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Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5'-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5'-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.
Vitamin B6 is an important nutrient for optimal brain function, with deficiencies linked to impaired memory, learning and mood in various mental disorders. In older people, vitamin B6 deficiency is also associated with declining memory and dementia. Although this has been known for years, the precise role of vitamin B6 in these disorders and whether supplements can be used to treat or prevent them remained unclear. This is partly because vitamin B6 is actually an umbrella term for a small number of very similar and interchangeable molecules. Only one of these is 'bioactive', meaning it has a biological role in cells. However, therapeutic strategies aimed at increasing only the bioactive form of vitamin B6 are lacking. Previous work showed that disrupting the gene for an enzyme called pyridoxal phosphatase, which breaks down vitamin B6, improves memory and learning in mice. To investigate whether these effects could be mimicked by drug-like compounds, Brenner, Zink, Witzinger et al. used several biochemical and structural biology approaches to search for molecules that bind to and inhibit pyridoxal phosphatase. The experiments showed that a molecule called 7,8-dihydroxyflavone which was previously found to improve memory and learning in laboratory animals with brain disorders binds to pyridoxal phosphatase and inhibits its activity. This led to increased bioactive vitamin B6 levels in mouse brain cells involved in memory and learning. The findings of Brenner et al. suggest that inhibiting pyridoxal phosphatase to increase vitamin B6 levels in the brain could be used together with supplements. The identification of 7,8-dihydroxyflavone as a promising candidate drug is a first step in the discovery of more efficient pyridoxal phosphatase inhibitors. These will be useful experimental tools to directly study whether increasing the levels of bioactive vitamin B6 in the brain may help those with mental health conditions associated with impaired memory, learning and mood.
Assuntos
Inibidores Enzimáticos , Monoéster Fosfórico Hidrolases , Animais , Camundongos , Humanos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosfato de Piridoxal/metabolismo , Flavonas/farmacologia , Flavonas/metabolismo , Flavonas/química , Camundongos Endogâmicos C57BLRESUMO
Glycine receptor (GlyR) autoantibodies are associated with stiff-person syndrome and the life-threatening progressive encephalomyelitis with rigidity and myoclonus in children and adults. Patient histories show variability in symptoms and responses to therapeutic treatments. A better understanding of the autoantibody pathology is required to develop improved therapeutic strategies. So far, the underlying molecular pathomechanisms include enhanced receptor internalization and direct receptor blocking altering GlyR function. A common epitope of autoantibodies against the GlyRα1 has been previously defined to residues 1A-33G at the N-terminus of the mature GlyR extracellular domain. However, if other autoantibody binding sites exist or additional GlyR residues are involved in autoantibody binding is yet unknown. The present study investigates the importance of receptor glycosylation for binding of anti-GlyR autoantibodies. The glycine receptor α1 harbors only one glycosylation site at the amino acid residue asparagine 38 localized in close vicinity to the identified common autoantibody epitope. First, non-glycosylated GlyRs were characterized using protein biochemical approaches as well as electrophysiological recordings and molecular modeling. Molecular modeling of non-glycosylated GlyRα1 did not show major structural alterations. Moreover, non-glycosylation of the GlyRα1N38Q did not prevent the receptor from surface expression. At the functional level, the non-glycosylated GlyR demonstrated reduced glycine potency, but patient GlyR autoantibodies still bound to the surface-expressed non-glycosylated receptor protein in living cells. Efficient adsorption of GlyR autoantibodies from patient samples was possible by binding to native glycosylated and non-glycosylated GlyRα1 expressed in living not fixed transfected HEK293 cells. Binding of patient-derived GlyR autoantibodies to the non-glycosylated GlyRα1 offered the possibility to use purified non-glycosylated GlyR extracellular domain constructs coated on ELISA plates and use them as a fast screening readout for the presence of GlyR autoantibodies in patient serum samples. Following successful adsorption of patient autoantibodies by GlyR ECDs, binding to primary motoneurons and transfected cells was absent. Our results indicate that the glycine receptor autoantibody binding is independent of the receptor's glycosylation state. Purified non-glycosylated receptor domains harbouring the autoantibody epitope thus provide, an additional reliable experimental tool besides binding to native receptors in cell-based assays for detection of autoantibody presence in patient sera.
