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αB-crystallin (heat shock protein β5/HSPB5) is a member of the family of small heat shock proteins that is expressed in various organs of the human body including eye lenses and muscles. Therefore, mutations in the gene of this protein (CRYAB) might have many pathological consequences. A new mutation has recently been discovered in the α-crystallin domain of this chaperone protein which replaces aspartate 109 with alanine (D109A). This mutation can cause myofibrillar myopathy (MFM), cataracts, and cardiomyopathy. In the current study, several spectroscopic and microscopic analyses, as well as gel electrophoresis assessment were applied to elucidate the pathogenic contribution of human αB-crystallin bearing D109A mutation in development of eye lens cataract and myopathies. The protein oligomerization, chaperone-like activity and chemical/thermal stabilities of the mutant and wild-type protein were also investigated in the comparative assessments. Our results suggested that the D109A mutation has a significant impact on the important features of human αB-crystallin, including its structure, size of the protein oligomers, tendency to form amyloid fibrils, stability, and chaperone-like activity. Given the importance of aspartate 109 in maintaining the proper structure of the α-crystallin domain, its role in the dimerization and chaperone-like activity, as well as preserving protein stability through the formation of salt bridges; mutation at this important site might have critical consequences and can explain the genesis of myopathy and cataract disorders. Also, the formation of large light-scattering aggregates and disruption of the chaperone-like activity by D109A mutation might be considered as important contributing factors in development of the eye lens opacity.

Herpesviruses cause severe diseases particularly in immunocompromised patients. Both genome packaging and release from the capsid require a unique portal channel occupying one of the 12 capsid vertices. Here, we report the 2.6 Å crystal structure of the pentameric pORF19 of the γ-herpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV) resembling the portal cap that seals this portal channel. We also present the structure of its β-herpesviral ortholog, revealing a striking structural similarity to its α- and γ-herpesviral counterparts despite apparent differences in capsid association. We demonstrate pORF19 pentamer formation in solution and provide insights into how pentamerization is triggered in infected cells. Mutagenesis in its lateral interfaces blocked pORF19 pentamerization and severely affected KSHV capsid assembly and production of infectious progeny. Our results pave the way to better understand the role of pORF19 in capsid assembly and identify a potential novel drug target for the treatment of herpesvirus-induced diseases., In herpesviruses, genome packaging and release from the capsid require a unique portal channel. Here, the authors have resolved the crystal structure of a pentameric KSHV pORF19 assembly and find that it resembles the herpesviral portal cap and provides insights how the viral genome is retained within the capsid.

Tuberculosis causes the highest mortality among all single infections. Asymptomatic tuberculosis, afflicting one third of the global human population, is the major source as 5–10% of asymptomatic cases develop active tuberculosis during their lifetime. Thus it is one of important issues to develop diagnostic tools for accurately detecting asymptomatic infection. Mycobacterial DNA-binding protein 1 (MDP1) is a major protein in persistent Mycobacterium tuberculosis and has potential for diagnostic use in detecting asymptomatic infection. However, a previous ELISA-based study revealed a specificity problem; IgGs against MDP1 were detected in both M. tuberculosis-infected and uninfected individuals. Although the tertiary structures of an antigen are known to influence antibody recognition, the MDP1 structural details have not yet been investigated. The N-terminal half of MDP1, homologous to bacterial histone-like protein HU, is predicted to be responsible for DNA-binding, while the C-terminal half is assumed as totally intrinsically disordered regions. To clarify the relationship between the MDP1 tertiary structure and IgG recognition, we refined the purification method, which allow us to obtain a recombinant protein with the predicted structure. Furthermore, we showed that an IgG-ELISA using MDP1 purified by our refined method is indeed useful in the detection of asymptomatic tuberculosis.

Electron cryo-microscopy (Cryo-EM) is a powerful method for visualizing biological objects with up to near-angstrom resolution. Instead of chemical fixation, the method relies on very rapid freezing to immobilize the sample. Under these conditions, crystalline ice does not have time to form and distort structure. For many practical applications, the rate of cooling is fast enough to consider sample immobilization instantaneous, but in some cases, a more rigorous analysis of structure relaxation during freezing could be essential. This difficult yet important problem has been significantly under-reported in the literature, despite spectacular recent developments in Cryo-EM. Here we use Brownian dynamics modeling to examine theoretically the possible effects of cryo-immobilization on the apparent shapes of biological polymers. The main focus of our study is on tubulin protofilaments. These structures are integral parts of microtubules, which in turn are key elements of the cellular skeleton, essential for intracellular transport, maintenance of cell shape, cell division and migration. We theoretically examine the extent of protofilament relaxation within the freezing time as a function of the cooling rate, the filament’s flexural rigidity, and the effect of cooling on water’s viscosity. Our modeling suggests that practically achievable cooling rates are not rapid enough to capture tubulin protofilaments in conformations that are incompletely relaxed, suggesting that structures seen by cryo-EM are good approximations to physiological shapes. This prediction is confirmed by our analysis of curvatures of tubulin protofilaments, using samples, prepared and visualized with a variety of methods. We find, however, that cryofixation may capture incompletely relaxed shapes of more flexible polymers, and it may affect Cryo-EM-based measurements of their persistence lengths. This analysis will be valuable for understanding of structures of different types of biopolymers, observed with Cryo-EM.

