Cofactor Binding Research Articles

Structural Modifications Introduced by NS2B Cofactor Binding to the NS3 Protease of the Kyasanur Forest Disease Virus

Kyasanur Forest Disease virus (KFDV), a neglected human pathogenic virus, is a Flavivirus that causes severe hemorrhagic fever in humans. KFDV is transmitted to humans by the bite of the hard tick (Haemaphysalis spinigera), which acts as a reservoir of KFDV. The recent expansion of the endemic area of KFDV is of concern and requires the development of new preventive measures against KFDV. Currently, there is no antiviral therapy against KFDV, and the existing vaccine has limited efficacy. To develop a new antiviral therapy against KFDV, we focused on the nonstructural proteins NS2B and NS3 of KFDV, which are responsible for serine protease activity. Viral proteases have shown to be suitable therapeutic targets in the development of antiviral drugs against many diseases. However, success has been limited in flaviviruses, mainly because of the important features of the active site, which is flat and highly charged. In this context, the present study focuses on the dynamics of NS2B and NS3 to identify potential allosteric sites in the NS2B/NS3 protease of KDFV. To our knowledge, there are no reports on the dynamics of NS2B and NS3 in KFDV, and the crystal structure of the NS2B/NS3 protease of KFDV has not yet been solved. Overall, we created the structure of the NS2B/NS3 protease of KFDV using AlphaFold and performed molecular dynamics simulations with and without NS2B cofactor to investigate structural rearrangements due to cofactor binding and to identify alternative allosteric sites. The identified allosteric site is promising due to its geometric and physicochemical properties and druggability and can be used for new drug development. The applicability of the proposed allosteric binding sites was verified for the best-hit molecules from the virtual screening and MD simulations.

Crystal Structures of 6-Phosphogluconate Dehydrogenase from Corynebacterium glutamicum.

Corynebacterium glutamicum (C. glutamicum) has been considered a very important and meaningful industrial microorganism for the production of amino acids worldwide. To produce amino acids, cells require nicotinamide adenine dinucleotide phosphate (NADPH), which is a biological reducing agent. The pentose phosphate pathway (PPP) can supply NADPH in cells via the 6-phosphogluconate dehydrogenase (6PGD) enzyme, which is an oxidoreductase that converts 6-phosphogluconate (6PG) to ribulose 5-phosphate (Ru5P), to produce NADPH. In this study, we identified the crystal structure of 6PGD_apo and 6PGD_NADP from C. glutamicum ATCC 13032 (Cg6PGD) and reported our biological research based on this structure. We identified the substrate binding site and co-factor binding site of Cg6PGD, which are crucial for understanding this enzyme. Based on the findings of our research, Cg6PGD is expected to be used as a NADPH resource in the food industry and as a drug target in the pharmaceutical industry.

1589-P: Portal Serotonin Is Critical for Efficient Hepatic Control of Systemic Glycemia

There is growing evidence for a portal factor, released following a meal, that combined with insulin, induces hepatic glycogen synthesis. We have developed an in vitro model that demonstrates that serotonin could be the portal factor. Using rat primary hepatocyte cultures in 6 well plates with 750,000 cells per well and a 4-hour treatment time, we observed that 0.1 mU/ml of human insulin resulted in generation of 8.8 ± 2.3 ng glycogen/ug total protein compared to 6.7 ± 1.0 ng glycogen/ug total protein, with 5 uM 5HT-serotonin. Interestingly, the combination of 0.1 mU insulin/ml and 5 uM 5HT-serotonin resulted in 22.5 ± 3.8 ng glycogen/ug total protein, a 256% increase in glycogen synthesis compared to insulin alone. In the absence of serotonin, it takes approximately 10-fold greater insulin to achieve the same levels of glycogen synthesis as insulin with serotonin. These data combined with the observation that 95% of serotonin is made in the gut, that gastrointestinal serotonin is released upon food ingestion and the growing evidence that obese subjects have decreasing levels of circulating serotonin, suggest that gastrointestinal serotonin could be the portal factor necessary for efficient hepatic control of system glycemia. These data further suggest that insulin insensitivity is due to loss of a critical co-factor binding, and that this co-factor is serotonin. In summary, type 1 diabetes is due to a deficiency of insulin and type 2 diabetes may be due to a deficiency of its co-factor, portal serotonin. Disclosure M.Kiedrowski: None. C.El sanadi: None. M.S.Penn: Consultant; Diasome, Quest Diagnostics. W.Geho: None.