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Formation of the Aurora-A-MYCN complex increases levels of the oncogenic transcription factor MYCN in neuroblastoma cells by abrogating its degradation through the ubiquitin proteasome system. While some small-molecule inhibitors of Aurora-A were shown to destabilize MYCN, clinical trials have not been satisfactory to date. MYCN itself is considered to be `undruggable' due to its large intrinsically disordered regions. Targeting the Aurora-A-MYCN complex rather than Aurora-A or MYCN alone will open new possibilities for drug development and screening campaigns. To overcome the challenges that a ternary system composed of Aurora-A, MYCN and a small molecule entails, a covalently cross-linked construct of the Aurora-A-MYCN complex was designed, expressed and characterized, thus enabling screening and design campaigns to identify selective binders.
Assuntos
Neuroblastoma , Humanos , Proteína Proto-Oncogênica N-Myc/metabolismo , Proteína Proto-Oncogênica N-Myc/uso terapêutico , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Linhagem Celular TumoralRESUMO
The CTLH (C-terminal to lissencephaly-1 homology motif) complex is a multisubunit RING E3 ligase with poorly defined substrate specificity and flexible subunit composition. Two key subunits, muskelin and Wdr26, specify two alternative CTLH complexes that differ in quaternary structure, thereby allowing the E3 ligase to presumably target different substrates. With the aid of different biophysical and biochemical techniques, we characterized CTLH complex assembly pathways, focusing not only on Wdr26 and muskelin but also on RanBP9, Twa1, and Armc8ß subunits, which are critical to establish the scaffold of this E3 ligase. We demonstrate that the ability of muskelin to tetramerize and the assembly of Wdr26 into dimers define mutually exclusive oligomerization modules that compete with nanomolar affinity for RanBP9 binding. The remaining scaffolding subunits, Armc8ß and Twa1, strongly interact with each other and with RanBP9, again with nanomolar affinity. Our data demonstrate that RanBP9 organizes subunit assembly and prevents higher order oligomerization of dimeric Wdr26 and the Armc8ß-Twa1 heterodimer through its tight binding. Combined, our studies define alternative assembly pathways of the CTLH complex and elucidate the role of RanBP9 in governing differential oligomeric assemblies, thereby advancing our mechanistic understanding of CTLH complex architectures.
Assuntos
Multimerização Proteica , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Multimerização Proteica/genética , Estrutura Quaternária de Proteína , Polimerização , Ligação ProteicaRESUMO
Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates.
Assuntos
Neoplasias , Monoéster Fosfórico Hidrolases , Humanos , Monoéster Fosfórico Hidrolases/metabolismo , GlicóliseRESUMO
Efficient neuronal signaling depends on the proper assembly of the postsynaptic neurotransmitter machinery. The majority of inhibitory synapses feature γ-aminobutyric acid type A (GABAA) receptors. The function of these GABAergic synapses is controlled by the scaffolding protein gephyrin and collybistin, a Dbl family guanine nucleotide exchange factor and neuronal adaptor protein. Specifically, collybistin interacts with small GTPases, cell adhesion proteins, and phosphoinositides to recruit gephyrin and GABAA receptors to postsynaptic membrane specializations. Collybistin usually contains an N-terminal SH3 domain and exists in closed/inactive or open/active states. Here, we elucidate the molecular basis of the gephyrin-collybistin interaction with newly designed collybistin Förster resonance energy transfer (FRET) sensors. Using fluorescence lifetime-based FRET measurements, we deduce the affinity of the gephyrin-collybistin complex, thereby confirming that the C-terminal dimer-forming E domain binds collybistin, an interaction that does not require E domain dimerization. Simulations based on fluorescence lifetime and sensor distance distributions reveal at least a two-state equilibrium of the SH3 domain already in the free/unbound collybistin, thereby illustrating the accessible volume of the SH3 domain. Finally, our data provide strong evidence for a tightly regulated collybistin-gephyrin interplay, where, unexpectedly, switching of collybistin from closed/inactive to open/active states is efficiently triggered by gephyrin.
RESUMO
Interneuronal synaptic transmission relies on the proper spatial organization of presynaptic neurotransmitter release and its reception on the postsynaptic side by cognate neurotransmitter receptors. Neurotransmitter receptors are incorporated into and arranged within the plasma membrane with the assistance of scaffolding and adaptor proteins. At inhibitory GABAergic postsynapses, collybistin, a neuronal adaptor protein, recruits the scaffolding protein gephyrin and interacts with various neuronal factors including cell adhesion proteins of the neuroligin family, the GABA A receptor α2-subunit and the closely related small GTPases Cdc42 and TC10 (RhoQ). Most collybistin splice variants harbor an N-terminal SH3 domain and exist in an autoinhibited/closed state. Cdc42 and TC10, despite sharing 67.4% amino acid sequence identity, interact differently with collybistin. Here, we delineate the molecular basis of the collybistin conformational activation induced by TC10 with the aid of recently developed collybistin FRET sensors. Time-resolved fluorescence-based FRET measurements reveal that TC10 binds to closed/inactive collybistin leading to relief of its autoinhibition, contrary to Cdc42, which only interacts with collybistin when forced into an open state by the introduction of mutations destabilizing the closed state of collybistin. Taken together, our data describe a TC10-driven signaling mechanism in which collybistin switches from its autoinhibited closed state to an open/active state.
