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.

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.

Recombinant Protein Production Using the Baculovirus Expression Vector System (BEVS).

The baculovirus expression vector system (BEVS) is one of the most popular eukaryotic systems for recombinant protein production. The focus of our protein production platform is the provision of recombinant proteins for research use, where generally only small quantities are required, in the range of tens of micrograms to a few hundred milligrams. Here, we present methods that reflect our standard operating procedures and setup to be able to frequently, and often repeatedly, produce many different types of proteins.

New Insights into the Interaction of Class II Dihydroorotate Dehydrogenases with Ubiquinone in Lipid Bilayers as a Function of Lipid Composition.

The fourth enzymatic reaction in the de novo pyrimidine biosynthesis, the oxidation of dihydroorotate to orotate, is catalyzed by dihydroorotate dehydrogenase (DHODH). Enzymes belonging to the DHODH Class II are membrane-bound proteins that use ubiquinones as their electron acceptors. We have designed this study to understand the interaction of an N-terminally truncated human DHODH (HsΔ29DHODH) and the DHODH from Escherichia coli (EcDHODH) with ubiquinone (Q10) in supported lipid membranes using neutron reflectometry (NR). NR has allowed us to determine in situ, under solution conditions, how the enzymes bind to lipid membranes and to unambiguously resolve the location of Q10. Q10 is exclusively located at the center of all of the lipid bilayers investigated, and upon binding, both of the DHODHs penetrate into the hydrophobic region of the outer lipid leaflet towards the Q10. We therefore show that the interaction between the soluble enzymes and the membrane-embedded Q10 is mediated by enzyme penetration. We can also show that EcDHODH binds more efficiently to the surface of simple bilayers consisting of 1-palmitoyl, 2-oleoyl phosphatidylcholine, and tetraoleoyl cardiolipin than HsΔ29DHODH, but does not penetrate into the lipids to the same degree. Our results also highlight the importance of Q10, as well as lipid composition, on enzyme binding.

Protein-lipid interactions of human dihydroorotate dehydrogenase and three mutants associated with Miller syndrome.

Human dihydroorotate dehydrogenase (DHODH) catalyzes the fourth step of the de novo pyrimidine biosynthesis pathway and uses ubiquinone Q10, a lipophilic molecule located in the inner mitochondrial membrane (IMM), as its co-substrate. DHODH is anchored to the IMM by a single transmembrane helix located at its N-terminus. Nevertheless, how DHODH function is determined by its surrounding membrane environment and protein-lipid interactions, as well as the mechanism by which ubiquinone Q10 accesses the active site of DHODH from within the membrane are still largely unknown. Here, we describe the interaction between wild-type DHODH and three DHODH mutants associated with Miller syndrome and lipids using enzymatic assays, thermal stability assays and Quartz Crystal Microbalance with Dissipation monitoring (QCM-D). Our results provide evidence indicating that the N-terminal part of human DHODH is not only a structural element for mitochondrial import and location of DHODH, but also influences enzymatic activity and utilization of ubiquinone Q10 and ubiquinone analogues in in vitro assays. They also support the role of tetraoleoyl cardiolipin as a lipid interacting with DHODH. Additionally, the results from QCM-D show that the Miller syndrome mutants studied differ in their interactions with supported lipid bilayers compared to wild-type DHODH. These altered interactions add another dimension to the effects of mutations found in Miller syndrome. To the best of our knowledge, this is the first investigation of the protein-lipid interactions of DHODH variants associated with Miller syndrome.

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.

Protein production, kinetic and biophysical characterization of three human dihydroorotate dehydrogenase mutants associated with Miller syndrome.

Miller syndrome is a rare Mendelian disorder caused by mutations in the gene encoding human dihydroorotate dehydrogenase (DHODH). Human DHODH, a Class II DHODH, is an integral protein of the inner mitochondrial membrane (IMM) catalyzing the fourth step of the de novo pyrimidine biosynthesis pathway. Here we present a summary of the state of knowledge regarding Miller syndrome in the absence of any current review on the topic. We then describe the production and characterization of three distinct DHODH missense mutations (G19E, E52G, R135C) associated with Miller syndrome by means of enzyme kinetics and biophysical techniques. These human DHODH mutants were produced both in E. coli and in insect cells using the baculovirus expression vector system. We can show that the effects of these mutations differ from each other and the wild-type enzyme with respect to decreased enzymatic activity, decreased protein stability and probably disturbance of the correct import into the IMM. In addition, our results show that the N-terminus of human DHODH is not only a structural element necessary for correct mitochondrial import and location of DHODH on the outer side of the IMM, but also influences thermal stability, enzymatic activity and affects the kinetic parameters.Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.2023749 .