Identifying the Molecular Drivers of Pathogenic Aldehyde Dehydrogenase Missense Mutations in Cancer and Non-Cancer Diseases.

Human aldehyde dehydrogenases (ALDHs) comprising 19 isoenzymes play a vital role on both endogenous and exogenous aldehyde metabolism. This NAD(P)-dependent catalytic process relies on the intact structural and functional activity of the cofactor binding, substrate interaction, and the oligomerization of ALDHs. Disruptions on the activity of ALDHs, however, could result in the accumulation of cytotoxic aldehydes, which have been linked with a wide range of diseases, including both cancers as well as neurological and developmental disorders. In our previous works, we have successfully characterised the structure-function relationships of the missense variants of other proteins. We, therefore, applied a similar analysis pipeline to identify potential molecular drivers of pathogenic ALDH missense mutations. Variants data were first carefully curated and labelled as cancer-risk, non-cancer diseases, and benign. We then leveraged various computational biophysical methods to describe the changes caused by missense mutations, informing a bias of detrimental mutations with destabilising effects. Cooperating with these insights, several machine learning approaches were further utilised to investigate the combination of features, revealing the necessity of the conservation of ALDHs. Our work aims to provide important biological perspectives on pathogenic consequences of missense mutations of ALDHs, which could be invaluable resources in the development of cancer treatment.

ALAN as an omic tool to build gene signatures and to stratify patients.

1563 Background: Understanding aberrant gene behavior is critical towards devising clinical therapeutics or gene based diagnostic panels. Clustering and AI/ML have clinical utility, but they do not contextualize changes in gene behavior that result from differences in cell type, microenvironment, or prior treatments. Here, we developed the Algorithm for Linking Activity Networks (ALAN), an informatics algorithm that allows rapid functional comparisons of all detectable genes between clinically annotated patient cohorts. ALAN outputs quantify gene behavior without any additional inputs and can be applied on all forms of clinical omic data. We tested the utility of ALAN to identify the molecular features of resistance and novel precision therapeutics. Methods: We applied the ALAN algorithm in prostate tissue using whole-transcriptome sequencing (WTS) data from SU2C/PCF (n = 209, metastatic prostate cancer), TCGA (n = 493, primary prostate cancer), and GTEX (n = 245, benign prostate tissue). We conducted informatics analysis of whole-exome sequencing (WES) data from SU2C/PCF (n = 444 mPC), Fred Hutch (n = 130 mPC), and MCTP (n = 61 mPC) to identify mPC patients with amplifications in RSPO2. We scored an ALAN gene signature in a patient with single-cell RNA sequencing pre and post AR inhibition (n = 112, 83). We examined ChIP-seq data to evaluate binding of AR and Co-factors at the CD276 transcription regulatory sequences. Results: In mPCs, ALAN unbiasedly identified genes that prior studies have implicated as resistance mechanisms towards enzalutamide. In this process, ALAN nominated RSPO2, a secreted protein, as a potential resistant gene that has never been targeted in mPC. In recent studies, we found RSPO2 amplifications in 19.7-28.5% of mPC. We utilized ALAN outputs to build a novel gene signature that was exclusively enriched (p-value < 0.0001) in the transcriptome collected from single cells of a post-resistant mPC sample. We also utilized ALAN to stratify the optimal mPC patients in receiving precision therapies against B7-H3 ( CD276). B7-H3 is an immune regulatory molecule in the B7 superfamily that includes PD-L1 and CTLA-4 and has not been targeted in mPC. B7-H3 was unlike other B7 family members and exhibited robust association with AR co-factors and AR signaling. We confirmed ALAN findings as multiple B7-H3 transcriptional enhancers and promoters were bound by AR and AR-cofactors. Conclusions: ALAN is an algorithm that can be readily deployed on existing clinical omic data to build gene signatures, identify resistant genes, and risk stratify patients. ALAN nominated a novel therapeutic target, RSPO2, which is recurrently amplified in mPC patients. ALAN predicted that mPCs with high baseline levels of AR activity would be optimal candidates for B7-H3 precision therapeutics. Altogether, ALAN outputs should be considered for making clinical decisions that would benefit by comprehensive comparisons of all detectable genes.