RESUMO
The covalent modification of target proteins with ubiquitin or ubiquitin-like modifiers is initiated by E1 activating enzymes, which typically transfer a single modifier onto cognate conjugating enzymes. UBA6 is an unusual E1 since it activates two highly distinct modifiers, ubiquitin and FAT10. Here, we report crystal structures of UBA6 in complex with either ATP or FAT10. In the UBA6-FAT10 complex, the C-terminal domain of FAT10 binds to where ubiquitin resides in the UBA1-ubiquitin complex, however, a switch element ensures the alternate recruitment of either modifier. Simultaneously, the N-terminal domain of FAT10 interacts with the 3-helix bundle of UBA6. Site-directed mutagenesis identifies residues permitting the selective activation of either ubiquitin or FAT10. These results pave the way for studies investigating the activation of either modifier by UBA6 in physiological and pathophysiological settings.
Assuntos
Ubiquitina , Ubiquitinas , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinas/metabolismoRESUMO
RNA-catalyzed RNA methylation was recently shown to be part of the catalytic repertoire of ribozymes. The methyltransferase ribozyme MTR1 catalyzes the site-specific synthesis of 1-methyladenosine (m1A) in RNA, using O6-methylguanine (m6G) as a methyl group donor. Here, we report the crystal structure of MTR1 at a resolution of 2.8 Å, which reveals a guanine-binding site reminiscent of natural guanine riboswitches. The structure represents the postcatalytic state of a split ribozyme in complex with the m1A-containing RNA product and the demethylated cofactor guanine. The structural data suggest the mechanistic involvement of a protonated cytidine in the methyl transfer reaction. A synergistic effect of two 2'-O-methylated ribose residues in the active site results in accelerated methyl group transfer. Supported by these results, it seems plausible that modified nucleotides may have enhanced early RNA catalysis and that metabolite-binding riboswitches may resemble inactivated ribozymes that have lost their catalytic activity during evolution.
Assuntos
RNA Catalítico , Sítios de Ligação , Catálise , Guanina , Metiltransferases/genética , Conformação de Ácido Nucleico , RNA Catalítico/metabolismoRESUMO
The strict human pathogen Streptococcus pyogenes causes infections of varying severity, ranging from self-limiting suppurative infections to life-threatening diseases like necrotizing fasciitis or streptococcal toxic shock syndrome. Here, we show that the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase GapN is an essential enzyme for S. pyogenes. GapN converts glyceraldehyde 3-phosphate into 3-phosphoglycerate coupled to the reduction of NADP to NADPH. The knock-down of gapN by antisense peptide nucleic acids (asPNA) significantly reduces viable bacterial counts of S. pyogenes laboratory and macrolide-resistant clinical strains in vitro. As S. pyogenes lacks the oxidative part of the pentose phosphate pathway, GapN appears to be the major NADPH source for the bacterium. Accordingly, other streptococci that carry a complete pentose phosphate pathway are not prone to asPNA-based gapN knock-down. Determination of the crystal structure of the S. pyogenes GapN apo-enzyme revealed an unusual cis-peptide in proximity to the catalytic binding site. Furthermore, using a structural modeling approach, we correctly predicted competitive inhibition of S. pyogenes GapN by erythrose 4-phosphate, indicating that our structural model can be used for in silico screening of specific GapN inhibitors. In conclusion, the data provided here reveal that GapN is a potential target for antimicrobial substances that selectively kill S. pyogenes and other streptococci that lack the oxidative part of the pentose phosphate pathway.
RESUMO
Biosensor techniques have become increasingly important for fragment-based drug discovery during the last years. The AAA+ ATPase p97 is an essential protein with key roles in protein homeostasis and a possible target for cancer chemotherapy. Currently available p97 inhibitors address its ATPase activity and globally impair p97-mediated processes. In contrast, inhibition of cofactor binding to the N-domain by a protein-protein-interaction inhibitor would enable the selective targeting of specific p97 functions. Here, we describe a biolayer interferometry-based fragment screen targeting the N-domain of p97 and demonstrate that a region known as SHP-motif binding site can be targeted with small molecules. Guided by molecular dynamics simulations, the binding sites of selected screening hits were postulated and experimentally validated using protein- and ligand-based NMR techniques, as well as X-ray crystallography, ultimately resulting in the first structure of a small molecule in complex with the N-domain of p97. The identified fragments provide insights into how this region could be targeted and present first chemical starting points for the development of a protein-protein interaction inhibitor preventing the binding of selected cofactors to p97.