The Antifungal Mechanism of Amphotericin B Elucidated in Ergosterol and Cholesterol-Containing Membranes Using Neutron Reflectometry.

We have characterized and compared the structures of ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes before and after interaction with the amphiphilic antifungal drug amphotericin B (AmB) using neutron reflection. AmB inserts into both pure POPC and sterol-containing membranes in the lipid chain region and does not significantly perturb the structure of pure POPC membranes. By selective per-deuteration of the lipids/sterols, we show that AmB extracts ergosterol but not cholesterol from the bilayers and inserts to a much higher degree in the cholesterol-containing membranes. Ergosterol extraction by AmB is accompanied by membrane thinning. Our results provide new insights into the mechanism and antifungal effect of AmB in these simple models of fungal and mammalian membranes and help understand the molecular origin of its selectivity and toxic side effects.

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.

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.

Biophysical Characterization of Cancer-Related Carbonic Anhydrase IX.

Upregulation of carbonic anhydrase IX (CA IX) is associated with several aggressive forms of cancer and promotes metastasis. CA IX is normally constitutively expressed at low levels in selective tissues associated with the gastrointestinal tract, but is significantly upregulated upon hypoxia in cancer. CA IX is a multi-domain protein, consisting of a cytoplasmic region, a single-spanning transmembrane helix, an extracellular CA catalytic domain, and a proteoglycan-like (PG) domain. Considering the important role of CA IX in cancer progression and the presence of the unique PG domain, little information about the PG domain is known. Here, we report biophysical characterization studies to further our knowledge of CA IX. We report the 1.5 Å resolution crystal structure of the wild-type catalytic domain of CA IX as well as small angle X-ray scattering and mass spectrometry of the entire extracellular region. We used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to characterize the spontaneous degradation of the CA IX PG domain and confirm that it is only the CA IX catalytic domain that forms crystals. Small angle X-ray scattering analysis of the intact protein indicates that the PG domain is not randomly distributed and adopts a compact distribution of shapes in solution. The observed dynamics of the extracellular domain of CA IX could have physiological relevance, including observed cleavage and shedding of the PG domain.

Identification and analyses of inhibitors targeting apolipoprotein(a) kringle domains KIV-7, KIV-10, and KV provide insight into kringle domain function.

Increased plasma concentrations of lipoprotein(a) (Lp(a)) are associated with an increased risk for cardiovascular disease. Lp(a) is composed of apolipoprotein(a) (apo(a)) covalently bound to apolipoprotein B of low-density lipoprotein (LDL). Many of apo(a)'s potential pathological properties, such as inhibition of plasmin generation, have been attributed to its main structural domains, the kringles, and have been proposed to be mediated by their lysine-binding sites. However, available small-molecule inhibitors, such as lysine analogs, bind unselectively to kringle domains and are therefore unsuitable for functional characterization of specific kringle domains. Here, we discovered small molecules that specifically bind to the apo(a) kringle domains KIV-7, KIV-10, and KV. Chemical synthesis yielded compound AZ-05, which bound to KIV-10 with a Kd of 0.8 µm and exhibited more than 100-fold selectivity for KIV-10, compared with the other kringle domains tested, including plasminogen kringle 1. To better understand and further improve ligand selectivity, we determined the crystal structures of KIV-7, KIV-10, and KV in complex with small-molecule ligands at 1.6-2.1 Å resolutions. Furthermore, we used these small molecules as chemical probes to characterize the roles of the different apo(a) kringle domains in in vitro assays. These assays revealed the assembly of Lp(a) from apo(a) and LDL, as well as potential pathophysiological mechanisms of Lp(a), including (i) binding to fibrin, (ii) stimulation of smooth-muscle cell proliferation, and (iii) stimulation of LDL uptake into differentiated monocytes. Our results indicate that a small-molecule inhibitor targeting the lysine-binding site of KIV-10 can combat the pathophysiological effects of Lp(a).

The pathway to pyrimidines: The essential focus on dihydroorotate dehydrogenase, the mitochondrial enzyme coupled to the respiratory chain.

This paper is based on the Anne Simmonds Memorial Lecture, given by Monika Löffler at the International Symposium on Purine and Pyrimidine Metabolism in Man, Lyon 2019. It is dedicated to H. Anne Simmonds (died 2010) - a founding member of the ESSPPMM, since 2003 Purine and Pyrimidine Society - and her outstanding contributions to the identification and study of inborn errors of purine and pyrimidine metabolism. The distinctive intracellular arrangement of pyrimidine de novo synthesis in higher eukaryotes is important to cells with a high demand for nucleic acid synthesis. The proximity of the enzyme active sites and the resulting channeling in CAD and UMP synthase is of kinetic benefit. The intervening enzyme dihydroorotate dehydrogenase (DHODH) is located in the mitochondrion with access to the ubiquinone pool, thus ensuring efficient removal of redox equivalents through the constitutive activity of the respiratory chain, also a mechanism through which the input of 2 ATP for carbamylphosphate synthesis is balanced by Oxphos. The obligatory contribution of O2 to de novo UMP synthesis means that DHODH has a pivotal role in adapting the proliferative capacity of cells to different conditions of oxygenation, such as hypoxia in growing tumors. DHODH also is a validated drug target in inflammatory diseases. This survey of selected topics of personal interest and reflection spans some 40 years of our studies from tumor cell cultures under hypoxia to in vitro assays including purification from mitochondria, localization, cloning, expression, biochemical characterization, crystallisation, kinetics and inhibition patterns of eukaryotic DHODH enzymes.

Preparation of human dihydroorotate dehydrogenase for interaction studies with lipid bilayers.

Human dihydroorotate dehydrogenase (DHODH) is an integral protein of the inner mitochondrial membrane (IMM) that catalyzes the fourth step of the de novo pyrimidine biosynthesis and is functionally connected to the respiratory chain via its lipophilic co-substrate, ubiquinone Q10. DHODH is the target for drugs approved for the treatment of rheumatoid arthritis and multiple sclerosis, and mutations in its sequence have been identified as the cause of Miller syndrome, a rare genetic disorder. The N-terminus of DHODH consists of a signal peptide for mitochondrial import (MS), a transmembrane domain (TM), followed by a microdomain which interacts with the lipids of the IMM and has been proposed to form the binding site for ubiquinone Q10. However, the mechanism by which DHODH interacts with the membrane-embedded Q10 and the lipids of the IMM remains unknown. We present the preparation and characterization of proteins necessary for investigating the structural interactions of DHODH with the lipids of the IMM, including expression and purification of full-length and N-terminally truncated (without MS and TM) DHODH. We characterized the interaction of truncated DHODH with lipid bilayers containing some key lipids of the IMM using Quartz Crystal Microbalance with Dissipation monitoring and compared it to the DHODH from E. coli, a DHODH that naturally lacks a TM. Our results suggest that although cardiolipin enhances the interaction of truncated DHODH with lipid bilayers, the presence of the TM in human DHODH is necessary for stable binding to and securing its location at the outer surface of the IMM.

Effect of ergosterol on the interlamellar spacing of deuterated yeast phospholipid multilayers.

Sterols regulate several physico-chemical properties of biological membranes that are considered to be linked to function. Ergosterol is the main sterol molecule found in the cell membranes of yeasts and other fungi. Like the cholesterol found in mammalian cells, ergosterol has been proposed to have an ordering and condensing effect on saturated phospholipid membranes. The effects of cholesterol have been investigated extensively and result in an increase in the membrane thickness and the lipid acyl chain order. Less information is available on the effects of ergosterol on phospholipid membranes. Neutron Diffraction (ND) was used to characterize the effect of ergosterol on lipid multilayers prepared with deuterated natural phospholipids extracted from the yeast Pichia pastoris. The data show that the effect of ergosterol on membranes prepared from the natural phospholipid extract rich in unsaturated acyl chains, differs from what has been observed previously in membranes rich in saturated phospholipids. In contrast to cholesterol in synthetic phospholipid membranes, the presence of ergosterol up to 30 mol % in yeast phospholipid membranes only slightly altered the multilayer structure. In particular, only a small decrease in the multilayer d-spacing was observed as function of increasing ergosterol concentrations. This result highlights the need for further investigation to elucidate the effects of ergosterol in biological lipid mixtures.