Structural and functional insights into the flexible β-hairpin of glycerol dehydrogenase.

During glycerol metabolism, the initial step of glycerol oxidation is catalysed by glycerol dehydrogenase (GDH), which converts glycerol to dihydroxyacetone in a NAD+ -dependent manner via an ordered Bi-Bi kinetic mechanism. Structural studies conducted with GDH from various species have mainly elucidated structural details of the active site and ligand binding. However, the structure of the full GDH complex with both cofactor and substrate bound is not determined, and thus, the structural basis of the kinetic mechanism of GDH remains unclear. Here, we report the crystal structures of Escherichia coli GDH with a substrate analogue bound in the absence or presence of NAD+ . Structural analyses including molecular dynamics simulations revealed that GDH possesses a flexible β-hairpin, and that during the ordered progression of the kinetic mechanism, the flexibility of the β-hairpin is reduced after NAD+ binding. It was also observed that this alterable flexibility of the β-hairpin contributes to the cofactor binding and possibly to the catalytic efficiency of GDH. These findings suggest the importance of the flexible β-hairpin to GDH enzymatic activity and shed new light on the kinetic mechanism of GDH.

The FAM86 domain of FAM86A confers substrate specificity to promote EEF2-Lys525 methylation

FAM86A is a class I lysine methyltransferase (KMT) that generates trimethylation on the eukaryotic translation elongation factor 2 (EEF2) at Lys525. Publicly available data from The Cancer Dependency Map project indicate high dependence of hundreds of human cancer cell lines on FAM86A expression. This classifies FAM86A among numerous other KMTs as potential targets for future anticancer therapies. However, selective inhibition of KMTs by small molecules can be challenging due to high conservation within the S-adenosyl methionine (SAM) cofactor binding domain among KMT subfamilies. Therefore, understanding the unique interactions within each KMT-substrate pair can facilitate developing highly specific inhibitors. The FAM86A gene encodes an N-terminal FAM86 domain of unknown function in addition to its C-terminal methyltransferase domain. Here, we used a combination of X-ray crystallography, the AlphaFold algorithms, and experimental biochemistry to identify an essential role of the FAM86 domain in mediating EEF2 methylation by FAM86A. To facilitate our studies, we also generated a selective EEF2K525 methyl antibody. Overall, this is the first report of a biological function for the FAM86 structural domain in any species and an example of a noncatalytic domain participating in protein lysine methylation. The interaction between the FAM86 domain and EEF2 provides a new strategy for developing a specific FAM86A small molecule inhibitor, and our results provide an example in which modeling a protein-protein interaction with AlphaFold expedites experimental biology.

Structural basis for CEP192-mediated regulation of centrosomal AURKA

Aurora kinase A (AURKA) performs critical functions in mitosis. Thus, the activity and subcellular localization of AURKA are tightly regulated and depend on diverse factors including interactions with the multiple binding cofactors. How these different cofactors regulate AURKA to elicit different levels of activity at distinct subcellular locations and times is poorly understood. Here, we identified a conserved region of CEP192, the major cofactor of AURKA, that mediates the interaction with AURKA. Quantitative binding studies were performed to map the interactions of a conserved helix (Helix-1) within CEP192. The crystal structure of Helix-1 bound to AURKA revealed a distinct binding site that is different from other cofactor proteins such as TPX2. Inhibiting the interaction between Helix-1 and AURKA in cells led to the mitotic defects, demonstrating the importance of the interaction. Collectively, we revealed a structural basis for the CEP192-mediated AURKA regulation at the centrosome, which is distinct from TPX2-mediated regulation on the spindle microtubule.

Transcriptomic analysis reveals GA3 is involved in regulating flavonoid metabolism in grape development for facility cultivation.

