hyperthermophilic Sac10b proteins ( Table 1) and only one study of the binding of a Sac10b protein to DNA and RNA using NMR have been published [46]. In order to answer the major open questions concerning the physicochemical properties and physiological activities of the Sac10b proteins, more X-ray and NMR structures of individual hyperthermophilic, thermophilic and mesophilic Sac10b proteins and of various Sac10b proteinnucleic acid complexes must be available.…”

Transcription Regulation in Archaea

“…Crenarchaeonencoded nucleoid-associated proteins have been shown to influence transcription output through the acetylation/deaceytlation of Alba in vitro (76), although Alba has not yet been shown to influence transcription in vivo. It is possible that Alba regulates transcription, given that Alba proteins can loop, condense, bridge, and even saturate DNA in vitro, but the in vivo dynamics remain unknown (178)(179)(180)(181)(182). In the euryarchaeal organism Methanococcus voltae the deletion of the gene encoding Alba resulted in the upregulation of only a small number of genes, implying that Albabased regulation may be limited in scope (173).…”