Little is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, fission protein B (FisB), is expressed during sporulation in Bacillus subtilis and is required to release the developing spore into the mother cell cytoplasm. Here, we characterized the requirements for FisB-mediated membrane fission. FisB forms mobile clusters of approximately 12 molecules that give way to an immobile cluster at the engulfment pole containing approximately 40 proteins at the time of membrane fission. Analysis of FisB mutants revealed that binding to acidic lipids and homo-oligomerization are both critical for targeting FisB to the engulfment pole and membrane fission. Experiments using artificial membranes and filamentous cells suggest that FisB does not have an intrinsic ability to sense or induce membrane curvature but can bridge membranes. Finally, modeling suggests that homo-oligomerization and trans-interactions with membranes are sufficient to explain FisB accumulation at the membrane neck that connects the engulfment membrane to the rest of the mother cell membrane during late stages of engulfment. Together, our results show that FisB is a robust and unusual membrane fission protein that relies on homo-oligomerization, lipid binding, and the unique membrane topology generated during engulfment for localization and membrane scission, but surprisingly, not on lipid microdomains, negative-curvature lipids, or curvature sensing., Little is known about how membrane fission occurs in bacteria; this study suggests that the membrane fission protein FisB exploits the unique cellular geometry encountered during sporulation to enable its localization to the fission site through a novel mechanism, where it catalyzes membrane scission.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by the aberrant production and accumulation of amyloid-β (Aβ) peptides in the brain. Accumulated Aβ in soluble oligomer and insoluble plaque forms are considered to be a pathological culprit and biomarker of the disorder. Here, we report a fluorescent universal Aβ-indicator YI-13, 5-(4-fluorobenzoyl)-7,8-dihydropyrrolo[1,2-b]isoquinolin-9(6H)-one, which detects Aβ monomers, dimers, and plaques. We synthesized a library of 26 fluorescence chemicals with the indolizine core and screen them through a series of in vitro tests utilizing Aβ as a target and YI-13 was selected as the final imaging candidate. YI-13 was found to stain and visualize insoluble Aβ plaques in the brain tissue, of a transgenic mouse model with five familial AD mutations (5XFAD), by a histochemical approach and to label soluble Aβ oligomers within brain lysates of the mouse model under a fluorescence plate reader. Among oligomers aggregated from monomers and synthetic dimers from chemically conjugated monomers, YI-13 preferred the dimeric Aβ.

Matrix proteins are encoded by many enveloped viruses, including influenza viruses, herpes viruses, and coronaviruses. Underneath the viral envelope of influenza virus, matrix protein 1 (M1) forms an oligomeric layer critical for particle stability and pH-dependent RNA genome release. However, high-resolution structures of full-length monomeric M1 and the matrix layer have not been available, impeding antiviral targeting and understanding of the pH-dependent transitions involved in cell entry. Here, purification and extensive mutagenesis revealed protein–protein interfaces required for the formation of multilayered helical M1 oligomers similar to those observed in virions exposed to the low pH of cell entry. However, single-layered helical oligomers with biochemical and ultrastructural similarity to those found in infectious virions before cell entry were observed upon mutation of a single amino acid. The highly ordered structure of the single-layered oligomers and their likeness to the matrix layer of intact virions prompted structural analysis by cryo-electron microscopy (cryo-EM). The resulting 3.4-Å–resolution structure revealed the molecular details of M1 folding and its organization within the single-shelled matrix. The solution of the full-length M1 structure, the identification of critical assembly interfaces, and the development of M1 assembly assays with purified proteins are crucial advances for antiviral targeting of influenza viruses., Multi-subunit shells of matrix proteins line the interior of infectious influenza virus particles. In this study, biochemical purification of wild-type and mutant influenza M1 proteins allows the structural determination of an oligomer whose shape corresponds to that of infectious virions and suggests mechanisms for its formation and dismantling during infection.

The induction of broad and potent immunity by vaccines is the key focus of research efforts aimed at protecting against HIV-1 infection. Soluble native-like HIV-1 envelope glycoproteins have shown promise as vaccine candidates as they can induce potent autologous neutralizing responses in rabbits and non-human primates. In this study, monoclonal antibodies were isolated and characterized from rhesus macaques immunized with the BG505 SOSIP.664 trimer to better understand vaccine-induced antibody responses. Our studies reveal a diverse landscape of antibodies recognizing immunodominant strain-specific epitopes and non-neutralizing neo-epitopes. Additionally, we isolated a subset of mAbs against an epitope cluster at the gp120-gp41 interface that recognize the highly conserved fusion peptide and the glycan at position 88 and have characteristics akin to several human-derived broadly neutralizing antibodies., Author summary Major efforts are currently directed towards developing vaccine strategies to successfully elicit broadly neutralizing antibodies (bNAbs) against HIV-1. However, how to achieve this goal remains a critical problem. Soluble native-like HIV-1 envelope glycoproteins (Env) have shown promise as vaccine candidates in pre-clinical studies. Here, the antibody response against the BG505 SOSIP.664 Env trimer was studied through the isolation of monoclonal antibodies (mAbs) in rhesus macaques (RMs) after immunization. Overall, a diverse landscape of mAbs was elicited that target the Env trimer in highly similar ways in two animals. By mapping the target epitopes of all of the mAbs isolated from the immunized RMs, rather than focusing only on the neutralizing mAbs, we were able to identify several non-neutralizing and potentially immunodominant epitopes that would ideally be eliminated in future immunization studies. In addition, we identified neutralizing mAbs that recognize the highly conserved fusion peptide in a similar way as human broadly neutralizing antibodies. These insights can be further used to develop immunogens and immunization strategies able to induce bNAb-like responses that can protect against infection.

Oligomeric amyloid β (Aβ) is currently considered the most neurotoxic form of the Aβ peptide implicated in Alzheimer’s disease (AD). The molecular structures of the oligomers have remained mostly unknown due to their transient nature. As a result, the molecular mechanisms of interactions between conformation-specific antibodies and their Aβ oligomer (AβO) cognates are not well understood. A monoclonal conformation-specific antibody, m5E3, was raised against a structural epitope of Aβ oligomers. m5E3 binds to AβOs with high affinity, but not to Aβ monomers or fibrils. In this study, a computational model of the variable fragment (Fv) of the m5E3 antibody (Fv5E3) is introduced. We further employ docking and molecular dynamics simulations to determine the molecular details of the antibody-oligomer interactions, and to classify the AβOs as Fv5E3-positives and negatives, and to provide a rationale for the low affinity of Fv5E3 for fibrils. This information will help us to perform site-directed mutagenesis on the m5E3 antibody to improve its specificity and affinity toward oligomeric Aβ species. We also provide evidence for the possible capability of the m5E3 antibody to disaggregate AβOs and to fragment protofilaments.