Gibberellin, as one of the pivotal plant growth regulators, can improve fruit quality by altering fruit size and secondary metabolite content. Flavonoids are the most abundant secondary metabolites in grapes, which influence the color and quality of the fruit. However, the molecular mechanism of whether and how GA3 affects flavonoid metabolism has not been reported, especially for the 'Red globe' grape with delayed cultivation in Hexi corridor. In the present study, the 'Red globe' grape grown in delayed facilities was sprayed with 20, 40, 60, 80 and 100mg/L GA3 at berries pea size (BPS), veraison (V) and berries ripe (BR), respectively. The results showed that the berry weight, soluble sugar content and secondary metabolite content (the flavonoid content, anthocyanin content and polyphenol content) at BR under 80mg/L GA3 treatment were remarkably increased compared with other concentration treatments. Therefore, RNA sequencing (RNA-seq) was used to analyze the differentially expressed genes (DEGS) and pathways under 80mg/L GA3 treatment at three periods. GO analysis showed that DEGs were closely related to transporter activity, cofactor binding, photosynthetic membrane, thylakoid, ribosome biogenesis and other items. The KEGG enrichment analysis found that the DEGs were mainly involved in flavonoid biosynthesis and phenylpropanoid biosynthesis, indicating GA3 exerted an impact on the color and quality of berries through these pathways. In conclusion, GA3 significantly increased the expression of genes related to flavonoid synthesis, enhanced the production of secondary metabolites, and improved fruit quality. In addition, these findings can provide a theoretical basis for GA3 to modulate the accumulation and metabolism of flavonoids in grape fruit.

Feedback Inhibition of DszC, a Crucial Enzyme for Crude Oil Biodessulfurization

The Rhodococcus erythropolis (strain IGTS8) bacterium has a tremendous industrial interest as it can remove sulfur from crude oil through its four-enzyme (DszA-D) 4S metabolic pathway. DszC is one of the rate-limiting enzymes of the pathway and the one that most suffers from feedback inhibition. We have combined molecular docking and molecular dynamics simulations to identify binding sites through which two products of the 4S pathway, 2-hydroxybiphenyl and 2′-hydroxybiphenyl-2-sulfinate, induce DszC feedback inhibition. We have identified four potential binding sites: two adjacent binding sites close to the 280–295 lid loop proposed to contribute to DszC oligomerization and proper binding of the flavin mononucleotide cofactor, and two other close to the active site of DszC and the substrate binding site. By considering (i) the occupancy of the binding sites and (ii) the similar inhibitor poses, we propose that the mechanism of feedback inhibition of DszC occurs through disturbance of the DszC oligomerization and consequent binding of the flavin mononucleotide due to the weakening of the interactions between the 280–295 lid loop, and both the 131–142 loop and the C-terminal tail. Nevertheless, inhibitor binding close to the active site or the substrate binding sites also compromises critical interactions within the active site of DszC. The disclosed molecular details provide valuable insight for future rational enzyme engineering protocols to develop DszC mutants more resistant against the observed feedback inhibition mechanism.

Two structural switches in HIV-1 capsid regulate capsid curvature and host factor binding

The mature HIV-1 capsid protects the viral genome and interacts with host proteins to travel from the cell periphery into the nucleus. To achieve this, the capsid protein, CA, constructs conical capsids from a lattice of hexamers and pentamers, and engages in and then relinquishes multiple interactions with cellular proteins in an orchestrated fashion. Cellular host factors including Nup153, CPSF6, and Sec24C engage the same pocket within CA hexamers. How CA assembles pentamers and hexamers of different curvatures, how CA oligomerization states or curvature might modulate host-protein interactions, and how binding of multiple cofactors to a single site is coordinated, all remain to be elucidated. Here, using single-particle cryoEM, we have determined the structure of the mature HIV-1 CA pentamer and hexamer from conical CA-IP6 polyhedra to ~3Å resolution. We also determined structures of hexamers in the context of multiple lattice curvatures and number of pentamer contacts. Comparison of these structures, bound or not to host protein peptides, revealed two structural switches within HIV-1 CA that modulate peptide binding according to CA lattice curvature and whether CA is hexameric or pentameric. These observations suggest that the conical HIV-1 capsid has different host-protein binding properties at different positions on its surface, which may facilitate cell entry and represent an evolutionary advantage of conical morphology.

