Molecular determinants of chaperone interactions on MHC-I for folding and antigen repertoire selection.

The interplay between a highly polymorphic set of MHC-I alleles and molecular chaperones shapes the repertoire of peptide antigens displayed on the cell surface for T cell surveillance. Here, we demonstrate that the molecular chaperone TAP-binding protein related (TAPBPR) associates with a broad range of partially folded MHC-I species inside the cell. Bimolecular fluorescence complementation and deep mutational scanning reveal that TAPBPR recognition is polarized toward the α2 domain of the peptide-binding groove, and depends on the formation of a conserved MHC-I disulfide epitope in the α2 domain. Conversely, thermodynamic measurements of TAPBPR binding for a representative set of properly conformed, peptide-loaded molecules suggest a narrower MHC-I specificity range. Using solution NMR, we find that the extent of dynamics at "hotspot" surfaces confers TAPBPR recognition of a sparsely populated MHC-I state attained through a global conformational change. Consistently, restriction of MHC-I groove plasticity through the introduction of a disulfide bond between the α1/α2 helices abrogates TAPBPR binding, both in solution and on a cellular membrane, while intracellular binding is tolerant of many destabilizing MHC-I substitutions. Our data support parallel TAPBPR functions of 1) chaperoning unstable MHC-I molecules with broad allele-specificity at early stages of their folding process, and 2) editing the peptide cargo of properly conformed MHC-I molecules en route to the surface, which demonstrates a narrower specificity. Our results suggest that TAPBPR exploits localized structural adaptations, both near and distant to the peptide-binding groove, to selectively recognize discrete conformational states sampled by MHC-I alleles, toward editing the repertoire of displayed antigens

EstDZ3:a new esterolytic enzyme exhibiting remarkable thermostability

Lipolytic enzymes that retain high levels of catalytic activity when exposed to a variety of denaturing conditions are of high importance for a number of biotechnological applications. In this study, we aimed to identify new lipolytic enzymes, which are highly resistant to prolonged exposure at elevated temperatures. To achieve this, we searched for genes encoding for such proteins in the genomes of a microbial consortium residing in a hot spring located in China. After performing a functional genomic screening on a bacterium of the genus Dictyoglomus, which was isolated from this hot spring after in situ enrichment, we identified a new esterolytic enzyme, termed EstDZ3. Detailed biochemical characterization of the recombinant enzyme, revealed that it constitutes a slightly alkalophilic and highly active esterase against esters of fatty acids with short to medium chain lengths. Importantly, EstDZ3 exhibits remarkable thermostability, as it retained high levels of catalytic activity after exposure to temperatures as high as 95 oC for several hours. Interestingly, EstDZ3 was found to have very little similarity to previously characterized esterolytic enzymes. Computational modelling of the three-dimensional structure of this new enzyme predicted that it exhibits a typical α/β hydrolase fold, which seems to include a subdomain insertion. This insertion is similar to the one present in its closest homologue of known function and structure, the cinnamoyl esterase Lj0536 from Lactobacillus johnsonii. As it was found in the case of Lj0536, this structural feature is expected to be an important determinant of the catalytic properties of EstDZ3. The high levels of esterolytic activity of EstDZ3, combined with its remarkable thermostability and good stability against a wide range of metal ions, organic solvents, and other denaturing agents, render this new enzyme a candidate biocatalyst for high-temperature biotechnological applications

EstDZ3: A New Esterolytic Enzyme Exhibiting Remarkable Thermostability

Lipolytic enzymes that retain high levels of catalytic activity when exposed to a variety of denaturing conditions are of high importance for a number of biotechnological applications. In this study, we aimed to identify new lipolytic enzymes, which are highly resistant to prolonged exposure at elevated temperatures. To achieve this, we searched for genes encoding for such proteins in the genomes of a microbial consortium residing in a hot spring located in China. After performing a functional genomic screening on a bacterium of the genus Dictyoglomus, which was isolated from this hot spring after in situ enrichment, we identified a new esterolytic enzyme, termed EstDZ3. Detailed biochemical characterization of the recombinant enzyme, revealed that it constitutes a slightly alkalophilic and highly active esterase against esters of fatty acids with short to medium chain lengths. Importantly, EstDZ3 exhibits remarkable thermostability, as it retained high levels of catalytic activity after exposure to temperatures as high as 95 oC for several hours. Interestingly, EstDZ3 was found to have very little similarity to previously characterized esterolytic enzymes. Computational modelling of the three-dimensional structure of this new enzyme predicted that it exhibits a typical α/β hydrolase fold, which seems to include a subdomain insertion. This insertion is similar to the one present in its closest homologue of known function and structure, the cinnamoyl esterase Lj0536 from Lactobacillus johnsonii. As it was found in the case of Lj0536, this structural feature is expected to be an important determinant of the catalytic properties of EstDZ3. The high levels of esterolytic activity of EstDZ3, combined with its remarkable thermostability and good stability against a wide range of metal ions, organic solvents, and other denaturing agents, render this new enzyme a candidate biocatalyst for high-temperature biotechnological applications