Cytokine signaling is transmitted by cell surface receptors which act as natural biological switches to control cellular functions such as immune reactions. Recently, we have designed synthetic cytokine receptors (SyCyRs) consisting of green fluorescent protein (GFP)- and mCherry-nanobodies fused to the transmembrane and intracellular domains of cytokine receptors. Following stimulation with homo- and heterodimeric GFP-mCherry fusion proteins, the resulting receptors phenocopied signaling induced by physiologically occurring cytokines. GFP and mCherry fusion proteins were produced in E. coli or CHO-K1 cells, but the overall yield and stability was low. Therefore, we applied two alternative multimerization strategies and achieved immunoglobulin Fc-mediated dimeric and coiled-coil GCN4pII-mediated trimeric assemblies. GFP- and/or mCherry-Fc homodimers activated synthetic gp130 cytokine receptors, which naturally respond to Interleukin 6 family cytokines. Activation of these synthetic gp130 receptors resulted in STAT3 and ERK phosphorylation and subsequent proliferation of Ba/F3-gp130 cells. Half-maximal effective concentrations (EC50) of 8.1 ng/ml and 0.64 ng/ml were determined for dimeric GFP-Fc and mCherry-Fc, respectively. This is well within the expected EC50 range of the native cytokines. Moreover, we generated tetrameric and hexameric GFP-mCherry-Fc fusion proteins, which were also biologically active. This highlighted the importance of close juxtaposition of two cytokine receptors for efficient receptor activation. Finally, we used a trimeric GCN4pII motif to generate homo-trimeric GFP and mCherry complexes. These synthetic cytokines showed improved EC50 values (GFP3: 0.58 ng/ml; mCherrry3: 0.37 ng/ml), over dimeric Fc fused variants. In conclusion, we successfully generated highly effective and stable multimeric synthetic cytokine receptor ligands for activation of synthetic cytokine receptors.

Lactate oxidases belong to a group of FMN-dependent enzymes and they catalyze a conversion of lactate to pyruvate with a release of hydrogen peroxide. Hydrogen peroxide is also utilized as a read out in biosensors to quantitate lactate levels in biological samples.Aerococcus viridanslactate oxidase is the best characterized lactate oxidase and our knowledge of lactate oxidases relies largely to studies conducted with that particular enzyme.Pediococcus acidilacticilactate oxidase is also commercially available for e.g. lactate measurements, but this enzyme has not been characterized before in detail. Here we report structural characterization of the recombinant enzyme and its co-factor dependent oligomerization. The crystal structures revealed two distinct conformations in the loop closing the active site, consistent with previous biochemical studies implicating the role of loop in catalysis. Despite the structural conservation of active site residues when compared toAerococcus viridanslactate oxidase we were not able to detect either oxidase or monooxygenase activity when L-lactate or other potential alpha hydroxyl acids were used as a substrate.Pediococcus acidilacticilactate oxidase is therefore an example of a misannotation of an FMN-dependent enzyme, which catalyzes likely a so far unknown oxidation reaction.

Plasmodium falciparumcauses the most lethal form of malaria. The cooperation of heat shock protein (Hsp) 70 and 90 is important for folding of a select number of cellular proteins that are crucial for cyto-protection and development of the parasites. Hsp70 and Hsp90 are brought into a functional complex that allows substrate exchange by stress inducible protein 1 (STI1), also known as Hsp70-Hsp90 organizing protein (Hop).P. falciparumHop (PfHop) co-localises and occurs in complex with the parasite cytosolic chaperones, PfHsp70-1 and PfHsp90. Here, we characterised the structure of recombinant PfHop using synchrotron radiation circular dichroism (SRCD) and small-angle X-ray scattering. Structurally, PfHop is a monomeric, elongated but folded protein, in agreement with its predicted TPR domain structure. Using SRCD, we established that PfHop is unstable at temperatures higher than 40 °C. This suggests that PfHop is less stable at elevated temperatures compared to its functional partner, PfHsp70-1, that is reportedly stable at temperatures as high as 80 °C. These findings contribute towards our understanding of the role of the Hop-mediated functional partnership between Hsp70 and Hsp90.

Centriolar satellites are dynamic, membraneless granules composed of over 200 proteins. They store, modify, and traffic centrosome and primary cilium proteins, and help to regulate both the biogenesis and some functions of centrosomes and cilium. In most cell types, satellites cluster around the perinuclear centrosome, but their integrity and cellular distribution are dynamically remodeled in response to different stimuli, such as cell cycle cues. Dissecting the specific and temporal functions and mechanisms of satellites and how these are influenced by their cellular positioning and dynamics has been challenging using genetic approaches, particularly in ciliated and proliferating cells. To address this, we developed a chemical-based trafficking assay to rapidly and efficiently redistribute satellites to either the cell periphery or center, and fuse them into stable clusters in a temporally controlled way. Induced satellite clustering at either the periphery or center resulted in antagonistic changes in the pericentrosomal levels of a subset of proteins, revealing a direct and selective role for their positioning in protein targeting and sequestration. Systematic analysis of the interactome of peripheral satellite clusters revealed enrichment of proteins implicated in cilium biogenesis and mitosis. Importantly, induction of peripheral satellite targeting in ciliated cells revealed a function for satellites not just for efficient cilium assembly but also in the maintenance of steady-state cilia and in cilia disassembly by regulating the structural integrity of the ciliary axoneme. Finally, perturbing satellite distribution and dynamics inhibited their mitotic dissolution, and mitotic progression was perturbed only in cells with centrosomal satellite clustering. Collectively, our results for the first time showed a direct link between satellite functions and their pericentrosomal clustering, suggested new mechanisms underlying satellite functions during cilium assembly, and provided a new tool for probing temporal satellite functions in different contexts, What happens when centriolar satellites are not in the right place at the right time? By redistributing satellites to the periphery or center of the cell and assessing the consequences of their mispositioning, this study reveals novel functions for satellites during mitosis, cilium maintenance, and cilium disassembly and suggests new mechanisms.

At some point early in the vertebrate lineage, two whole genome duplication events (1R, 2R) took place that allowed for the diversification and sub-/neo-functionalization of the glycoprotein hormones (GpHs). All jawed vertebrates possess the GpHs luteinizing hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH), each of which are heterodimers with a common alpha subunit and unique beta subunits. In 2002, a novel glycoprotein hormone named thyrostimulin was described to have unique GpA2 and GpB5 subunits that were homologous to the vertebrate alpha and beta subunits. The presence of GpA2 and GpB5 in representative protostomes and deuterostomes indicates their ancestry in the GpH family. There are several reports of GpH subunit evolution, but none have included GpA2 and GpB5 for species in each major vertebrate class. Thus, we addressed the ancestry of two paralogous GpB5 subunits (GpB5a and GpB5b) that were previously only recognized in two teleost species. Our search for orthologous GpB5a and GpB5b sequences in representative vertebrates and phylogenetic analysis, in addition to the currently published evolutionary scenarios of the GpH family, supports that GpB5a and GpB5b are paralogs that arose from the first vertebrate whole genome duplication event (1R). Syntenic analysis supports lineage specific losses of GpB5a in chondrichthyes, basal actinopterygians, and tetrapods, and retention in coelacanth and teleosts. Additionally, we were unable to identify GpA2 transcripts from tilapia mRNA, suggesting that this species does not produce heterodimeric thyrostimulin. While the conserved or even species-specific functional role of thyrostimulin or its individual subunits are still unknown in vertebrates, the analyses presented here provide context for future studies on the functional divergence of the GpH family.