Abstract 4718: Analyses of epigenetic marks and mechanisms in disease: Your guide to a successful CUT&RUN assay

Abstract Like the chromatin immunoprecipitation (ChIP) assay, Cleavage Under Targets & Release Using Nuclease (CUT&RUN) is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell. The CUT&RUN assay can be combined with downstream qPCR or NG-seq to analyze histone modifications and binding of transcription factors, DNA replication factors, or DNA repair proteins at specific target genes or across the entire genome. CUT&RUN provides a rapid, robust, and true low cell number assay for detection of protein-DNA interactions in the cell. Unlike the ChIP assay, CUT&RUN is free from formaldehyde cross-linking, chromatin fragmentation, and immunoprecipitation. Previously, we have shown that, compared to ChIP, CUT&RUN requires fewer starting cells (100K), has a much faster protocol (one day from cells to DNA), generates lower background signal (requires less sequencing depth), and offers spike-in control DNA for effective normalization of signal between samples and between experiments. We recently updated the CST CUT&RUN Assay Kit for use with 5,000-20,000 cells and added protocols for fixed cells and tissue. I will discuss the basics of the CUT&RUN assay and important factors to consider when setting up your experiment. In addition, I will provide data showing the versatility of this assay for mapping various histone modifications, transcription factor, and transcription cofactor binding across multiple sample types. Finally, I will discuss how the general protocol is optimized for greater signal to noise ratio, reduced number of starting cells, and provide an alternative digestion method to prepare the input DNA as a critical control of the CUT&RUN experiment. Citation Format: Angela H. Guo, Christopher R. Comeau, Fang Chen, Christopher J. Fry. Analyses of epigenetic marks and mechanisms in disease: Your guide to a successful CUT&RUN assay. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4718.

Abstract 3079: Characterization of ER-AF2 inhibitors in breast cancer

Abstract Up to 80% of breast cancers (BCa) rely on the estrogen receptor (ER) for their growth and progression. This dependence on ER has led to many hormonal therapies that target this receptor. However, almost 40% of these cancers will acquire resistance over the course of the treatment period. One potential cause of resistance is mutations in the estrogen binding site (EBS) of ER. As such, there is an increasing need for novel inhibitors that targets ER at a site separate from the EBS. Here, we propose targeting the activation-function (AF2) pocket of ER that is important for cofactor binding and transcription activation. Billions of compounds were screened through an in-silico deep docking method, and potential AF2 inhibitors were then validated in cell-based and biophysical assays. We tested the effect of potential AF2 inhibitors on ER transcriptional activity using luciferase reporter assay in ER-positive T47D-kbluc cells. We then evaluated the effect of molecules on cell viability of ER-positive T47D and ER-negative MDA-MB-231 cells using PrestoBlue assays in order to exclude off-target effects. From these cell-based assays, we identified several inhibitors that effectively reduced transcriptional activity and viability in ER-positive T47D cells at low micromolar concentrations. We conducted PGC-1α peptide displacement assay to confirm their AF2 binding and estradiol displacement assays to exclude any binding to the EBS. Proximity ligation assay (PLA) showed disruption of the interaction between ER and coactivator SRC-3 upon treatment with ER-AF2 inhibitors in T47D cells. Current work focuses on confirming the direct binding between the compounds and recombinant ER-ligand binding domain by various biophysical assays (MST, BLI, and ITC). Future work aims to solve the structure of ER-LBD in a complex with our lead compound by X-ray crystallography. We predict that the use of potent ER-AF2 inhibitors along with current treatments, will provide a novel tactic that can act as a complementary therapeutic to target treatment resistance in ER+ BCa. Citation Format: Jane Foo, Francesco Gentile, Joseph Lee, Helene Morin, Shabnam Massah, Maria Guo, Jason Smith, Fuqiang Ban, Artem Cherkasov, Nada Lallous. Characterization of ER-AF2 inhibitors in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3079.

Exploration of seryl tRNA synthetase to identify potent inhibitors against leishmanial parasites

Aminoacyl-tRNA synthetases are crucial enzymes for cellular protein metabolism and have been considered as an attractive target for development of new antimicrobials. In the current study, seryl tRNA synthetase of Leishmania donovani (LdSerRS) and its mutants were purified and characterized through biochemical and structural methods. Purified LdSerRS was found to be enzymatically active and exhibited more alpha helices in secondary structure. The enzymatic activity of purified protein was observed as highest near physiological temperature and pH. Mutation in ATP binding residues (R295 and E297) demonstrated reduction in the affinity for cofactor with no significant deviation in secondary structure. In vitro inhibition studies with ureidosulfocoumarin derivatives helped to identify Comp 5l as a specific inhibitor for leishmanial SerRS that showed lesser potency towards purified HsSerRS. The identified compound presented competitive mode of inhibition for LdSerRS and also revealed druglikeness along with very low toxicity for human macrophages. Structural analysis of protein and ligand complex depicted the binding of Comp 5l into the cofactor binding site of LdSerRS with high affinity succeeded by validation employing molecular dynamics simulations. Altogether, our study presents a promising scaffold to explore small molecules to target the enzymatic activity of leishmanial SerRS to develop the specific therapeutics.