RESUMO
Artemisinin, a major extract of the annual mugwort Artemisia annua, and its semisynthetic derivatives represent state-of-the-art antimalarial drugs. These compounds also target, via poorly understood mechanisms, various mammalian pathways, thereby exhibiting anticancer and immunomodulatory properties. Recently, crystal structures of artemisinins with two mammalian targets were determined, namely, gephyrin, the prime scaffolding protein at inhibitory postsynapses, and pyridoxal kinase, a central metabolic enzyme synthesizing vitamin B6. These structures and corresponding functional studies demonstrate that artemisinins play a dual role in modulating inhibitory synapses, acting on postsynaptic sites by impeding inhibitory neurotransmitter receptor clustering and on presynaptic terminals by limiting the biosynthesis of the inhibitory neurotransmitter γ-aminobutyric acid. These studies pave the way for further investigations of artemisinins as inhibitory neurotransmission modulators in humans.
Assuntos
Antimaláricos , Artemisininas , Animais , Antimaláricos/farmacologia , Artemisininas/farmacologia , Humanos , Neurotransmissores , Sinapses , Transmissão SinápticaRESUMO
Legionnaires' disease is caused by infection with the intracellularly replicating Gram-negative bacterium Legionella pneumophila. This pathogen uses an unconventional way of ubiquitinating host proteins by generating a phosphoribosyl linkage between substrate proteins and ubiquitin by making use of an ADPribosylated ubiquitin (UbADPr ) intermediate. The family of SidE effector enzymes that catalyze this reaction is counteracted by Legionella hydrolases, which are called Dups. This unusual ubiquitination process is important for Legionella proliferation and understanding these processes on a molecular level might prove invaluable in finding new treatments. Herein, a modular approach is used for the synthesis of triazole-linked UbADPr , and analogues thereof, and their affinity towards the hydrolase DupA is determined and hydrolysis rates are compared to natively linked UbADPr . The inhibitory effects of modified Ub on the canonical eukaryotic E1-enzyme Uba1 are investigated and rationalized in the context of a high-resolution crystal structure reported herein. Finally, it is shown that synthetic UbADPr analogues can be used to effectively pull-down overexpressed DupA from cell lysate.
Assuntos
ADP-Ribosilação , Legionella pneumophila/enzimologia , Doença dos Legionários/microbiologia , Ubiquitina/química , Ubiquitina/metabolismo , Proteínas de Bactérias/metabolismo , Células HEK293 , Humanos , Hidrolases/metabolismo , Legionella pneumophila/crescimento & desenvolvimento , Enzimas Ativadoras de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
The antimalarial artemisinins have also been implicated in the regulation of various cellular pathways including immunomodulation of cancers and regulation of pancreatic cell signaling in mammals. Despite their widespread application, the cellular specificities and molecular mechanisms of target recognition by artemisinins remain poorly characterized. We recently demonstrated how these drugs modulate inhibitory postsynaptic signaling by direct binding to the postsynaptic scaffolding protein gephyrin. Here, we report the crystal structure of the central metabolic enzyme pyridoxal kinase (PDXK), which catalyzes the production of the active form of vitamin B6 (also known as pyridoxal 5'-phosphate [PLP]), in complex with artesunate at 2.4-Šresolution. Partially overlapping binding of artemisinins with the substrate pyridoxal inhibits PLP biosynthesis as demonstrated by kinetic measurements. Electrophysiological recordings from hippocampal slices and activity measurements of glutamic acid decarboxylase (GAD), a PLP-dependent enzyme synthesizing the neurotransmitter γ-aminobutyric acid (GABA), define how artemisinins also interfere presynaptically with GABAergic signaling. Our data provide a comprehensive picture of artemisinin-induced effects on inhibitory signaling in the brain.
Assuntos
Artemisininas/farmacologia , Regulação para Baixo , Inibição Neural/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridoxal Quinase/antagonistas & inibidores , Transmissão Sináptica/efeitos dos fármacos , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Artemisininas/química , Sítios de Ligação , Regulação para Baixo/efeitos dos fármacos , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Feminino , Neurônios GABAérgicos/efeitos dos fármacos , Neurônios GABAérgicos/metabolismo , Glutamato Descarboxilase/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Piridoxal Quinase/química , Piridoxal Quinase/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Ácido gama-Aminobutírico/biossínteseRESUMO
Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. