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The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key enzyme, which extensively digests CoV replicase polyproteins essential for viral replication and transcription, making it an attractive target for antiviral drug development. However, the molecular mechanism of how Mpro of SARS-CoV-2 digests replicase polyproteins, releasing the nonstructural proteins (nsps), and its substrate specificity remain largely unknown. Here, we determine the high-resolution structures of SARS-CoV-2 Mpro in its resting state, precleavage state, and postcleavage state, constituting a full cycle of substrate cleavage. The structures show the delicate conformational changes that occur during polyprotein processing. Further, we solve the structures of the SARS-CoV-2 Mpro mutant (H41A) in complex with six native cleavage substrates from replicase polyproteins, and demonstrate that SARS-CoV-2 Mpro can recognize sequences as long as 10 residues but only have special selectivity for four subsites. These structural data provide a basis to develop potent new inhibitors against SARS-CoV-2.

Thousands of proteins have been validated genetically as therapeutic targets for human diseases 1 . However, very few have been successfully targeted, and many are considered ‘undruggable’. This is particularly true for proteins that function via protein–protein interactions—direct inhibition of binding interfaces is difficult and requires the identification of allosteric sites. However, most proteins have no known allosteric sites, and a comprehensive allosteric map does not exist for any protein. Here we address this shortcoming by charting multiple global atlases of inhibitory allosteric communication in KRAS. We quantified the effects of more than 26,000 mutations on the folding of KRAS and its binding to six interaction partners. Genetic interactions in double mutants enabled us to perform biophysical measurements at scale, inferring more than 22,000 causal free energy changes. These energy landscapes quantify how mutations tune the binding specificity of a signalling protein and map the inhibitory allosteric sites for an important therapeutic target. Allosteric propagation is particularly effective across the central β-sheet of KRAS, and multiple surface pockets are genetically validated as allosterically active, including a distal pocket in the C-terminal lobe of the protein. Allosteric mutations typically inhibit binding to all tested effectors, but they can also change the binding specificity, revealing the regulatory, evolutionary and therapeutic potential to tune pathway activation. Using the approach described here, it should be possible to rapidly and comprehensively identify allosteric target sites in many proteins. Analysis of the effects of more than 26,000 KRAS mutations on abundance and interactions with six other proteins is used to construct an energy landscape of KRAS and identify allosteric drug target sites.

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.

In eukaryotes, hydrogen sulphide acts as a signalling molecule and cytoprotectant. Hydrogen sulphide is known to be produced from L- cysteine by cystathionine β-synthase, cystathionine γ-lyase and 3-mercaptopyruvate sulfurtransferase coupled with cysteine aminotransferase. Here we report an additional biosynthetic pathway for the production of hydrogen sulphide from D- cysteine involving 3-mercaptopyruvate sulfurtransferase and D -amino acid oxidase. Unlike the L- cysteine pathway, this D -cysteine-dependent pathway operates predominantly in the cerebellum and the kidney. Our study reveals that administration of D -cysteine protects primary cultures of cerebellar neurons from oxidative stress induced by hydrogen peroxide and attenuates ischaemia-reperfusion injury in the kidney more than L- cysteine. This study presents a novel pathway of hydrogen sulphide production and provides a new therapeutic approach to deliver hydrogen sulphide to specific tissues. Hydrogen sulphide is a signalling molecule with cytoprotective activity in mammals. Here, Kimura and colleagues identify a new biosynthetic pathway for the production of hydrogen sulphide from D -cysteine, which is shown to protect mouse kidneys from oxidative stress after ischaemia/reperfusion injury.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic toxicity associated with prominent lipid accumulation in humans. Here, the authors report that the lysosomal copper transporter SLC46A3 is induced by TCDD and underlies the hepatic lipid accumulation in mice, potentially via effects on mitochondrial function. SLC46A3 was localized to the lysosome where it modulated intracellular copper levels. Forced expression of hepatic SLC46A3 resulted in decreased mitochondrial membrane potential and abnormal mitochondria morphology consistent with lower copper levels. SLC46A3 expression increased hepatic lipid accumulation similar to the known effects of TCDD exposure in mice and humans. The TCDD-induced hepatic triglyceride accumulation was significantly decreased in Slc46a3 −/− mice and was more pronounced when these mice were fed a high-fat diet, as compared to wild-type mice. These data are consistent with a model where lysosomal SLC46A3 induction by TCDD leads to cytosolic copper deficiency resulting in mitochondrial dysfunction leading to lower lipid catabolism, thus linking copper status to mitochondrial function, lipid metabolism and TCDD-induced liver toxicity. The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic toxicity associated with prominent lipid accumulation in humans. Here, the authors report that the lysosomal copper transporter SLC46A3 is induced by TCDD and underlies the hepatic lipid accumulation in mice, potentially via effects on mitochondrial function.