RNAi as a Tool to Study Virulence in the Pathogenic Yeast Candida glabrata.

The yeast Candida glabrata is a major opportunistic pathogen causing mucosal and systemic infections in humans. Systemic infections caused by this yeast have high mortality rates and are difficult to treat due to this yeast's intrinsic and frequently adapting antifungal resistance. To understand and treat C. glabrata infections, it is essential to investigate the molecular basis of C. glabrata virulence and resistance. We established an RNA interference (RNAi) system in C. glabrata by expressing the Dicer and Argonaute genes from Saccharomyces castellii (a budding yeast with natural RNAi). Our experiments with reporter genes and putative virulence genes showed that the introduction of RNAi resulted in 30 and 70% gene-knockdown for the construct-types antisense and hairpin, respectively. The resulting C. glabrata RNAi strain was used for the screening of a gene library for new virulence-related genes. Phenotypic profiling with a high-resolution quantification of growth identified genes involved in the maintenance of cell integrity, antifungal drugs, and ROS resistance. The genes identified by this approach are promising targets for the treatment of C. glabrata infections.

Using neutron crystallography to elucidate the basis of selective inhibition of carbonic anhydrase by saccharin and a derivative.

Up-regulation of carbonic anhydrase IX (CA IX) expression is an indicator of metastasis and associated with poor cancer patient prognosis. CA IX has emerged as a cancer drug target but development of isoform-specific inhibitors is challenging due to other highly conserved CA isoforms. In this study, a CA IXmimic construct was used (CA II with seven point mutations introduced, to mimic CA IX active site) while maintaining CA II solubility that make it amenable to crystallography. The structures of CA IXmimic unbound and in complex with saccharin (SAC) and a saccharin-glucose conjugate (SGC) were determined using joint X-ray and neutron protein crystallography. Previously, SAC and SGC have been shown to display CA isoform inhibitor selectivity in assays and X-ray crystal structures failed to reveal the basis of this selectivity. Joint X-ray and neutron crystallographic studies have shown active site residues, solvent, and H-bonding re-organization upon SAC and SGC binding. These observations highlighted the importance of residues 67 (Asn in CA II, Gln in CA IX) and 130 (Asp in CA II, Arg in CA IX) in selective CA inhibitor targeting.

Improvement of thermotolerance in Lachancea thermotolerans using a bacterial selection pressure.

The use of thermotolerant yeast strains is an important attribute for a cost-effective high temperature biofermentation processes. However, the availability of thermotolerant yeast strains remains a major challenge. Isolation of temperature resistant strains from extreme environments or the improvements of current strains are two major strategies known to date. We hypothesised that bacteria are potential "hurdles" in the life cycle of yeasts, which could influence the evolution of extreme phenotypes, such as thermotolerance. We subjected a wild-type yeast, Lachancea thermotolerans to six species of bacteria sequentially for several generations. After coevolution, we observed that three replicate lines of yeasts grown in the presence of bacteria grew up to 37 °C whereas the controls run in parallel without bacteria could only grow poorly at 35 °C retaining the ancestral mesophilic trait. In addition to improvement of thermotolerance, our results show that the fermentative ability was also elevated, making the strains more ideal for the alcoholic fermentation process because the overall productivity and ethanol titers per unit volume of substrate consumed during the fermentation process was increased. Our unique method is attractive for the development of thermotolerant strains or to augment the available strain development approaches for high temperature industrial biofermentation.

Genome dynamics and evolution in yeasts: A long-term yeast-bacteria competition experiment.

There is an enormous genetic diversity evident in modern yeasts, but our understanding of the ecological basis of such diversifications in nature remains at best fragmented so far. Here we report a long-term experiment mimicking a primordial competitive environment, in which yeast and bacteria co-exist and compete against each other. Eighteen yeasts covering a wide phylogenetic background spanning approximately 250 million years of evolutionary history were used to establish independent evolution lines for at most 130 passages. Our collection of hundreds of modified strains generated through such a rare two-species cross-kingdom competition experiment re-created the appearance of large-scale genomic rearrangements and altered phenotypes important in the diversification history of yeasts. At the same time, the methodology employed in this evolutionary study would also be a non-gene-technological method of reprogramming yeast genomes and then selecting yeast strains with desired traits. Cross-kingdom competition may therefore be a method of significant value to generate industrially useful yeast strains with new metabolic traits.