The transient receptor potential Ankyrin-1 (TRPA1) ion channel is modulated by myriad noxious stimuli that interact with multiple regions of the channel, including cysteine-reactive natural extracts from onion and garlic which modify residues in the cytoplasmic domains. The way in which TRPA1 cytoplasmic domain modification is coupled to opening of the ion-conducting pore has yet to be elucidated. The cryo-EM structure of TRPA1 revealed a tetrameric C-terminal coiled-coil surrounded by N-terminal ankyrin repeat domains (ARDs), an architecture shared with the canonical transient receptor potential (TRPC) ion channel family. Similarly, structures of the TRP melastatin (TRPM) ion channel family also showed a C-terminal coiled-coil surrounded by N-terminal cytoplasmic domains. This conserved architecture may indicate a common gating mechanism by which modification of cytoplasmic domains can transduce conformational changes to open the ion-conducting pore. We developed an in vitro system in which N-terminal ARDs and C-terminal coiled-coil domains can be expressed in bacteria and maintain the ability to interact. We tested three gating regulators: temperature; the polyphosphate compound IP6; and the covalent modifier allyl isothiocyanate to determine whether they alter N-and C-terminal interactions. We found that none of the modifiers tested abolished ARD-coiled-coil interactions, though there was a significant reduction at 37°C. We found that coiled-coils tetramerize in a concentration dependent manner, with monomers and trimers observed at lower concentrations. Our system provides a method for examining the mechanism of oligomerization of TRPA1 cytoplasmic domains as well as a system to study the transmission of conformational changes resulting from covalent modification.

Lysozyme is a type of enzymatic protein found in a wide range of organisms. Among the many applications of lysozyme, the antibacterial activity features caused by the hydrolysis of 1-4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine of gram-positive bacteria are beneficial in the food industry, medicine, trade, and pharmacology. Studies have indicated that specific modifications of lysozyme cause oligomerization of the enzyme, and the resulting dimer, which also undergoes changes in physicochemical properties, shows greater total antibacterial activity. Among these modifications, thermo-chemical methods are one of the most important groups. In this study, the microwave method of the enzyme heating with the associated process of enzyme oxidation was used as a novel thermo-chemical method to induce lysozyme oligomerization. The research shows that using this new method can produce enzymatic preparations composed of approximately 58.9% oligomers, including 33.5% dimer and 25.4% trimer under a hydrogen peroxide concentration of 4% and pH of 8. The maximum percentage of lysozyme dimer of 39.4% was obtained at pH 6.0 with the addition of 2% oxidant. In addition, as a result of the modification process, the hydrolytic activity and surface hydrophobicity of the enzyme were changed.

The native octameric structure of streptococcal enolase from Streptococcus pyogenes increasingly dissociates as amino acid residues are removed one by one from the carboxy-terminus. These truncations gradually convert native octameric enolase into monomers and oligomers. In this work, we investigated how these truncations influence the interaction between Streptococcal enolase and canine plasminogen. We used dual polarization interferometry (DPI), localized surface plasmon resonance (LSPR), and sedimentation velocity analytical ultracentrifugation (AUC) to study the interaction. The DPI was our first technique, was performed on all the truncations and used one exclusive kind of chip. The LSRP was used to show that the DPI results were not dependent on the type of chip used. The AUC was required to show that our surface results were not the result of selecting a minority population in any given sample; the majority of the protein was responsible for the binding phenomenon we observed. By comparing results from these techniques we identified one detail that is essential for streptococcal enolase to bind plasminogen: In our hands the individual monomers bind plasminogen; dimers, trimers, tetramers may or may not bind, the fully intact, native, octamer does not bind plasminogen. We also evaluated the contribution to the equilibrium constant made by surface binding as well as in solution. On a surface, the association coefficient is about twice that in solution. The difference is probably not significant. Finally, the fully octameric form of the protein that does not contain a hexahis N-terminal peptide does not bind to a silicon oxynitride surface, does not bind to a Au-nanoparticle surface, does not bind to a surface coated with Ni-NTA nor does it bind to a surface coated with DPgn. The likelihood is great that the enolase species on the surface of Streptococcus pyogenes is an x-mer of the native octamer.

Mevalonate kinase (MK) is a key enzyme of the mevalonate pathway, which produces the biosynthetic precursors for steroids, including cholesterol, and isoprenoids, the largest class of natural products. Currently available crystal structures of MK from different organisms depict the enzyme in its unbound, substrate-bound, and inhibitor-bound forms; however, until now no structure has yet been determined of MK bound to its product, 5-phosphomevalonate. Here, we present crystal structures of mevalonate-bound and 5-phosphomevalonate-bound MK from Methanosarcina mazei (MmMK), a methanogenic archaeon. In contrast to the prior structure of a eukaryotic MK bound with mevalonate, we find a striking lack of direct interactions between this archaeal MK and its substrate. Further, these two MmMK structures join the prior structure of the apoenzyme to complete the first suite of structural snapshots that depict unbound, substrate-bound, and product-bound forms of the same MK. With this collection of structures, we now provide additional insight into the catalytic mechanism of this biologically essential enzyme.

