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

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 Are and Phe(60). 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 Phe(60) 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.Publisher PDFPeer reviewe

Obligate Heterodimerization of the Archaeal Alba2 Protein with Alba1 Provides a Mechanism for Control of DNA Packaging

SummaryOrganisms growing at elevated temperatures face a particular challenge to maintain the integrity of their genetic material. All thermophilic and hyperthermophilic archaea encode one or more copies of the Alba (Sac10b) gene. Alba is an abundant, dimeric, highly basic protein that binds cooperatively and at high density to DNA. Sulfolobus solfataricus encodes a second copy of the Alba gene, and the Alba2 protein is expressed at ∼5% of the level of Alba1. We demonstrate by NMR, ITC, and crystallography that Alba2 exists exclusively as a heterodimer with Alba1 at physiological concentrations and that heterodimerization exerts a clear effect upon the DNA packaging, as observed by EM, potentially by changing the interface between adjacent Alba dimers in DNA complexes. A functional role for Alba2 in modulation of higher order chromatin structure and DNA condensation is suggested

ATRX dysfunction Induces replication defects in primary mouse cells

The chromatin remodeling protein ATRX, which targets tandem repetitive DNA, has been shown to be required for expression of the alpha globin genes, for proliferation of a variety of cellular progenitors, for chromosome congression and for the maintenance of telomeres. Mutations in ATRX have recently been identified in tumours which maintain their telomeres by a telomerase independent pathway involving homologous recombination thought to be triggered by DNA damage. It is as yet unknown whether there is a central underlying mechanism associated with ATRX dysfunction which can explain the numerous cellular phenomena observed. There is, however, growing evidence for its role in the replication of various repetitive DNA templates which are thought to have a propensity to form secondary structures. Using a mouse knockout model we demonstrate that ATRX plays a direct role in facilitating DNA replication. Ablation of ATRX alone, although leading to a DNA damage response at telomeres, is not sufficient to trigger the alternative lengthening of telomere pathway in mouse embryonic stem cells

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

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 Arg(59) and Phe(60). 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 Phe(60) 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

A method for the reversible trapping of proteins in non-native conformations

High-dilution equilibrium macrocyclization is developed as a general approach to trapping proteins in a non-native state with a synthetic cross-linking agent. The approach is illustrated using the N-terminal domain of phosphoglycerate kinase and a synthetic reagent containing two maleimide groups, for selective attachment to cysteines introduced onto the protein surface through mutagenesis, and an aromatic disulfide that can be chemically or photochemically cleaved. Following functionalization of the cysteine residues, thiol-disulfide exchange chemistry under strongly unfolding conditions was used to achieve intramolecular cyclization and a high yield of the cross-linked protein. H-1 NMR, CD, and fluorescence spectroscopies indicate that the conformation of the cross-linked protein is non-native. Chemical cleavage of the aromatic disulfide cross-link by a reducing agent results in the acquisition of a nativelike conformation for the reduced protein. Thus, the cross-link acts as a reversible switch of protein folding

Multiple forms of copper (II) co-ordination occur throughout the disordered N-terminal region of the prion protein at pH 7.4.

Although the physiological function of the prion protein remains unknown, in vitro experiments suggest that the protein may bind copper (II) ions and play a role in copper transport or homoeostasis in vivo. The unstructured N-terminal region of the prion protein has been shown to bind up to six copper (II) ions, with each of these ions co-ordinated by a single histidine imidazole and nearby backbone amide nitrogen atoms. Individually, these sites have micromolar affinities, which is weaker than would be expected of a true cuproprotein. In the present study, we show that with subsaturating levels of copper, different forms of co-ordination will occur, which have higher affinity. We have investigated the copper-binding properties of two peptides representing the known copper-binding regions of the prion protein: residues 57–91, which contains four tandem repeats of the octapeptide GGGWGQPH, and residues 91–115. Using equilibrium dialysis and spectroscopic methods, we unambiguously demonstrate that the mode of copper co-ordination in both of these peptides depends on the number of copper ions bound and that, at low copper occupancy, copper ions are co-ordinated with sub-micromolar affinity by multiple histidine imidazole groups. At pH 7.4, three different modes of copper co-ordination are accessible within the octapeptide repeats and two within the peptide comprising residues 91–115. The highest affinity copper (II)-binding modes cause self-association of both peptides, suggesting a role for copper (II) in controlling prion protein self-association in vivo