Oligomeric State of ß-Coronavirus Non-Structural Protein 10 Stimulators Studied by Small Angle X-ray Scattering.

The ß-coronavirus family, encompassing Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Severe Acute Respiratory Syndrome Coronavirus (SARS), and Middle East Respiratory Syndrome Coronavirus (MERS), has triggered pandemics within the last two decades. With the possibility of future pandemics, studying the coronavirus family members is necessary to improve knowledge and treatment. These viruses possess 16 non-structural proteins, many of which play crucial roles in viral replication and in other vital functions. One such vital protein is non-structural protein 10 (nsp10), acting as a pivotal stimulator of nsp14 and nsp16, thereby influencing RNA proofreading and viral RNA cap formation. Studying nsp10 of pathogenic coronaviruses is central to unraveling its multifunctional roles. Our study involves the biochemical and biophysical characterisation of full-length nsp10 from MERS, SARS and SARS-CoV-2. To elucidate their oligomeric state, we employed a combination of Multi-detection Size exclusion chromatography (Multi-detection SEC) with multi-angle static light scattering (MALS) and small angle X-ray scattering (SAXS) techniques. Our findings reveal that full-length nsp10s primarily exist as monomers in solution, while truncated versions tend to oligomerise. SAXS experiments reveal a globular shape for nsp10, a trait conserved in all three coronaviruses, although MERS nsp10, diverges most from SARS and SARS-CoV-2 nsp10s. In summary, unbound nsp10 proteins from SARS, MERS, and SARS-CoV-2 exhibit a globular and predominantly monomeric state in solution.

Crystal Structure of Non-Structural Protein 10 from Severe Acute Respiratory Syndrome Coronavirus-2.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing Coronavirus Disease 19 (COVID-19), emerged at the end of 2019 and quickly spread to cause a global pandemic with severe socio-economic consequences. The early sequencing of its RNA genome revealed its high similarity to SARS, likely to have originated from bats. The SARS-CoV-2 non-structural protein 10 (nsp10) displays high sequence similarity with its SARS homologue, which binds to and stimulates the 3'-to-5' exoribonuclease and the 2'-O-methlytransferase activities of nsps 14 and 16, respectively. Here, we report the biophysical characterization and 1.6 Å resolution structure of the unbound form of nsp10 from SARS-CoV-2 and compare it to the structures of its SARS homologue and the complex-bound form with nsp16 from SARS-CoV-2. The crystal structure and solution behaviour of nsp10 will not only form the basis for understanding the role of SARS-CoV-2 nsp10 as a central player of the viral RNA capping apparatus, but will also serve as a basis for the development of inhibitors of nsp10, interfering with crucial functions of the replication-transcription complex and virus replication.

Crystal Structure of an Engineered LRRTM2 Synaptic Adhesion Molecule and a Model for Neurexin Binding.

Synaptic adhesion molecules are key components in development of the brain, and in the formation of neuronal circuits, as they are central in the assembly and maturation of chemical synapses. Several families of neuronal adhesion molecules have been identified such as the neuronal cell adhesion molecules, neurexins and neuroligins, and in particular recently several leucine-rich repeat proteins, e.g., Netrin G-ligands, SLITRKs, and LRRTMs. The LRRTMs form a family of four proteins. They have been implicated in excitatory glutamatergic synapse function and were specifically characterized as ligands for neurexins in excitatory synapse formation and maintenance. In addition, LRRTM3 and LRRTM4 have been found to be ligands for heparan sulfate proteoglycans, including glypican. We report here the crystal structure of a thermostabilized mouse LRRTM2, with a Tm 30 °C higher than that of the wild-type protein. We localized the neurexin binding site to the concave surface based on protein engineering, sequence conservation, and prior information about the interaction of the ligand with neurexins, which allowed us to propose a tentative model for the LRRTM-neurexin interaction complex. We also determined affinities of the thermostabilized LRRTM2 and wild-type LRRTM1 and LRRTM2 for neurexin-ß1 with and without Ca(2+). Cell culture studies and binding experiments show that the engineered protein is functional and capable of forming synapselike contacts. The structural and functional data presented here provide the first structure of an LRRTM protein and allow us to propose a model for the molecular mechanism of LRRTM function in the synaptic adhesion.

Low-resolution structure of vaccinia virus DNA replication machinery.

Smallpox caused by the poxvirus variola virus is a highly lethal disease that marked human history and was eradicated in 1979 thanks to a worldwide mass vaccination campaign. This virus remains a significant threat for public health due to its potential use as a bioterrorism agent and requires further development of antiviral drugs. The viral genome replication machinery appears to be an ideal target, although very little is known about its structure. Vaccinia virus is the prototypic virus of the Orthopoxvirus genus and shares more than 97% amino acid sequence identity with variola virus. Here we studied four essential viral proteins of the replication machinery: the DNA polymerase E9, the processivity factor A20, the uracil-DNA glycosylase D4, and the helicase-primase D5. We present the recombinant expression and biochemical and biophysical characterizations of these proteins and the complexes they form. We show that the A20D4 polymerase cofactor binds to E9 with high affinity, leading to the formation of the A20D4E9 holoenzyme. Small-angle X-ray scattering yielded envelopes for E9, A20D4, and A20D4E9. They showed the elongated shape of the A20D4 cofactor, leading to a 150-Å separation between the polymerase active site of E9 and the DNA-binding site of D4. Electron microscopy showed a 6-fold rotational symmetry of the helicase-primase D5, as observed for other SF3 helicases. These results favor a rolling-circle mechanism of vaccinia virus genome replication similar to the one suggested for tailed bacteriophages.

New insights into complex formation by SARS-CoV-2 nsp10 and nsp14.

SARS-CoV-2 non-structural protein 10 (nsp10) is essential for the stimulation of enzymatic activities of nsp14 and nsp16, acting as both an activator and scaffolding protein. Nsp14 is a bifunctional enzyme with the N-terminus containing a 3'-5' exoribonuclease (ExoN) domain that allows the excision of nucleotide mismatches at the virus RNA 3'-end, and a C-terminal N7-methyltransferase (N7-MTase) domain. Nsp10 is required for stimulating both ExoN proofreading and the nsp16 2'-O-methyltransferase activities. This makes nsp10 a central player in both viral resistance to nucleoside-based drugs and the RNA cap methylation machinery that helps the virus evade innate immunity. We characterised the interactions between full-length nsp10 (139 residues), N- and C-termini truncated nsp10 (residues 10-133), and nsp10 with a C-terminal truncation (residues 1-133) with nsp14 using microscale thermophoresis, multi-detection SEC, and hydrogen-deuterium (H/D) exchange mass spectrometry. We describe the functional role of the C-terminal region of nsp10 for binding to nsp14 and show that full N- and C-termini of nsp10 are important for optimal binding. In addition, our H/D exchange experiments suggest an intermediary interaction of nsp10 with the N7-MTase domain of nsp14. In summary, our results suggest intermediary steps in the process of association or dissociation of the nsp10-nsp14 complex, involving contacts between the two proteins in regions not identifiable by X-ray crystallography alone.

La synthèse du génome des poxvirus.

Poxviruses are distinguished from other DNA viruses by replicating exclusively in the cytoplasm of the infected host cell. Replication of the linear double-stranded DNA genome takes place in the perinuclear area, in cytoplasmic foci called viral factories. Poxvirus genome organization evolved in order to prevent the virus from being dependent on nuclear enzymes. Therefore, they encode most, if not all, of the proteins required for efficient replication of their genome. Some of these proteins are essential for virus growth (i.e., enzymes directly involved in DNA synthesis). In contrast, others are dispensable for virus propagation in cell culture (i.e., proteins involved in nucleotide metabolism). Most of our knowledge concerning poxvirus replication comes from studies performed on vaccinia virus, the virus used as vaccine to eradicate smallpox more than 30 years ago. This article reviews our current knowledge of the molecular mechanisms governing poxvirus genome synthesis, with a particular focus on the viral proteins involved in this process. A working model for poxvirus DNA replication is also presented.

Identification of fragments binding to SARS-CoV-2 nsp10 reveals ligand-binding sites in conserved interfaces between nsp10 and nsp14/nsp16.

Since the emergence of SARS-CoV-2 in 2019, Covid-19 has developed into a serious threat to our health, social and economic systems. Although vaccines have been developed in a tour-de-force and are now increasingly available, repurposing of existing drugs has been less successful. There is a clear need to develop new drugs against SARS-CoV-2 that can also be used against future coronavirus infections. Non-structural protein 10 (nsp10) is a conserved stimulator of two enzymes crucial for viral replication, nsp14 and nsp16, exhibiting exoribonuclease and methyltransferase activities. Interfering with RNA proofreading or RNA cap formation represents intervention strategies to inhibit replication. We applied fragment-based screening using nano differential scanning fluorometry and X-ray crystallography to identify ligands targeting SARS-CoV-2 nsp10. We identified four fragments located in two distinct sites: one can be modelled to where it would be located in the nsp14-nsp10 complex interface and the other in the nsp16-nsp10 complex interface. Microscale thermophoresis (MST) experiments were used to quantify fragment affinities for nsp10. Additionally, we showed by MST that the interaction by nsp14 and 10 is weak and thereby that complex formation could be disrupted by small molecules. The fragments will serve as starting points for the development of more potent analogues using fragment growing techniques and structure-based drug design.

FragMAX: the fragment-screening platform at the MAX IV Laboratory.

Advances in synchrotron storage rings and beamline automation have pushed data-collection rates to thousands of data sets per week. With this increase in throughput, massive projects such as in-crystal fragment screening have become accessible to a larger number of research groups. The quality of support offered at large-scale facilities allows medicinal chemistry-focused or biochemistry-focused groups to supplement their research with structural biology. Preparing the experiment, analysing multiple data sets and prospecting for interesting complexes of protein and fragments require, for both newcomers and experienced users, efficient management of the project and extensive computational power for data processing and structure refinement. Here, FragMAX, a new complete platform for fragment screening at the BioMAX beamline of the MAX IV Laboratory, is described. The ways in which users are assisted in X-ray-based fragment screenings and in which the fourth-generation storage ring available at the facility is best exploited are also described.

The structure of SALM5 suggests a dimeric assembly for the presynaptic RPTP ligand recognition.

Synaptic adhesion molecules play a crucial role in the regulation of synapse development and maintenance. Recently, several families of leucine-rich repeat (LRR) domain-containing neuronal adhesion molecules have been characterised, including netrin-G ligands, LRRTMs and the synaptic adhesion-like molecule (SALM) family proteins. Most of these are expressed at the excitatory glutamatergic synapses, and dysfunctions of these genes are genetically linked with cognitive disorders, such as autism spectrum disorders and schizophrenia. The SALM family proteins SALM3 and SALM5, similar to SLITRKs, have been shown to bind to the presynaptic receptor protein tyrosine phosphatase (RPTP) family ligands. Here, we present the 3.1 Å crystal structure of the SALM5 LRR-Ig-domain construct and biophysical studies that verify the crystallographic results. We show that SALM1, SALM3 and SALM5 form similar dimeric structures, in which the LRR domains form the dimer interface. Both SALM3 and SALM5 bind to RPTP immunoglobulin domains with micromolar affinity. SALM3 shows a clear preference for the RPTP ligands with the meB splice insert. Our structural studies and sequence conservation analysis suggests a ligand-binding site and mechanism for RPTP binding via the dimeric LRR domain region.