Sorting nexin 5 (SNX5), a member of sorting nexin family, plays an important role in membrane trafficking, including the retrograde trafficking of the cation independent mannose 6-phosphate receptor (CI-M6PR) and macropinocytosis. Using ESI-LCMS/MS analysis, we confirmed that SNX5 serine 226 is phosphorylated. Since SNX5 forms heterodimers with SNX1 or SNX2, we examined the effect of phosphorylation at S226 on the heterodimer formations. Wild-type and mutants of SNX5, in which S226 was mutated to a glutamic acid or an alanine, were expressed in 8505C cells. In pull-down assays using SNX5 as bait, only the S226E mutant failed to precipitate both SNX1 and SNX2. Confocal microscopy data indicated that the wild type and S226A mutant were colocalized with SNX1 and SNX2 in endosomes, but the S226E was not. SNX5 and SNX6 support each other's functions and are involved with CI-M6PR retrograde trafficking. In SNX5 and SNX6 double knockdown cells, CI-M6PR was dispersed and colocalized with the endosomal marker EEA1. In a rescue experiment using SNX5 mutants, the S226A rescued CI-M6PR localization, similar to control cells, but S226E did not. Furthermore, the decrease in the uptake of dextran by macropinocytosis in SNX5 knockdown cells was recovered by the expression of rescue-wild type or S226A mutant, but not by the rescue-S226E mutant. These observations indicate that SNX5 constitutive phosphorylation that mimics the mutant S226E decreases the active SNX5 in these cells. The phosphorylation of SNX5 regulates the dimerization with SNX1 or SNX2, and this suggests that it controls membrane trafficking and protein sorting.

Chemical-genetic interactions–observed when the treatment of mutant cells with chemical compounds reveals unexpected phenotypes–contain rich functional information linking compounds to their cellular modes of action. To systematically identify these interactions, an array of mutants is challenged with a compound and monitored for fitness defects, generating a chemical-genetic interaction profile that provides a quantitative, unbiased description of the cellular function(s) perturbed by the compound. Genetic interactions, obtained from genome-wide double-mutant screens, provide a key for interpreting the functional information contained in chemical-genetic interaction profiles. Despite the utility of this approach, integrative analyses of genetic and chemical-genetic interaction networks have not been systematically evaluated. We developed a method, called CG-TARGET (Chemical Genetic Translation via A Reference Genetic nETwork), that integrates large-scale chemical-genetic interaction screening data with a genetic interaction network to predict the biological processes perturbed by compounds. In a recent publication, we applied CG-TARGET to a screen of nearly 14,000 chemical compounds in Saccharomyces cerevisiae, integrating this dataset with the global S. cerevisiae genetic interaction network to prioritize over 1500 compounds with high-confidence biological process predictions for further study. We present here a formal description and rigorous benchmarking of the CG-TARGET method, showing that, compared to alternative enrichment-based approaches, it achieves similar or better accuracy while substantially improving the ability to control the false discovery rate of biological process predictions. Additional investigation of the compatibility of chemical-genetic and genetic interaction profiles revealed that one-third of observed chemical-genetic interactions contributed to the highest-confidence biological process predictions and that negative chemical-genetic interactions overwhelmingly formed the basis of these predictions. We also present experimental validations of CG-TARGET-predicted tubulin polymerization and cell cycle progression inhibitors. Our approach successfully demonstrates the use of genetic interaction networks in the high-throughput functional annotation of compounds to biological processes., Author summary Understanding how chemical compounds affect biological systems is of paramount importance as pharmaceutical companies strive to develop life-saving medicines, governments seek to regulate the safety of consumer products and agrichemicals, and basic scientists continue to study the fundamental inner workings of biological organisms. One powerful approach to characterize the effects of chemical compounds in living cells is chemical-genetic interaction screening. Using this approach, a collection of cells–each with a different defined genetic perturbation–is tested for sensitivity or resistance to the presence of a compound, resulting in a quantitative profile describing the functional effects of that compound on the cells. The work presented here describes our efforts to integrate compounds’ chemical-genetic interaction profiles with reference genetic interaction profiles containing information on gene function to predict the cellular processes perturbed by the compounds. We focused on specifically developing a method that could scale to perform these functional predictions for large collections of thousands of screened compounds and robustly control the false discovery rate. With chemical-genetic and genetic interaction screens now underway in multiple species including human cells, the method described here can be generally applied to enable the characterization of compounds’ effects across the tree of life.

B cell follicles in secondary lymphoid tissues represent an immune privileged sanctuary for AIDS viruses, in part because cytotoxic CD8+ T cells are mostly excluded from entering the follicles that harbor infected T follicular helper (TFH) cells. We studied the effects of native heterodimeric IL-15 (hetIL-15) treatment on uninfected rhesus macaques and on macaques that had spontaneously controlled SHIV infection to low levels of chronic viremia. hetIL-15 increased effector CD8+ T lymphocytes with high granzyme B content in blood, mucosal sites and lymph nodes, including virus-specific MHC-peptide tetramer+ CD8+ cells in LN. Following hetIL-15 treatment, multiplexed quantitative image analysis (histo-cytometry) of LN revealed increased numbers of granzyme B+ T cells in B cell follicles and SHIV RNA was decreased in plasma and in LN. Based on these properties, hetIL-15 shows promise as a potential component in combination immunotherapy regimens to target AIDS virus sanctuaries and reduce long-term viral reservoirs in HIV-1 infected individuals.ClinicalTrials.gov NCT02452268.

Plants in arid zones are constantly exposed to drought stress. The ASR protein family (Abscisic, Stress, Ripening) -a subgroup of the late embryogenesis abundant superfamily-is involved in the water stress response and adaptation to dry environments. Tomato ASR1, as well as other members of this family, is an intrinsically disordered protein (IDP) that functions as a transcription factor and a chaperone. Here we employed different biophysical techniques to perform a deep in vitro characterization of ASR1 as an IDP and showed how both environmental factors and in vivo targets modulate its folding. We report that ASR1 adopts different conformations such as α-helix or polyproline type II in response to environmental changes. Low temperatures and low pH promote the polyproline type II conformation (PII). While NaCl increases PII content and slightly destabilizes α-helix conformation, PEG and glycerol have an important stabilizing effect of α-helix conformation. The binding of Zn 2 + in the low micromolar range promotes α-helix folding, while extra Zn 2+ results in homo-dimerization. The ASR1-DNA binding is sequence specific and dependent on Zn 2+ . ASR1 chaperone activity does not change upon the structure induction triggered by the addition of Zn 2+ . Furthermore, trehalose, which has no effect on the ASR1 structure by itself, showed a synergistic effect on the ASR1-driven heat shock protection towards the reporter enzyme citrate synthase (CS). These observations prompted the development of a FRET reporter to sense ASR1 folding in vivo. Its performance was confirmed in Escherichia coli under saline and osmotic stress conditions, representing a promising probe to be used in plant cells. Overall, this work supports the notion that ASR1 plasticity is a key feature that facilitates its response to drought stress and its interaction with specific targets. Fil: Wetzler, Diana Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Fuchs Wightman, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Bucci, Hernán Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Rinaldi, Jimena Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Caramelo, Julio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Iusem, Norberto Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Ricardi, Martiniano María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina

The trimeric SARS coronavirus (SARS-CoV) surface spike (S) glycoprotein consisting of three S1-S2 heterodimers binds the cellular receptor angiotensin-converting enzyme 2 (ACE2) and mediates fusion of the viral and cellular membranes through a pre- to postfusion conformation transition. Here, we report the structure of the SARS-CoV S glycoprotein in complex with its host cell receptor ACE2 revealed by cryo-electron microscopy (cryo-EM). The complex structure shows that only one receptor-binding domain of the trimeric S glycoprotein binds ACE2 and adopts a protruding “up” conformation. In addition, we studied the structures of the SARS-CoV S glycoprotein and its complexes with ACE2 in different in vitro conditions, which may mimic different conformational states of the S glycoprotein during virus entry. Disassociation of the S1-ACE2 complex from some of the prefusion spikes was observed and characterized. We also characterized the rosette-like structures of the clustered SARS-CoV S2 trimers in the postfusion state observed on electron micrographs. Structural comparisons suggested that the SARS-CoV S glycoprotein retains a prefusion architecture after trypsin cleavage into the S1 and S2 subunits and acidic pH treatment. However, binding to the receptor opens up the receptor-binding domain of S1, which could promote the release of the S1-ACE2 complex and S1 monomers from the prefusion spike and trigger the pre- to postfusion conformational transition., Author summary The global outbreak of SARS in 2002–2003 was caused by infection by a human coronavirus, SARS-CoV. Although the virus has been extensively studied with regard to epidemiology, virology, clinical features and other aspects, there are still no approved antiviral drugs and vaccines to treat and prevent infections of SARS-CoV. The spike (S) glycoprotein of the coronavirus, responsible for host cell attachment and mediating host cell membrane and viral membrane fusion during infection, is key to the viral life cycle and a major target for antiviral drugs and vaccines. In this study, we report the structures of different conformational states of the SARS-CoV S glycoprotein during virus entry. Specifically, we found that the S glycoprotein retains the prefusion trimer structure after trypsin cleavage and low-pH treatment. Additionally, binding with host cell receptor ACE2 promotes the release of S1 subunits from the S trimer and triggers the pre- to postfusion conformational transition. Our results provide new insights for understanding the mechanisms involved in coronavirus S glycoprotein-mediated virus entry.

Oligomeric forms of the Aβ peptide represent the most probable neurotoxic agent in Alzheimer's disease. The dynamic and heterogeneous character of these oligomers makes their structural characterization by classic methods difficult. Native mass spectrometry, when supported by additional gas phase techniques, like ion mobility separation and hydrogen-deuterium exchange (IM-HDX-MS), enable analysis of different oligomers coexisting in the sample and may provide species-specific structural information for each oligomeric form populated in the gas phase. Here, we have combined these three techniques to obtain insight into the structural properties of oligomers of Aβ1-40 and two variants with scrambled sequences. Gas-phase HDX-MS revealed a sequence-specific engagement of the side-chains of residues located at the N-terminal part of the peptide in a network of oligomer-stabilizing interactions. Oligomer-specific interactions were no longer observed in the case of the fully scrambled sequence. Also, the ability to form alternative structures, observed for WT Aβ peptide, was lost upon scrambling. Our data underscore a role for the N-terminal residues in shaping the equilibria of oligomeric forms. Although the peptide lacking the N-terminal 1-16 residues (p3 peptide) is thought to be benign, the role of the N-terminus has not been sufficiently characterized yet. We speculate that the interaction networks revealed here may be crucial for enabling structural transitions necessary to obtain mature parallel cross-β structures from smaller antiparallel oligomers. We provide a hypothetical molecular model of the trajectory that allows a gradual conversion from antiparallel to parallel oligomers without decomposition of oligomers. Oligomer-defining interactions involving the Aβ peptide N-terminus may be important in production of the neurotoxic forms and thus should not be neglected.

The CHD3-like chromatin remodeling protein MOM1 and the PIAS-type SUMO E3 ligase-like protein PIAL2 are known to interact with each other and mediate transcriptional silencing in Arabidopsis. However, it is poorly understood whether and how the interaction is involved in transcriptional silencing. Here, we demonstrate that, while the PIAL2 interaction domain (PIAL2-IND) is required for PIAL2 dimerization, MOM-PIAL2 interaction, and transcriptional silencing, a transgene fusing the wild-type MOM1 protein with the PIAL2 protein defective in PIAL2-IND can completely restore transcriptional silencing in the mom1/pial2 double mutant, demonstrating that the artificial fusion of MOM1 and PIAL2 mimics the in vivo interaction of these two proteins so that PIAL2-IND is no longer required for transcriptional silencing in the fusion protein. Further, our yeast two-hybrid assay identifies a previously unrecognized SUMO interaction motif (SIM) in the conserved MOM1 motif CMM3 and demonstrates that the SIM is responsible for the interaction of MOM1 with SUMO. Given that eukaryotic PIAS-type SUMO E3 ligases have a conserved role in chromatin regulation, the findings reported in this study may represent a conserved chromatin regulatory mechanism in higher eukaryotes.

Flavin-binding photoreceptor proteins sense blue-light (BL) in diverse organisms and have become core elements in recent optogenetic applications. The light-oxygen-voltage (LOV) protein Vivid (VVD) from the filamentous fungus Neurospora crassa is a classic BL photoreceptor, characterized by effecting a photocycle based on light-driven formation and subsequent spontaneous decay of a flavin-cysteinyl adduct. Here we report that VVD presents alternative outcomes to light exposure that result in protein self-oxidation and, unexpectedly, rise of stability through kinetic control. Using optical absorbance and mass spectrometry we show that purified VVD develops amorphous aggregates with the presence of oxidized residues located at the cofactor binding pocket. Light exposure increases oxidative levels in VVD and specific probe analysis identifies singlet oxygen production by the flavin. These results indicate that VVD acts alternatively as a photosensitizer, inducing self-oxidative damage and subsequent aggregation. Surprisingly, BL illumination has an additional, opposite effect in VVD. We show that light-induced adduct formation establishes a stable state, delaying protein aggregation until photoadduct decay occurs. In accordance, repeated BL illumination suppresses VVD aggregation altogether. Furthermore, photoadduct formation confers VVD stability against chemical denaturation. Analysis of the aggregation kinetics and testing of stabilizers against aggregation reveal that aggregation in VVD proceeds through light-dependent kinetic control and dimer formation. These results uncover the aggregation pathway of a photosensor, where light induces a remarkable interplay between protein damage and stability.

