The cytoplasmic tail of myelin protein zero induces morphological changes in lipid membranes.

The major myelin protein expressed by the peripheral nervous system Schwann cells is protein zero (P0), which represents 50% of the total protein content in myelin. This 30-kDa integral membrane protein consists of an immunoglobulin (Ig)-like domain, a transmembrane helix, and a 69-residue C-terminal cytoplasmic tail (P0ct). The basic residues in P0ct contribute to the tight packing of myelin lipid bilayers, and alterations in the tail affect how P0 functions as an adhesion molecule necessary for the stability of compact myelin. Several neurodegenerative neuropathies are related to P0, including the more common Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS) as well as rare cases of motor and sensory polyneuropathy. We found that high P0ct concentrations affected the membrane properties of bicelles and induced a lamellar-to-inverted hexagonal phase transition, which caused bicelles to fuse into long, protein-containing filament-like structures. These structures likely reflect the formation of semicrystalline lipid domains with potential relevance for myelination. Not only is P0ct important for stacking lipid membranes, but time-lapse fluorescence microscopy also shows that it might affect membrane properties during myelination. We further describe recombinant production and low-resolution structural characterization of full-length human P0. Our findings shed light on P0ct effects on membrane properties, and with the successful purification of full-length P0, we have new tools to study the role of P0 in myelin formation and maintenance in vitro.

Arc self-association and formation of virus-like capsids are mediated by an N-terminal helical coil motif.

Activity-regulated cytoskeleton-associated protein (Arc) is a protein interaction hub with diverse roles in intracellular neuronal signaling, and important functions in neuronal synaptic plasticity, memory, and postnatal cortical development. Arc has homology to retroviral Gag protein and is capable of self-assembly into virus-like capsids implicated in the intercellular transfer of RNA. However, the molecular basis of Arc self-association and capsid formation is largely unknown. Here, we identified a 28-amino-acid stretch in the mammalian Arc N-terminal (NT) domain that is necessary and sufficient for self-association. Within this region, we identified a 7-residue oligomerization motif, critical for the formation of virus-like capsids. Purified wild-type Arc formed capsids as shown by transmission and cryo-electron microscopy, whereas mutant Arc with disruption of the oligomerization motif formed homogenous dimers. An atomic-resolution crystal structure of the oligomerization region peptide demonstrated an antiparallel coiled-coil interface, strongly supporting NT-NT domain interactions in Arc oligomerization. The NT coil-coil interaction was also validated in live neurons using fluorescence lifetime FRET imaging, and mutation of the oligomerization motif disrupted Arc-facilitated endocytosis. Furthermore, using single-molecule photobleaching, we show that Arc mRNA greatly enhances higher-order oligomerization in a manner dependent on the oligomerization motif. In conclusion, a helical coil in the Arc NT domain supports self-association above the dimer stage, mRNA-induced oligomerization, and formation of virus-like capsids. DATABASE: The coordinates and structure factors for crystallographic analysis of the oligomerization region were deposited at the Protein Data Bank with the entry code 6YTU.

A data-driven structural model of hSSB1 (NABP2/OBFC2B) self-oligomerization.

The maintenance of genome stability depends on the ability of the cell to repair DNA efficiently. Single-stranded DNA binding proteins (SSBs) play an important role in DNA processing events such as replication, recombination and repair. While the role of human single-stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) in the repair of double-stranded breaks has been well established, we have recently shown that it is also essential for the base excision repair (BER) pathway following oxidative DNA damage. However, unlike in DSB repair, the formation of stable hSSB1 oligomers under oxidizing conditions is an important prerequisite for its proper function in BER. In this study, we have used solution-state NMR in combination with biophysical and functional experiments to obtain a structural model of hSSB1 self-oligomerization. We reveal that hSSB1 forms a tetramer that is structurally similar to the SSB from Escherichia coli and is stabilized by two cysteines (C81 and C99) as well as a subset of charged and hydrophobic residues. Our structural and functional data also show that hSSB1 oligomerization does not preclude its function in DSB repair, where it can interact with Ints3, a component of the SOSS1 complex, further establishing the versatility that hSSB1 displays in maintaining genome integrity.

hSSB1 phosphorylation is dynamically regulated by DNA-PK and PPP-family protein phosphatases.

The maintenance of genomic stability is essential for cellular viability and the prevention of diseases such as cancer. Human single-stranded DNA-binding protein 1 (hSSB1) is a protein with roles in the stabilisation and restart of stalled DNA replication forks, as well as in the repair of oxidative DNA lesions and double-strand DNA breaks. In the latter process, phosphorylation of threonine 117 by the ATM kinase is required for hSSB1 stability and efficient DNA repair. The regulation of hSSB1 in other DNA repair pathways has however remained unclear. Here we report that hSSB1 is also directly phosphorylated by DNA-PK at serine residue 134. While this modification is largely suppressed in undamaged cells by PPP-family protein phosphatases, S134 phosphorylation is enhanced following the disruption of replication forks and promotes cellular survival. Together, these data thereby represent a novel mechanism for hSSB1 regulation following the inhibition of replication.

A structural analysis of DNA binding by hSSB1 (NABP2/OBFC2B) in solution.

Single-stranded DNA binding proteins (SSBs) play an important role in DNA processing events such as replication, recombination and repair. Human single-stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) contains a single oligosaccharide/oligonucleotide binding (OB) domain followed by a charged C-terminus and is structurally homologous to the SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus Recent work has revealed that hSSB1 is critical to homologous recombination and numerous other important biological processes such as the regulation of telomeres, the maintenance of DNA replication forks and oxidative damage repair. Since the ability of hSSB1 to directly interact with single-stranded DNA (ssDNA) is paramount for all of these processes, understanding the molecular details of ssDNA recognition is essential. In this study, we have used solution-state nuclear magnetic resonance in combination with biophysical and functional experiments to structurally analyse ssDNA binding by hSSB1. We reveal that ssDNA recognition in solution is modulated by base-stacking of four key aromatic residues within the OB domain. This DNA binding mode differs significantly from the recently determined crystal structure of the SOSS1 complex containing hSSB1 and ssDNA. Our findings elucidate the detailed molecular mechanism in solution of ssDNA binding by hSSB1, a major player in the maintenance of genomic stability.

hSSB1 (NABP2/OBFC2B) is regulated by oxidative stress.

The maintenance of genome stability is an essential cellular process to prevent the development of diseases including cancer. hSSB1 (NABP2/ OBFC2A) is a critical component of the DNA damage response where it participates in the repair of double-strand DNA breaks and in base excision repair of oxidized guanine residues (8-oxoguanine) by aiding the localization of the human 8-oxoguanine glycosylase (hOGG1) to damaged DNA. Here we demonstrate that following oxidative stress, hSSB1 is stabilized as an oligomer which is required for hSSB1 to function in the removal of 8-oxoguanine. Monomeric hSSB1 shows a decreased affinity for oxidized DNA resulting in a cellular 8-oxoguanine-repair defect and in the absence of ATM signaling initiation. While hSSB1 oligomerization is important for the removal of 8-oxoguanine from the genome, it is not required for the repair of double-strand DNA-breaks by homologous recombination. These findings demonstrate a novel hSSB1 regulatory mechanism for the repair of damaged DNA.

Backbone (1)H, (13)C and (15)N resonance assignments of the OB domain of the single stranded DNA-binding protein hSSB1 (NABP2/OBFC2B) and chemical shift mapping of the DNA-binding interface.

Single-stranded DNA-binding proteins (SSBs) are highly important in DNA metabolism and play an essential role in all major DNA repair pathways. SSBs are generally characterised by the presence of an oligonucleotide binding (OB) fold which is able to recognise single-stranded DNA (ssDNA) with high affinity. We discovered two news SSBs in humans (hSSB1 and hSSB2) that both contain a single OB domain followed by a divergent spacer region and a charged C-terminus. We have extensively characterised one of these, hSSB1 (NABP2/OBFC2B), in numerous important DNA processing events such as, in DNA double-stranded break repair and in the response to oxidative DNA damage. Although the structure of hSSB1 bound to ssDNA has recently been determined using X-ray crystallography, the detailed atomic level mechanism of the interaction of hSSB1 with ssDNA in solution has not been established. In this study we report the solution-state backbone chemical shift assignments of the OB domain of hSSB1. In addition, we have utilized NMR to map the DNA-binding interface of hSSB1, revealing major differences between recognition of ssDNA under physiological conditions and in the recently determined crystal structure. Our NMR data in combination with further biophysical and biochemical experiments will allow us to address these discrepancies and shed light onto the structural basis of DNA-binding by hSSB1 in solution.

hSSB1 (NABP2/ OBFC2B) is required for the repair of 8-oxo-guanine by the hOGG1-mediated base excision repair pathway.