The cofactor-dependent folding mechanism of Drosophila cryptochrome revealed by single-molecule pulling experiments

The link between cofactor binding and protein activity is well-established. However, how cofactor interactions modulate folding of large proteins remains unknown. We use optical tweezers, clustering and global fitting to dissect the folding mechanism of Drosophila cryptochrome (dCRY), a 542-residue protein that binds FAD, one of the most chemically and structurally complex cofactors in nature. We show that the first dCRY parts to fold are independent of FAD, but later steps are FAD-driven as the remaining polypeptide folds around the cofactor. FAD binds to largely unfolded intermediates, yet with association kinetics above the diffusion-limit. Interestingly, not all FAD moieties are required for folding: whereas the isoalloxazine ring linked to ribitol and one phosphate is sufficient to drive complete folding, the adenosine ring with phosphates only leads to partial folding. Lastly, we propose a dCRY folding model where regions that undergo conformational transitions during signal transduction are the last to fold.

Classifying metal-binding sites with neural networks.

To advance our ability to predict impacts of the protein scaffold on catalysis, robust classification schemes to define features of proteins that will influence reactivity are needed. One of these features is a protein's metal-binding ability, as metals are critical to catalytic conversion by metalloenzymes. As a step toward realizing this goal, we used convolutional neural networks (CNNs) to enable the classification of a metal cofactor binding pocket within a protein scaffold. CNNs enable images to be classified based on multiple levels of detail in the image, from edges and corners to entire objects, and can provide rapid classification. First, six CNN models were fine-tuned to classify the 20 standard amino acids to choose a performant model for amino acid classification. This model was then trained in two parallel efforts: to classify a 2D image of the environment within a given radius of the central metal binding site, either an Fe ion or a [2Fe-2S] cofactor, with the metal visible (effort 1) or the metal hidden (effort 2). We further used two sub-classifications of the [2Fe-2S] cofactor: (1) a standard [2Fe-2S] cofactor and (2) a Rieske [2Fe-2S] cofactor. The accuracy for the model correctly identifying all three defined features was >95%, despite our perception of the increased challenge of the metalloenzyme identification. This demonstrates that machine learning methodology to classify and distinguish similar metal-binding sites, even in the absence of a visible cofactor, is indeed possible and offers an additional tool for metal-binding site identification in proteins.

Ligand induced dimerization and topology of a knotted spout methyltransferase.

Understanding the complex knotted topology in protein folding is an important structural and functional indication as increasing families of protein are discovered to contain deeply knotted structure. Methyltransferases in SpoU-TrmD (SPOUT) family catalyze the post-translational methylation of RNA assisted by the cofactor, S-Adenosyl methionine (SAM), and have a deeply folded trefoil knot that associated with the dimer interface and ligand binding domain. Using the “minimalism” of knotted methyltransferase, 1vh0, which only contains α/β SPOUT domain and a 31 trefoil knot, the biological function and structural change have been studied by cofactor binding. The complexity of the trefoil knot and localized frustration bring up the discussion about dimeric enzymatic active states formation. The monomeric apo-state of 1vh0 in solution is indicated by HSQC spectrum and the unfolding curve monitored by CD and fluorescence spectroscopy. In comparison, isothermal titration calorimetry (ITC) and NMR spectroscopy indicate that the holo-state of 1vh0 forms dimer. With the observed allosteric conformational change of the frustrated region that implicates knot formation, we conclude that the cofactor binding leads to allosteric conformational change, which induces dimerization.

