A Biochemical and Biophysical Analysis of the Interaction of nsp9 with nsp12 from SARS-CoV-2-Implications for Future Drug Discovery Efforts.

The ongoing global pandemic of the coronavirus 2019 (COVID-19) disease is caused by the virus SARS-CoV-2, with very few highly effective antiviral treatments currently available. The machinery responsible for the replication and transcription of viral RNA during infection is made up of several important proteins. Two of these are nsp12, the catalytic subunit of the viral polymerase, and nsp9, a cofactor of nsp12 involved in the capping and priming of viral RNA. While several recent studies have determined the structural details of the interaction of nsp9 with nsp12 in the context of RNA capping, very few biochemical or biophysical details are currently available. In this study, we have used a combination of surface plasmon resonance (SPR) experiments, size exclusion chromatography (SEC) experiments, and biochemical assays to identify specific nsp9 residues that are critical for nsp12 binding as well as RNAylation, both of which are essential for the RNA capping process. Our data indicate that nsp9 dimerization is unlikely to play a significant functional role in the virus. We confirm that a set of recently discovered antiviral peptides inhibit nsp9-nsp12 interaction by specifically binding to nsp9; however, we find that these peptides do not impact RNAylation. In summary, our results have important implications for future drug discovery efforts to combat SARS-CoV-2 and any newly emerging coronaviruses.

Sequence- and Structure-Dependent Cytotoxicity of Phosphorothioate and 2'-O-Methyl Modified Single-Stranded Oligonucleotides.

Single-stranded oligonucleotides (SSOs) are a rapidly expanding class of therapeutics that comprises antisense oligonucleotides, microRNAs, and aptamers, with ten clinically approved molecules. Chemical modifications such as the phosphorothioate backbone and the 2'-O-methyl ribose can improve the stability and pharmacokinetic properties of therapeutic SSOs, but they can also lead to toxicity in vitro and in vivo through nonspecific interactions with cellular proteins, gene expression changes, disturbed RNA processing, and changes in nuclear structures and protein distribution. In this study, we screened a mini library of 277 phosphorothioate and 2'-O-methyl-modified SSOs, with or without mRNA complementarity, for cytotoxic properties in two cancer cell lines. Using circular dichroism, nucleic magnetic resonance, and molecular dynamics simulations, we show that phosphorothioate- and 2'-O-methyl-modified SSOs that form stable hairpin structures through Watson-Crick base pairing are more likely to be cytotoxic than those that exist in an extended conformation. In addition, moderate and highly cytotoxic SSOs in our dataset have a higher mean purine composition than pyrimidine. Overall, our study demonstrates a structure-cytotoxicity relationship and indicates that the formation of stable hairpins should be a consideration when designing SSOs toward optimal therapeutic profiles.

A structural analysis of the nsp9 protein from the coronavirus MERS CoV reveals a conserved RNA binding interface.

Middle East respiratory syndrome coronavirus (MERS CoV) and severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) are RNA viruses from the Betacoronavirus family that cause serious respiratory illness in humans. One of the conserved non-structural proteins encoded for by the coronavirus family is non-structural protein 9 (nsp9). Nsp9 plays an important role in the RNA capping process of the viral genome, where it is covalently linked to viral RNA (known as RNAylation) by the conserved viral polymerase, nsp12. Nsp9 also directly binds to RNA; we have recently revealed a distinct RNA recognition interface in the SARS CoV-2 nsp9 by using a combination of nuclear magnetic resonance spectroscopy and biolayer interferometry. In this study, we have utilized a similar methodology to determine a structural model of RNA binding of the related MERS CoV. Based on these data, we uncover important similarities and differences to SARS CoV-2 nsp9 and other coronavirus nsp9 proteins. Our findings that replacing key RNA binding residues in MERS CoV nsp9 affects RNAylation efficiency indicate that recognition of RNA may play a role in the capping process of the virus.

The molecular details of a novel phosphorylation-dependent interaction between MRN and the SOSS complex.

The repair of double-strand DNA breaks (DSBs) by homologous recombination is crucial in the maintenance of genome integrity. While the key role of the Mre11-Rad50-Nbs1 (MRN) complex in repair is well known, hSSB1 (SOSSB and OBFC2B), one of the main components of the sensor of single-stranded DNA (SOSS) protein complex, has also been shown to rapidly localize to DSB breaks and promote repair. We have previously demonstrated that hSSB1 binds directly to Nbs1, a component of the MRN complex, in a DNA damage-independent manner. However, recruitment of the MRN complex has also been demonstrated by an interaction between Integrator Complex Subunit 3 (INTS3; also known as SOSSA), another member of the SOSS complex, and Nbs1. In this study, we utilize a combined approach of in silico, biochemical, and functional experiments to uncover the molecular details of INTS3 binding to Nbs1. We demonstrate that the forkhead-associated domain of Nbs1 interacts with INTS3 via phosphorylation-dependent binding to INTS3 at Threonine 592, with contributions from Serine 590. Based on these data, we propose a model of MRN recruitment to a DSB via INTS3.