The maintenance of genome stability is essential to prevent loss of genetic information and the development of diseases such as cancer. One of the most common forms of damage to the genetic code is the oxidation of DNA by reactive oxygen species (ROS), of which 8-oxo-7,8-dihydro-guanine (8-oxoG) is the most frequent modification. Previous studies have established that human single-stranded DNA-binding protein 1 (hSSB1) is essential for the repair of double-stranded DNA breaks by the process of homologous recombination. Here we show that hSSB1 is also required following oxidative damage. Cells lacking hSSB1 are sensitive to oxidizing agents, have deficient ATM and p53 activation and cannot effectively repair 8-oxoGs. Furthermore, we demonstrate that hSSB1 forms a complex with the human oxo-guanine glycosylase 1 (hOGG1) and is important for hOGG1 localization to the damaged chromatin. In vitro, hSSB1 binds directly to DNA containing 8-oxoguanines and enhances hOGG1 activity. These results underpin the crucial role hSSB1 plays as a guardian of the genome.

The structural basis of DNA binding by the single-stranded DNA-binding protein from Sulfolobus solfataricus.

Canonical single-stranded DNA-binding proteins (SSBs) from the oligosaccharide/oligonucleotide-binding (OB) domain family are present in all known organisms and are critical for DNA replication, recombination and repair. The SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus (SsoSSB) has a 'simple' domain organization consisting of a single DNA-binding OB fold coupled to a flexible C-terminal tail, in contrast with other SSBs in this family that incorporate up to four OB domains. Despite the large differences in the domain organization within the SSB family, the structure of the OB domain is remarkably similar all cellular life forms. However, there are significant differences in the molecular mechanism of ssDNA binding. We have determined the structure of the SsoSSB OB domain bound to ssDNA by NMR spectroscopy. We reveal that ssDNA recognition is modulated by base-stacking of three key aromatic residues, in contrast with the OB domains of human RPA and the recently discovered human homologue of SsoSSB, hSSB1. We also demonstrate that SsoSSB binds ssDNA with a footprint of five bases and with a defined binding polarity. These data elucidate the structural basis of DNA binding and shed light on the molecular mechanism by which these 'simple' SSBs interact with ssDNA.

Backbone and side-chain ¹H, ¹³C and ¹5N resonance assignments of the OB domain of the single stranded DNA binding protein from Sulfolobus solfataricus and chemical shift mapping of the DNA-binding interface.

Single stranded DNA binding proteins (SSBs) are present in all known cellular organisms and are critical for DNA replication, recombination and repair. The SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus (SsoSSB) has an unusual domain structure with a single DNA-binding oligonucleotide binding (OB) fold coupled to a flexible C-terminal tail. This 'simple' domain organisation differs significantly from other known SSBs, such as human replication protein A (RPA). However, it is conserved in another important human SSB, hSSB1, which we have recently discovered and shown to be essential in the DNA damage response. In this study we report the solution-state backbone and side-chain chemical shift assignments of the OB domain of SsoSSB. In addition, using the recently determined crystal structure, we have utilized NMR to reveal the DNA-binding interface of SsoSSB. These data will allow us to elucidate the structural basis of DNA-binding and shed light onto the molecular mechanism by which these 'simple' SSBs interact with single-stranded DNA.

Semiquantitative and quantitative analysis of protein-DNA interactions using steady-state measurements in surface plasmon resonance competition experiments.

One method commonly used to characterize protein-DNA interactions is surface plasmon resonance (SPR). In a typical SPR experiment, chip-bound DNA is exposed to increasing concentrations of protein; the resulting binding data are used to calculate a dissociation constant for the interaction. However, in cases in which knowledge of the specificity of the interaction is required, a large set of DNA variants has to be tested; this is time consuming and costly, in part because of the requirement for multiple SPR chips. We have developed a new protocol that uses steady-state binding levels in SPR competition experiments to determine protein-binding dissociation constants for a set of DNA variants. This approach is rapid and straightforward and requires the use of only a single SPR chip. Additionally, in contrast to other methods, our approach does not require prior knowledge of parameters such as on or off rates, using an estimate of the wild-type interaction as the sole input. Utilizing relative steady-state responses, our protocol also allows for the rapid, reliable, and simultaneous determination of protein-binding dissociation constants of a large series of DNA mutants in a single experiment in a semiquantitative fashion. We compare our approach to existing methods, highlighting specific advantages as well as limitations.