Etude structurale et fonctionnelle de la protéine ORF3 du VHE par RMN en solution

Hepatitis E Virus (HEV) is the most common cause of acute viral hepatitis worldwide with over 20 million infections and around 44,000 deaths recorded annually. Until today, 8 HEV genotypes are reported with only the first four genotypes infecting humans. In developing countries, HEV is transmitted via the fecal-oral route through contaminated drinking water, whereas in developed countries, the transmission occurs by consumption of uncooked or undercooked meat from infected animals. There is no specific treatment for HEV infection, apart from a vaccine which is available only in China. Therefore, HEV represents a public health problem which is constantly growing around the world. HEV is small, icosahedral virus with 27 to 34 nm diameter and classified in the Hepeviridae family. Virions can be found as quasi-enveloped, by host-cell-derived membranes, in bloodstream or as non-enveloped in the faeces and bile of infected individuals. The virus contains a ~7.2 kb positive-sense, single-stranded RNA genome which comprises three open reading frames: ORF1, ORF2 and ORF3. ORF1 encodes a non-structural polyprotein that includes multiple functional domains responsible for the replication of the viral genome. ORF2 encodes the viral capsid protein that assembles to make the viral particles. ORF3 encodes a small regulatory multifunctional protein which is poorly characterized. Previous studies have shown that ORF3 is mainly involved in the release of the infectious viral particles and interacts with other viral and host proteins inside the cell. This small protein is thought to interact with the human Ubiquitin E2 variant (UEV) domain of Tsg101 protein (Tsg101 UEV), a member of endosomal sorting complex required for transport-I (ESCRT-I), which has been involved in the release of HIV, Ebola and other viruses. Previous studies have also shown that ORF3 protein is associated with the cellular membranes either via its oligomerization and transmembrane insertion, or via palmitoylation of its N-terminal Cysteine-rich region.In this study, a detailed molecular characterization of the ORF3 protein in order to decipher its functional role(s) during the HEV life cycle is achieved using NMR spectroscopy and other biophysical techniques. Firstly, we performed HEV ORF3 recombinant expression in. E. coli and purified the protein. Its structural characterization by solution-state NMR spectroscopy shown that it is an intrinsically disordered protein devoid of any stable 3D structure organization. Secondly, for the further determination of the ORF3 protein membrane association, the Nanodisc (ND) technology is used which mimics a bilayer membrane and thus, is closed to the native environment of membrane proteins. Although the attachment of the protein into a ND assembly was successful, a reliable conclusion about the membrane attachment of the protein cannot be drawn based on our experimental results. Finally, we in-depth characterized the molecular interaction between HEV ORF3 and Tsg101 UEV proteins. We identified the binding sites in both ORF3 and Tsg101 UEV proteins by NMR spectroscopy, we measured the affinity of the interaction using Isothermal Titration Calorimetry (ITC) and finally a high-resolution atomic structure of the complex between Tsg101 UEV protein and a 10-residues ORF3-derived peptide was solved by X-ray crystallography. In addition, we used our biochemical and structural data to start the setup of an experimental assay that could be used to detect potential antiviral compounds targeting the ORF3-Tsg101 interaction.Le virus de l'hépatite E (VHE) est la cause la plus fréquente d'hépatite virale aiguë dans le monde avec plus de 20 millions d'infections et environ 44 000 décès enregistrés chaque année. A ce jour, 8 génotypes de VHE ont été identifiés, seuls les quatre premiers génotypes infectent les humains. Dans les pays en développement, le VHE se transmet par voie fécale-orale principalement via de l'eau contaminée, alors que dans les pays développés, la transmission se fait par l'intermédiaire de la consommation de viande crue ou insuffisamment cuite provenant d'animaux infectés. Il n'existe pas de traitement spécifique pour l'infection par le VHE, à part un vaccin qui n'est disponible qu'en Chine. Le VHE représente par conséquent un problème de santé publique qui ne cesse de croître dans le monde. Le VHE est un petit virus icosaédrique de 27 à 34 nm de diamètre qui est classé dans la famille des Hepeviridae. Les virions peuvent être trouvés quasi-enveloppés, par des membranes dérivées de la cellule hôte, dans la circulation sanguine ou non enveloppés dans les selles et la bile des individus infectés. Le virus contient un génome à ARN simple brin positif d'environ 7.2 kb qui comprend trois cadres de lecture ouverts : ORF1, ORF2 et ORF3. ORF1 code pour une polyprotéine non structurale qui comprend plusieurs domaines fonctionnels responsables de la réplication du génome viral. ORF2 code la protéine de capside virale qui s'assemble pour former les particules virales. ORF3 code pour une petite protéine multifonctionnelle régulatrice qui est mal caractérisée. Des études antérieures ont montré que l'ORF3 est principalement impliquée dans la libération des particules virales infectieuses et qu'elle interagit avec d'autres protéines virales et hôtes à l'intérieur de la cellule. Cette petite protéine pourrait interagir avec le domaine de la variante humaine de l'ubiquitine E2 (UEV) de la protéine Tsg101 (Tsg101 UEV), un membre du ‘endosomal sorting complex required for transport-I' (ESCRT-I), qui a été impliqué dans la libération d'autres virus comme le VIH et Ebola. Des études antérieures ont également montré que la protéine ORF3 est associée aux membranes cellulaires soit via son oligomérisation et son insertion transmembranaire, soit via la palmitoylation de sa région N-terminale riche en cystéine.Dans cette étude, une caractérisation moléculaire détaillée de la protéine ORF3 afin de déchiffrer son ou ses rôles fonctionnels au cours du cycle de vie du VHE est réalisée à l'aide de la spectroscopie RMN et d'autres techniques biophysiques. Tout d'abord, nous avons produit l'ORF3 du VHE dans E. coli puis purifié cette protéine recombinante. Sa caractérisation structurale par spectroscopie RMN en solution a montré qu'il s'agit d'une protéine intrinsèquement désordonnée dépourvue de structure 3D stable. Ensuite, pour un examen plus poussé de l'association membranaire de la protéine ORF3, la technologie Nanodisque (ND) qui imite une membrane bicouche physiologique a été utilisée. Bien que l'attachement de la protéine dans un ND ait réussi, une conclusion fiable sur le mode de fixation membranaire de ORF3 n'a pu être tirée sur la base de nos résultats expérimentaux. Enfin, nous avons caractérisé en détail l'interaction moléculaire entre les protéines HEV ORF3 et Tsg101 UEV. Nous avons identifié les sites de liaison dans les protéines ORF3 et Tsg101 UEV par spectroscopie RMN, nous avons mesuré l'affinité de l'interaction à l'aide de la calorimétrie de titrage isotherme (ITC) et enfin une structure atomique à haute résolution du complexe entre la protéine Tsg101 UEV et un peptide de 10 résidus dérivé de ORF3 a été résolue par cristallographie aux rayons X. Finalement, nous avons utilisé nos données biochimiques et structurales pour mettre au point un test expérimental qui pourrait être utilisé pour détecter des composés antiviraux potentiels ciblant l'interaction ORF3-Tsg101