A distinct ssDNA/RNA binding interface in the Nsp9 protein from SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel, highly infectious RNA virus that belongs to the coronavirus family. Replication of the viral genome is a fundamental step in the virus life cycle and SARS-CoV-2 non-structural protein 9 (Nsp9) is shown to be essential for virus replication through its ability to bind RNA in the closely related SARS-CoV-1 strain. Two recent studies revealing the three-dimensional structure of Nsp9 from SARS-CoV-2 have demonstrated a high degree of similarity between Nsp9 proteins within the coronavirus family. However, the binding affinity to RNA is very low which, until now, has prevented the determination of the structural details of this interaction. In this study, we have utilized nuclear magnetic resonance spectroscopy (NMR) in combination with surface biolayer interferometry (BLI) to reveal a distinct binding interface for both ssDNA and RNA that is different to the one proposed in the recently solved SARS-CoV-2 replication and transcription complex (RTC) structure. Based on these data, we have proposed a structural model of a Nsp9-RNA complex, shedding light on the molecular details of these important interactions.

Expression, Purification, and Solution-State NMR Analysis of the Two Human Single-Stranded DNA-Binding Proteins hSSB1 (NABP2/OBFC2B) and hSSB2 (NAPB1/OBFC2A).

Single-stranded DNA-binding proteins (SSBs) are essential to all living organisms as protectors and guardians of the genome. Apart from the well-characterized RPA, humans have also evolved two further SSBs, termed hSSB1 and hSSB2. Over the last few years, we have used NMR spectroscopy to determine the molecular and structural details of both hSSBs and their interactions with DNA and RNA. Here we provide a detailed overview of how to express and purify recombinant versions of these important human proteins for the purpose of detailed structural analysis by high-resolution solution-state NMR.

The structural details of the interaction of single-stranded DNA binding protein hSSB2 (NABP1/OBFC2A) with UV-damaged DNA.

Single-stranded DNA-binding proteins (SSBs) are required for all known DNA metabolic events such as DNA replication, recombination and repair. While a wealth of structural and functional data is available on the essential human SSB, hSSB1 (NABP2/OBFC2B), the close homolog hSSB2 (NABP1/OBFC2A) remains relatively uncharacterized. Both SSBs possess a well-structured OB (oligonucleotide/oligosaccharide-binding) domain that is able to recognize single-stranded DNA (ssDNA) followed by a flexible carboxyl-tail implicated in the interaction with other proteins. Despite the high sequence similarity of the OB domain, several recent studies have revealed distinct functional differences between hSSB1 and hSSB2. In this study, we show that hSSB2 is able to recognize cyclobutane pyrimidine dimers (CPD) that form in cellular DNA as a consequence of UV damage. Using a combination of biolayer interferometry and NMR, we determine the molecular details of the binding of the OB domain of hSSB2 to CPD-containing ssDNA, confirming the role of four key aromatic residues in hSSB2 (W59, Y78, W82, and Y89) that are also conserved in hSSB1. Our structural data thus demonstrate that ssDNA recognition by the OB fold of hSSB2 is highly similar to hSSB1, indicating that one SSB may be able to replace the other in any initial ssDNA binding event. However, any subsequent recruitment of other repair proteins most likely depends on the divergent carboxyl-tail and as such is likely to be different between hSSB1 and hSSB2.

A Structural Perspective on the Regulation of Human Single-Stranded DNA Binding Protein 1 (hSSB1, OBFC2B) Function in DNA Repair.

Single-stranded DNA binding (SSB) proteins are essential to protect singe-stranded DNA (ssDNA) that exists as a result of several important DNA repair pathways in living cells. In humans, besides the well-characterised Replication Protein A (RPA) we have described another SSB termed human SSB1 (hSSB1, OBFC2B) and have shown that this protein is an important player in the maintenance of the genome. In this review we define the structural and biophysical details of how hSSB1 interacts with both DNA and other essential proteins. While the presence of the oligonucleotide/oligosaccharide (OB) domain ensures ssDNA binding by hSSB1, it has also been shown to self-oligomerise as well as interact with and being modified by several proteins highlighting the versatility that hSSB1 displays in the context of DNA repair. A detailed structural understanding of these processes will likely lead to the designs of tailored hSSB1 inhibitors as anti-cancer drugs in the near future.

Human single-stranded DNA binding protein 1 (hSSB1, OBFC2B), a critical component of the DNA damage response.

Our genomic DNA is found predominantly in a double-stranded helical conformation. However, there are a number of cellular transactions and DNA damage events that result in the exposure of single stranded regions of DNA. DNA transactions require these regions of single stranded DNA, but they are only transient in nature as they are particularly susceptible to further damage through chemical and enzymatic degradation, metabolic activation, and formation of secondary structures. To protect these exposed regions of single stranded DNA, all living organisms have members of the Single Stranded DNA Binding (SSB) protein family, which are characterised by a conserved oligonucleotide/oligosaccharide-binding (OB) domain. In humans, three such proteins members have been identified; namely the Replication Protein A (RPA) complex, hSSB1 and hSSB2. While RPA is extremely well characterised, the roles of hSSB1 and hSSB2 have only emerged recently. In this review, we discuss the critical roles that hSSB1 plays in the maintenance of genomic stability.

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.