SARS-CoV-2–encoded ORF8 protein possesses complement inhibitory properties

Hyperactivation of the complement system, a major component of innate immunity, has been recognized as one of the core clinical features in severe covid-19 patients. However, how the virus escapes the targeted elimination by the network of activated complement pathways still remains an enigma. Here, we identified SARS-CoV-2-encoded ORF8 protein as one of the major binding partners of human complement C3/C3b components and their metabolites. Our results demonstrated that preincubation of ORF8 with C3/C3b in the fluid phase has two immediate functional consequences in the alternative pathway; this preincubation inhibits factor I-mediated proteolysis and blocks factor B zymogen activation into active Bb. ORF8 binding results in the occlusion of both factor H and factor B from C3b, rendering the complexes resistant to factor I-mediated proteolysis and inhibition of pro-C3-convertase (C3bB) formation, respectively. We also confirmed the complement inhibitory activity of ORF8 in our hemolysis-based assay, where ORF8 prevented human serum-induced lysis of rabbit erythrocytes with an IC50 value of about 2.3μM. This inhibitory characteristic of ORF8 was also supported by in-silico protein-protein docking analysis, as it appeared to establish primary interactions with the β-chain of C3b, orienting itself near the C3b CUB (C1r/C1s, Uegf, Bmp1) domain like a peptidomimetic compound, sterically hindering the binding of essential cofactors required for complement amplification. Thus, ORF8 has characteristics to act as an inhibitor of critical regulatory steps in the alternative pathway, converging to hasten the decay of C3-convertase and thereby, attenuating the complement amplification loop.

Candida parapsilosis carbonyl reductase as a tool for preliminary screening of inhibitors for alcohol dehydrogenase induced skin sensitization

Human alcohol dehydrogenases, specifically ADH1 oxidize primary alcohols to aldehydes in the skin. These aldehydes act as haptens, leading to sensitization. Inhibition of these ADH enzymes may help in combating skin allergies. In the present study, the recombinantly purified stereospecific enzyme from Candida parapsilosis ATCC 7330, Candida parapsilosis Carbonyl Reductase (CpCR) has 23.31 % sequence similarity with human alcohol dehydrogenase ADH1B1. These two enzymes have perfectly overlapping cofactor and zinc binding domains. CpCR was found to oxidize primary alcohols to their corresponding aldehydes. Oxidation of primary alcohols by CpCR was further optimized with cinnamyl alcohol as the model substrate that initially showed a conversion of 56 % ± 0.25, and upon optimization increased to 98.2 % ± 0.23. An increase of 20–50 % in the conversion rate has been observed for various primary alcohols under the optimized reaction conditions. A simple and efficient model was designed for the screening of compounds that inhibit CpCR with the possibility of mitigating the action of skin allergens by inhibiting ADH1. p-Nitrophenylglyoxal (PNG) was found to be a good inhibitor for CpCR which showed inhibitory activity at very low concentration (IC50,100 mM ± 1.27) as compared to the standard inhibitor 4-methyl pyrazole (4MP) (IC50, 400 mM ± 2.05).

Structures and mechanisms of tRNA methylation by METTL1-WDR4.

Specific, regulated modification of RNAs is important for proper gene expression1,2. tRNAs are rich with various chemical modifications that affect their stability and function3,4. 7-Methylguanosine (m7G) at tRNA position 46 is a conserved modification that modulates steady-state tRNA levels to affect cell growth5,6. The METTL1-WDR4 complex generatesm7G46 in humans, and dysregulation of METTL1-WDR4 has been linked to brain malformation and multiple cancers7-22. Here we show how METTL1 and WDR4 cooperate to recognize RNA substrates and catalyse methylation. A crystal structure of METTL1-WDR4 and cryo-electron microscopy structures of METTL1-WDR4-tRNA show that the composite protein surface recognizes the tRNA elbow through shape complementarity. The cryo-electron microscopy structures of METTL1-WDR4-tRNA with S-adenosylmethionine or S-adenosylhomocysteine along with METTL1 crystal structures provide additional insights into the catalytic mechanism by revealing the active site in multiple states. The METTL1 N terminus couples cofactor binding with conformational changes in the tRNA, the catalytic loop and the WDR4 C terminus, acting as the switchto activatem7G methylation. Thus, our structural models explain how post-translational modifications of the METTL1 N terminus can regulate methylation. Together, our work elucidates the core and regulatory mechanisms underlying m7G modification by METTL1, providing the framework to understand its contribution to biology and disease.