Etude structurale et fonctionnelle de la protéine ORF3 du VHE par RMN en solution

Hepatitis E Virus (HEV) is the most common cause of acute viral hepatitis worldwide with over 20 million infections and around 44,000 deaths recorded annually. Until today, 8 HEV genotypes are reported with only the first four genotypes infecting humans. In developing countries, HEV is transmitted via the fecal-oral route through contaminated drinking water, whereas in developed countries, the transmission occurs by consumption of uncooked or undercooked meat from infected animals. There is no specific treatment for HEV infection, apart from a vaccine which is available only in China. Therefore, HEV represents a public health problem which is constantly growing around the world. HEV is small, icosahedral virus with 27 to 34 nm diameter and classified in the Hepeviridae family. Virions can be found as quasi-enveloped, by host-cell-derived membranes, in bloodstream or as non-enveloped in the faeces and bile of infected individuals. The virus contains a ~7.2 kb positive-sense, single-stranded RNA genome which comprises three open reading frames: ORF1, ORF2 and ORF3. ORF1 encodes a non-structural polyprotein that includes multiple functional domains responsible for the replication of the viral genome. ORF2 encodes the viral capsid protein that assembles to make the viral particles. ORF3 encodes a small regulatory multifunctional protein which is poorly characterized. Previous studies have shown that ORF3 is mainly involved in the release of the infectious viral particles and interacts with other viral and host proteins inside the cell. This small protein is thought to interact with the human Ubiquitin E2 variant (UEV) domain of Tsg101 protein (Tsg101 UEV), a member of endosomal sorting complex required for transport-I (ESCRT-I), which has been involved in the release of HIV, Ebola and other viruses. Previous studies have also shown that ORF3 protein is associated with the cellular membranes either via its oligomerization and transmembrane insertion, or via palmitoylation of its N-terminal Cysteine-rich region.In this study, a detailed molecular characterization of the ORF3 protein in order to decipher its functional role(s) during the HEV life cycle is achieved using NMR spectroscopy and other biophysical techniques. Firstly, we performed HEV ORF3 recombinant expression in. E. coli and purified the protein. Its structural characterization by solution-state NMR spectroscopy shown that it is an intrinsically disordered protein devoid of any stable 3D structure organization. Secondly, for the further determination of the ORF3 protein membrane association, the Nanodisc (ND) technology is used which mimics a bilayer membrane and thus, is closed to the native environment of membrane proteins. Although the attachment of the protein into a ND assembly was successful, a reliable conclusion about the membrane attachment of the protein cannot be drawn based on our experimental results. Finally, we in-depth characterized the molecular interaction between HEV ORF3 and Tsg101 UEV proteins. We identified the binding sites in both ORF3 and Tsg101 UEV proteins by NMR spectroscopy, we measured the affinity of the interaction using Isothermal Titration Calorimetry (ITC) and finally a high-resolution atomic structure of the complex between Tsg101 UEV protein and a 10-residues ORF3-derived peptide was solved by X-ray crystallography. In addition, we used our biochemical and structural data to start the setup of an experimental assay that could be used to detect potential antiviral compounds targeting the ORF3-Tsg101 interaction.Le virus de l'hépatite E (VHE) est la cause la plus fréquente d'hépatite virale aiguë dans le monde avec plus de 20 millions d'infections et environ 44 000 décès enregistrés chaque année. A ce jour, 8 génotypes de VHE ont été identifiés, seuls les quatre premiers génotypes infectent les humains. Dans les pays en développement, le VHE se transmet par voie fécale-orale principalement via de l'eau contaminée, alors que dans les pays développés, la transmission se fait par l'intermédiaire de la consommation de viande crue ou insuffisamment cuite provenant d'animaux infectés. Il n'existe pas de traitement spécifique pour l'infection par le VHE, à part un vaccin qui n'est disponible qu'en Chine. Le VHE représente par conséquent un problème de santé publique qui ne cesse de croître dans le monde. Le VHE est un petit virus icosaédrique de 27 à 34 nm de diamètre qui est classé dans la famille des Hepeviridae. Les virions peuvent être trouvés quasi-enveloppés, par des membranes dérivées de la cellule hôte, dans la circulation sanguine ou non enveloppés dans les selles et la bile des individus infectés. Le virus contient un génome à ARN simple brin positif d'environ 7.2 kb qui comprend trois cadres de lecture ouverts : ORF1, ORF2 et ORF3. ORF1 code pour une polyprotéine non structurale qui comprend plusieurs domaines fonctionnels responsables de la réplication du génome viral. ORF2 code la protéine de capside virale qui s'assemble pour former les particules virales. ORF3 code pour une petite protéine multifonctionnelle régulatrice qui est mal caractérisée. Des études antérieures ont montré que l'ORF3 est principalement impliquée dans la libération des particules virales infectieuses et qu'elle interagit avec d'autres protéines virales et hôtes à l'intérieur de la cellule. Cette petite protéine pourrait interagir avec le domaine de la variante humaine de l'ubiquitine E2 (UEV) de la protéine Tsg101 (Tsg101 UEV), un membre du ‘endosomal sorting complex required for transport-I' (ESCRT-I), qui a été impliqué dans la libération d'autres virus comme le VIH et Ebola. Des études antérieures ont également montré que la protéine ORF3 est associée aux membranes cellulaires soit via son oligomérisation et son insertion transmembranaire, soit via la palmitoylation de sa région N-terminale riche en cystéine.Dans cette étude, une caractérisation moléculaire détaillée de la protéine ORF3 afin de déchiffrer son ou ses rôles fonctionnels au cours du cycle de vie du VHE est réalisée à l'aide de la spectroscopie RMN et d'autres techniques biophysiques. Tout d'abord, nous avons produit l'ORF3 du VHE dans E. coli puis purifié cette protéine recombinante. Sa caractérisation structurale par spectroscopie RMN en solution a montré qu'il s'agit d'une protéine intrinsèquement désordonnée dépourvue de structure 3D stable. Ensuite, pour un examen plus poussé de l'association membranaire de la protéine ORF3, la technologie Nanodisque (ND) qui imite une membrane bicouche physiologique a été utilisée. Bien que l'attachement de la protéine dans un ND ait réussi, une conclusion fiable sur le mode de fixation membranaire de ORF3 n'a pu être tirée sur la base de nos résultats expérimentaux. Enfin, nous avons caractérisé en détail l'interaction moléculaire entre les protéines HEV ORF3 et Tsg101 UEV. Nous avons identifié les sites de liaison dans les protéines ORF3 et Tsg101 UEV par spectroscopie RMN, nous avons mesuré l'affinité de l'interaction à l'aide de la calorimétrie de titrage isotherme (ITC) et enfin une structure atomique à haute résolution du complexe entre la protéine Tsg101 UEV et un peptide de 10 résidus dérivé de ORF3 a été résolue par cristallographie aux rayons X. Finalement, nous avons utilisé nos données biochimiques et structurales pour mettre au point un test expérimental qui pourrait être utilisé pour détecter des composés antiviraux potentiels ciblant l'interaction ORF3-Tsg101

Etude structurale et fonctionnelle de la protéine ORF3 du VHE par RMN en solution

Le virus de l'hépatite E (VHE) est la cause la plus fréquente d'hépatite virale aiguë dans le monde avec plus de 20 millions d'infections et environ 44 000 décès enregistrés chaque année. A ce jour, 8 génotypes de VHE ont été identifiés, seuls les quatre premiers génotypes infectent les humains. Dans les pays en développement, le VHE se transmet par voie fécale-orale principalement via de l'eau contaminée, alors que dans les pays développés, la transmission se fait par l'intermédiaire de la consommation de viande crue ou insuffisamment cuite provenant d'animaux infectés. Il n'existe pas de traitement spécifique pour l'infection par le VHE, à part un vaccin qui n'est disponible qu'en Chine. Le VHE représente par conséquent un problème de santé publique qui ne cesse de croître dans le monde. Le VHE est un petit virus icosaédrique de 27 à 34 nm de diamètre qui est classé dans la famille des Hepeviridae. Les virions peuvent être trouvés quasi-enveloppés, par des membranes dérivées de la cellule hôte, dans la circulation sanguine ou non enveloppés dans les selles et la bile des individus infectés. Le virus contient un génome à ARN simple brin positif d'environ 7.2 kb qui comprend trois cadres de lecture ouverts : ORF1, ORF2 et ORF3. ORF1 code pour une polyprotéine non structurale qui comprend plusieurs domaines fonctionnels responsables de la réplication du génome viral. ORF2 code la protéine de capside virale qui s'assemble pour former les particules virales. ORF3 code pour une petite protéine multifonctionnelle régulatrice qui est mal caractérisée. Des études antérieures ont montré que l'ORF3 est principalement impliquée dans la libération des particules virales infectieuses et qu'elle interagit avec d'autres protéines virales et hôtes à l'intérieur de la cellule. Cette petite protéine pourrait interagir avec le domaine de la variante humaine de l'ubiquitine E2 (UEV) de la protéine Tsg101 (Tsg101 UEV), un membre du ‘endosomal sorting complex required for transport-I' (ESCRT-I), qui a été impliqué dans la libération d'autres virus comme le VIH et Ebola. Des études antérieures ont également montré que la protéine ORF3 est associée aux membranes cellulaires soit via son oligomérisation et son insertion transmembranaire, soit via la palmitoylation de sa région N-terminale riche en cystéine.Dans cette étude, une caractérisation moléculaire détaillée de la protéine ORF3 afin de déchiffrer son ou ses rôles fonctionnels au cours du cycle de vie du VHE est réalisée à l'aide de la spectroscopie RMN et d'autres techniques biophysiques. Tout d'abord, nous avons produit l'ORF3 du VHE dans E. coli puis purifié cette protéine recombinante. Sa caractérisation structurale par spectroscopie RMN en solution a montré qu'il s'agit d'une protéine intrinsèquement désordonnée dépourvue de structure 3D stable. Ensuite, pour un examen plus poussé de l'association membranaire de la protéine ORF3, la technologie Nanodisque (ND) qui imite une membrane bicouche physiologique a été utilisée. Bien que l'attachement de la protéine dans un ND ait réussi, une conclusion fiable sur le mode de fixation membranaire de ORF3 n'a pu être tirée sur la base de nos résultats expérimentaux. Enfin, nous avons caractérisé en détail l'interaction moléculaire entre les protéines HEV ORF3 et Tsg101 UEV. Nous avons identifié les sites de liaison dans les protéines ORF3 et Tsg101 UEV par spectroscopie RMN, nous avons mesuré l'affinité de l'interaction à l'aide de la calorimétrie de titrage isotherme (ITC) et enfin une structure atomique à haute résolution du complexe entre la protéine Tsg101 UEV et un peptide de 10 résidus dérivé de ORF3 a été résolue par cristallographie aux rayons