The human dopamine transporter (hDAT) is located on presynaptic neurons, where it plays an essential role in limiting dopaminergic signaling by temporarily curtailing high neurotransmitter concentration through rapid re-uptake. Transport by hDAT is energized by transmembrane ionic gradients. Dysfunction of this transporter leads to disease states, such as Parkinson’s disease, bipolar disorder or depression. It has been shown that hDAT and other members of the monoamine transporter family exist in oligomeric forms at the plasma membrane. Several residues are known to be involved in oligomerization, but interaction interfaces, oligomer orientation and the quarternary arrangement in the plasma membrane remain poorly understood. Here we examine oligomeric forms of hDAT using a direct approach, by following dimerization of two randomly-oriented hDAT transporters in 512 independent simulations, each being 2 μs in length. We employed the DAFT (docking assay for transmembrane components) approach, which is an unbiased molecular dynamics simulation method to identify oligomers, their conformations and populations. The overall ensemble of a total of >1 ms simulation time revealed a limited number of symmetric and asymmetric dimers. The identified dimer interfaces include all residues known to be involved in dimerization. Importantly, we find that the surface of the bundle domain is largely excluded from engaging in dimeric interfaces. Such an interaction would typically lead to inhibition by stabilization of one conformation, while substrate transport relies on a large scale rotation between the inward-facing and the outward-facing state., Author summary The human dopamine transporter efficiently removes the neurotransmitter dopamine from the synaptic cleft. Alteration of dopamine transporter function is associated with several neurological diseases, including mood disorders or attention-deficit hyperactivity disorder, but is also a major player in addiction and drug abuse. Functional studies have revealed that not only is transporter oligomerization involved in surface expression and endocytosis, but, more importantly, in reverse transport (efflux) of dopamine that is triggered by amphetamine-like drugs of abuse. Structural knowledge of transporter oligomerization is largely missing. We performed a large scale comprehensive computational study on transporter oligomerization to reveal dimer geometries and the residues involved in the interfaces. The dimer conformations we find in our dataset are fully consistent with all available experimental data in the literature, but also show novel interfaces. We further verified all dimer geometries by free energy calculations. Our results identified an unpredicted—but for the mechanism of substrate transport essential—property: the bundle domain, which moves during the transport cycle, is excluded from contributing to dimer interfaces, thereby allowing for unrestrained movements necessary to translocate substrates through the membrane.

Dementia associated with the Alzheimer's disease is thought to be correlated with the conversion of the β - Amyloid (Aβ) peptides from soluble monomers to aggregated oligomers and insoluble fibrils. We present a discrete-time mathematical model for the aggregation of Aβ monomers into oligomers using concepts from chemical kinetics and population dynamics. Conditions for the stability and instability of the equilibria of the model are established. A formula for the number of monomers that is required for producing oligomers is also given. This may provide compound designers a mechanism to inhibit the Aβ aggregation.

We report a computational model for the assembly of HIV-1 Gag into immature viral particles at the plasma membrane. To reproduce experimental structural and kinetic properties of assembly, a process occurring on the order of minutes, a coarse-grained representation consisting of a single particle per Gag molecule is developed. The model uses information relating the functional interfaces implicated in Gag assembly, results from cryo electron-tomography, and biophysical measurements from fluorescence microscopy, such as the dynamics of Gag assembly at single virions. These experimental constraints eliminated many classes of potential interactions, and narrowed the model to a single interaction scheme with two non-equivalent interfaces acting to form Gags into a hexamer, and a third interface acting to link hexamers together. This model was able to form into a hexameric structure with correct lattice spacing and reproduced biologically relevant growth rates. We explored the effect of genomic RNA seeding punctum growth, finding that RNA may be a factor in locally concentrating Gags to initiate assembly. The simulation results infer that completion of assembly cannot be governed simply by Gag binding kinetics. However the addition of membrane curvature suggests that budding of the virion from the plasma membrane could factor into slowing incorporation of Gag at an assembly site resulting in virions of the same size and number of Gag molecules independent of Gag concentration or the time taken to complete assembly. To corroborate the results of our simulation model, we developed an analytic model for Gag assembly finding good agreement with the simulation results.

SUMOylation is a post-translational modification conserved in eukaryotic organisms that involves the covalent attachment of the small ubiquitin-like protein SUMO to internal lysine residues in target proteins. This tag usually alters the interaction surface of the modified protein and can be translated into changes in its biological activity, stability or subcellular localization, among other possible outputs. SUMO can be attached as a single moiety or as SUMO polymers in case there are internal acceptor sites in SUMO itself. These chains have been shown to be important for proteasomal degradation as well as for the formation of subnuclear structures such as the synaptonemal complex in Saccharomyces cerevisiae or promyelocytic leukemia nuclear bodies in mammals. In this work, we have examined SUMO chain formation in the protozoan parasite Trypanosoma brucei. Using a recently developed bacterial strain engineered to produce SUMOylated proteins we confirmed the ability of TbSUMO to form polymers and determined the type of linkage using site-directed mutational analysis. By generating transgenic procyclic parasites unable to form chains we demonstrated that although not essential for normal growth, SUMO polymerization determines the localization of the modified proteins in the nucleus. In addition, FISH analysis of telomeres showed a differential positioning depending on the polySUMOylation abilities of the cells. Thus, our observations suggest that TbSUMO chains might play a role in establishing interaction platforms contributing to chromatin organization. Fil: Iribarren, Paula Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Di Marzio, Lucía Ayelén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Berazategui, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: de Gaudenzi, Javier Gerardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Alvarez, Vanina Eder. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina

Hypoxia inducible factors (HIFs) are transcription factors belonging to the basic helix−loop−helix PER-ARNT-SIM (bHLH-PAS) protein family with a role in sensing oxygen levels in the cell. Under hypoxia, the HIF-α degradation pathway is blocked and dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT) makes HIF-α transcriptionally active. Due to the common hypoxic environment of tumors, inhibition of this mechanism by destabilization of HIF-α:ARNT dimerization has been proposed as a promising therapeutic strategy. Following the discovery of a druggable cavity within the PAS-B domain of HIF-2α, research efforts have been directed to identify artificial ligands that can impair heterodimerization. Although the crystallographic structures of the HIF-2α:ARNT complex have elucidated the dimer architecture and the 0X3-inhibitor placement within the HIF-2α PAS-B, unveiling the inhibition mechanism requires investigation of how ligand-induced perturbations could dynamically propagate through the structure and affect dimerization. To this end, we compared evolutionary features, intrinsic dynamics and energetic properties of the dimerization interfaces of HIF-2α:ARNT in both the apo and holo forms. Residue conservation analysis highlighted inter-domain connecting elements that have a role in dimerization. Analysis of domain contributions to the dimerization energy demonstrated the importance of bHLH and PAS-A of both partners and of HIF-2α PAS-B domain in dimer stabilization. Among quaternary structure oscillations revealed by Molecular Dynamics simulations, the hinge-bending motion of the ARNT PAS-B domain around the flexible PAS-A/PAS-B linker supports a general model for ARNT dimerization in different heterodimers. Comparison of the HIF-2α:ARNT dynamics in the apo and 0X3-bound forms indicated a model of inhibition where the HIF-2α-PAS-B interfaces are destabilised as a result of water-bridged ligand-protein interactions and these local effects allosterically propagate to perturb the correlated motions of the domains and inter-domain communication. These findings will guide the design of improved inhibitors to contrast cell survival in tumor masses., Author summary A low oxygen condition, called hypoxia, often occurs in tumor masses and generally correlates with worse prognosis. Cells in a tumor react to low oxygen levels with a metabolism modification induced by the activation of hypoxia inducible factors (HIFs) through dimerization with a partner protein and binding to a DNA target. Disrupting this protein-protein interaction could be a potential therapeutic strategy, but directly interfering with dimer formation can be troublesome because of the difficulty to design drugs that bind to protein interfaces. However, ligands that bind internal protein cavities can indirectly perturb the interfaces reducing dimers stability. Albeit protein crystallography had offered a detailed static picture of a HIF dimer bound to candidate inhibitors, it is not able to describe either the perturbation caused by binding or the molecular mechanism of dimer destabilization. Here we exploit molecular dynamics to identify the crucial interfaces in the HIF dimer stabilization and, by comparing the results obtained in the bound and unbound forms, we reveal the mechanism of ligand inhibition at atomic detail. All these findings will guide toward the design of improved dimerization inhibitors, to contrast cell survival in tumor masses.

The ecto-5’-nucleotidase CD73 plays an important role in the production of immune-suppressive adenosine in tumor micro-environment, and has become a validated drug target in oncology. Indeed, the anticancer immune response involves extracellular ATP to block cell proliferation through T-cell activation. However, in the tumor micro-environment, two extracellular membrane-bound enzymes (CD39 and CD73) are overexpressed and hydrolyze efficiently ATP into AMP then further into immune-suppressive adenosine. To circumvent the impact of CD73-generated adenosine, we applied an original bioinformatics approach to identify new allosteric inhibitors targeting the dimerization interface of CD73, which should impair the large dynamic motions required for its enzymatic function. Several hit compounds issued from virtual screening campaigns showed a potent inhibition of recombinant CD73 with inhibition constants in the low micromolar range and exhibited a non-competitive inhibition mode. The structure-activity relationships studies indicated that several amino acid residues (D366, H456, K471, Y484 and E543 for polar interactions and G453-454, I455, H456, L475, V542 and G544 for hydrophobic contacts) located at the dimerization interface are involved in the tight binding of hit compounds and likely contributed for their inhibitory activity. Overall, the gathered information will guide the upcoming lead optimization phase that may lead to potent and selective CD73 inhibitors, able to restore the anticancer immune response., Author summary Nucleotidases play a central role in maintaining the nucleotide pool homeostasis and the only extracellular member of this family, CD73, has become an attractive target in oncology because of its high expression level on immune and cancer cells. In the tumor microenvironment, CD73-generated adenosine prevents the pro-inflammatory response and is considered as a potent immune suppressor. The current study aimed at developing new CD73 inhibitors by targeting an allosteric binding site in order to block the enzyme dynamics and therefore its enzymatic function. Most of the existing inhibitors have been elaborated on the basis of the substrate skeleton and act as competitive inhibitors. Here, four non-competitive compounds are presented with an inhibition constant in the low micromolar range. This study confirms the existence of an allosteric binding site located at the dimerization interface allowing modulation of the enzyme activity by small molecules, similarly to a previously described monoclonal antibody.

GabR from Bacillus subtilis is a transcriptional regulator belonging to the MocR subfamily of the GntR regulators. The structure of the MocR regulators is characterized by the presence of two domains: i) a N-terminal domain, about 60 residue long, possessing the winged-Helix-Turn-Helix (wHTH) architecture with DNA recognition and binding capability; ii) a C-terminal domain (about 350 residue) folded as the pyridoxal 5'-phosphate (PLP) dependent aspartate aminotransferase (AAT) with dimerization and effector binding functions. The two domains are linked to each other by a peptide bridge. Although structural and functional characterization of MocRs is proceeding at a fast pace, virtually nothing is know about the molecular changes induced by the effector binding and on how these modifications influence the properties of the regulator. An extensive molecular dynamics simulation on the crystallographic structure of the homodimeric B. subtilis GabR has been undertaken with the aim to envisage the role and the importance of conformational flexibility in the action of GabR. Molecular dynamics has been calculated for the apo (without PLP) and holo (with PLP bound) forms of the GabR. A comparison between the molecular dynamics trajectories calculated for the two GabR forms suggested that one of the wHTH domain detaches from the AAT-like domain in the GabR PLP-bound form. The most evident conformational change in the holo PLP-bound form is represented by the rotation and the subsequent detachment from the subunit surface of one of the wHTH domains. The movement is mediated by a rearrangement of the linker connecting the AAT domain possibly triggered by the presence of the negative charge of the PLP cofactor. This is the second most significant conformational modification. The C-terminal section of the linker docks into the "active site" pocket and establish stabilizing contacts consisting of hydrogen-bonds, salt-bridges and hydrophobic interactions.