Citrate is best known as an intermediate in the tricarboxylic acid cycle of the cell. In addition to this essential role in energy metabolism, the tricarboxylate anion also acts as both a precursor and a regulator of fatty acid synthesis 1 – 3 . Thus, the rate of fatty acid synthesis correlates directly with the cytosolic concentration of citrate 4 , 5 . Liver cells import citrate through the sodium-dependent citrate transporter NaCT (encoded by SLC13A5 ) and, as a consequence, this protein is a potential target for anti-obesity drugs. Here, to understand the structural basis of its inhibition mechanism, we determined cryo-electron microscopy structures of human NaCT in complexes with citrate or a small-molecule inhibitor. These structures reveal how the inhibitor—which binds to the same site as citrate—arrests the transport cycle of NaCT. The NaCT–inhibitor structure also explains why the compound selectively inhibits NaCT over two homologous human dicarboxylate transporters, and suggests ways to further improve the affinity and selectivity. Finally, the NaCT structures provide a framework for understanding how various mutations abolish the transport activity of NaCT in the brain and thereby cause epilepsy associated with mutations in SLC13A5 in newborns (which is known as SLC13A5-epilepsy) 6 – 8 . Structures of the human sodium-dependent citrate transporter NaCT in complexes with citrate or a small-molecule inhibitor reveal how the inhibitor—which binds to the same site as citrate—arrests the transport cycle of NaCT.

The altered activity of the fructose transporter GLUT5, an isoform of the facilitated-diffusion glucose transporter family, has been linked to disorders such as type 2 diabetes and obesity. GLUT5 is also overexpressed in certain tumour cells, and inhibitors are potential drugs for these conditions. Here we describe the crystal structures of GLUT5 from Rattus norvegicus and Bos taurus in open outward- and open inward-facing conformations, respectively. GLUT5 has a major facilitator superfamily fold like other homologous monosaccharide transporters. On the basis of a comparison of the inward-facing structures of GLUT5 and human GLUT1, a ubiquitous glucose transporter, we show that a single point mutation is enough to switch the substrate-binding preference of GLUT5 from fructose to glucose. A comparison of the substrate-free structures of GLUT5 with occluded substrate-bound structures of Escherichia coli XylE suggests that, in addition to global rocker-switch-like re-orientation of the bundles, local asymmetric rearrangements of carboxy-terminal transmembrane bundle helices TM7 and TM10 underlie a ‘gated-pore’ transport mechanism in such monosaccharide transporters. This study has determined the X-ray crystal structures of GLUT5 from Rattus norvegicus in an open, outward-facing conformation and GLUT5 from Bos taurus in an open, inward-facing conformation; comparison of these structures with previously published structures of the related Escherichia coli d -xylose:H + symporter XylE suggests that transport in GLUT5 is controlled by both a global ‘rocker-switch’-type motion and a local ‘gated-pore’-type transport mechanism. Structure of fructose transporter GLUT5 SLC2 family glucose transporters (GLUTs) facilitate the transport of glucose and other monosaccharides across biological membranes. GLUT5, which is fructose-specific, has been linked to disorders such as type 2 diabetes and obesity and is overexpressed in certain tumour cells. The authors solve X-ray crystal structures of GLUT5 from the brown rat in an open, outward-facing conformation and GLUT5 from cattle in an open, inward-facing conformation. Comparison of these structures with previously published structures of the related XylE, a proton-coupled sugar transporter from Escherichia coli , suggest that transport in GLUT5 is controlled by both 'rocker-switch' and 'gated-pore' type transport mechanisms. Also in this issue of Nature , Dong Deng et al . solve the X-ray crystal structures of human GLUT3 in outward-open and outward-occluded conformations.

Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo–electron microscopy of apo human LAT2/CD98hc and a multi-disciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies.

β-Glucosidases (3.2.1.21) are found in all domains of living organisms, where they play essential roles in the removal of nonreducing terminal glucosyl residues from saccharides and glycosides. β-Glucosidases function in glycolipid and exogenous glycoside metabolism in animals, defense, cell wall lignification, cell wall β-glucan turnover, phytohormone activation, and release of aromatic compounds in plants, and biomass conversion in microorganisms. These functions lead to many agricultural and industrial applications. β-Glucosidases have been classified into glycoside hydrolase (GH) families GH1, GH3, GH5, GH9, and GH30, based on their amino acid sequences, while other β-glucosidases remain to be classified. The GH1, GH5, and GH30 β-glucosidases fall in GH Clan A, which consists of proteins with (β/α) 8 -barrel structures. In contrast, the active site of GH3 enzymes comprises two domains, while GH9 enzymes have (α/α) 6 barrel structures. The mechanism by which GH1 enzymes recognize and hydrolyze substrates with different specificities remains an area of intense study.

Substrate specificity is a defining feature of enzyme function, but its molecular underpinnings remain difficult to decode and engineer. Here, we leverage enzyme proximity sequencing (EP-Seq) to systematically map how single-point and combinatorial mutations reshape the substrate preferences of D-amino acid oxidase (DAOx) from Rhodotorula gracilis , a model promiscuous enzyme. We generate ~40,000 sequence–phenotype pairs, enabling us to profile the activities of ~6,500 unique DAOx variants against five D-amino acid substrates with distinct physicochemical properties. Our analysis reveals that substrate-specific mutations are distributed throughout the enzyme structure. Mutations near the active site drive strong specificity shifts but also incur catalytic penalties, while distal mutations subtly rewire intramolecular contacts in order to modulate specificity with minimal loss of activity. We identify and validate positional hotspots that act allosterically to influence specificity, and characterize key variants that acquire exclusive substrate specificity or exhibit up to 230-fold changes in substrate preference. Combining mutations with complementary effects further sharpens substrate discrimination, enabling rational design of highly selective biocatalysts. This work establishes a powerful framework for decoding enzyme specificity and provides foundational datasets to advance AI-guided enzyme engineering. Using mutational scanning, this study maps how thousands of mutations alter substrate specificity in the promiscuous enzyme D-amino acid oxidase, and reveals catalytic-site and long-range effects that inform the design of highly selective biocatalysts.