X. Finalement, nous avons utilisé nos données biochimiques et structurales pour mettre au point un test expérimental qui pourrait être utilisé pour détecter des composés antiviraux potentiels ciblant l'interaction ORF3-Tsg101.Hepatitis E Virus (HEV) is the most common cause of acute viral hepatitis worldwide with over 20 million infections and around 44,000 deaths recorded annually. Until today, 8 HEV genotypes are reported with only the first four genotypes infecting humans. In developing countries, HEV is transmitted via the fecal-oral route through contaminated drinking water, whereas in developed countries, the transmission occurs by consumption of uncooked or undercooked meat from infected animals. There is no specific treatment for HEV infection, apart from a vaccine which is available only in China. Therefore, HEV represents a public health problem which is constantly growing around the world. HEV is small, icosahedral virus with 27 to 34 nm diameter and classified in the Hepeviridae family. Virions can be found as quasi-enveloped, by host-cell-derived membranes, in bloodstream or as non-enveloped in the faeces and bile of infected individuals. The virus contains a ~7.2 kb positive-sense, single-stranded RNA genome which comprises three open reading frames: ORF1, ORF2 and ORF3. ORF1 encodes a non-structural polyprotein that includes multiple functional domains responsible for the replication of the viral genome. ORF2 encodes the viral capsid protein that assembles to make the viral particles. ORF3 encodes a small regulatory multifunctional protein which is poorly characterized. Previous studies have shown that ORF3 is mainly involved in the release of the infectious viral particles and interacts with other viral and host proteins inside the cell. This small protein is thought to interact with the human Ubiquitin E2 variant (UEV) domain of Tsg101 protein (Tsg101 UEV), a member of endosomal sorting complex required for transport-I (ESCRT-I), which has been involved in the release of HIV, Ebola and other viruses. Previous studies have also shown that ORF3 protein is associated with the cellular membranes either via its oligomerization and transmembrane insertion, or via palmitoylation of its N-terminal Cysteine-rich region.In this study, a detailed molecular characterization of the ORF3 protein in order to decipher its functional role(s) during the HEV life cycle is achieved using NMR spectroscopy and other biophysical techniques. Firstly, we performed HEV ORF3 recombinant expression in. E. coli and purified the protein. Its structural characterization by solution-state NMR spectroscopy shown that it is an intrinsically disordered protein devoid of any stable 3D structure organization. Secondly, for the further determination of the ORF3 protein membrane association, the Nanodisc (ND) technology is used which mimics a bilayer membrane and thus, is closed to the native environment of membrane proteins. Although the attachment of the protein into a ND assembly was successful, a reliable conclusion about the membrane attachment of the protein cannot be drawn based on our experimental results. Finally, we in-depth characterized the molecular interaction between HEV ORF3 and Tsg101 UEV proteins. We identified the binding sites in both ORF3 and Tsg101 UEV proteins by NMR spectroscopy, we measured the affinity of the interaction using Isothermal Titration Calorimetry (ITC) and finally a high-resolution atomic structure of the complex between Tsg101 UEV protein and a 10-residues ORF3-derived peptide was solved by X-ray crystallography. In addition, we used our biochemical and structural data to start the setup of an experimental assay that could be used to detect potential antiviral compounds targeting the ORF3-Tsg101 interaction

NMR spectroscopy of the main protease of SARS‐CoV‐2 and fragment‐based screening identify three protein hotspots and an antiviral fragment

International audienceThe main protease (3CLp) of the SARS-CoV-2, the causative agent for the COVID-19 pandemic, is one of the main targets for drug development. To be active, 3CLp relies on a complex interplay between dimerization, active site flexibility, and allosteric regulation. The deciphering of these mechanisms is a crucial step to enable the search for inhibitors. In this context, using NMR spectroscopy, we studied the conformation of dimeric 3CLp from the SARS-CoV-2 and monitored ligand binding, based on NMR signal assignments. We performed a fragment-based screening that led to the identification of 38 fragment hits. Their binding sites showed three hotspots on 3CLp, two in the substrate binding pocket and one at the dimer interface. F01 is a non-covalent inhibitor of the 3CLp and has antiviral activity in SARS-CoV-2 infected cells. This study sheds light on the complex structure-function relationships of 3CLp, and constitutes a strong basis to assist in developing potent 3CLp inhibitors

Computational design of closely related proteins that adopt two well-defined but structurally divergent folds.

The plasticity of naturally occurring protein structures, which can change shape considerably in response to changes in environmental conditions, is critical to biological function. While computational methods have been used for de novo design of proteins that fold to a single state with a deep free-energy minimum [P.-S. Huang, S. E. Boyken, D. Baker, Nature 537, 320-327 (2016)], and to reengineer natural proteins to alter their dynamics [J. A. Davey, A. M. Damry, N. K. Goto, R. A. Chica, Nat. Chem. Biol. 13, 1280-1285 (2017)] or fold [P. A. Alexander, Y. He, Y. Chen, J. Orban, P. N. Bryan, Proc. Natl. Acad. Sci. U.S.A. 106, 21149-21154 (2009)], the de novo design of closely related sequences which adopt well-defined but structurally divergent structures remains an outstanding challenge. We designed closely related sequences (over 94% identity) that can adopt two very different homotrimeric helical bundle conformations-one short (∼66 Å height) and the other long (∼100 Å height)-reminiscent of the conformational transition of viral fusion proteins. Crystallographic and NMR spectroscopic characterization shows that both the short- and long-state sequences fold as designed. We sought to design bistable sequences for which both states are accessible, and obtained a single designed protein sequence that populates either the short state or the long state depending on the measurement conditions. The design of sequences which are poised to adopt two very different conformations sets the stage for creating large-scale conformational switches between structurally divergent forms

Computational design of closely related proteins that adopt two well-defined but structurally divergent folds.

The plasticity of naturally occurring protein structures, which can change shape considerably in response to changes in environmental conditions, is critical to biological function. While computational methods have been used for de novo design of proteins that fold to a single state with a deep free-energy minimum [P.-S. Huang, S. E. Boyken, D. Baker, Nature 537, 320-327 (2016)], and to reengineer natural proteins to alter their dynamics [J. A. Davey, A. M. Damry, N. K. Goto, R. A. Chica, Nat. Chem. Biol. 13, 1280-1285 (2017)] or fold [P. A. Alexander, Y. He, Y. Chen, J. Orban, P. N. Bryan, Proc. Natl. Acad. Sci. U.S.A. 106, 21149-21154 (2009)], the de novo design of closely related sequences which adopt well-defined but structurally divergent structures remains an outstanding challenge. We designed closely related sequences (over 94% identity) that can adopt two very different homotrimeric helical bundle conformations-one short (∼66 Å height) and the other long (∼100 Å height)-reminiscent of the conformational transition of viral fusion proteins. Crystallographic and NMR spectroscopic characterization shows that both the short- and long-state sequences fold as designed. We sought to design bistable sequences for which both states are accessible, and obtained a single designed protein sequence that populates either the short state or the long state depending on the measurement conditions. The design of sequences which are poised to adopt two very different conformations sets the stage for creating large-scale conformational switches between structurally divergent forms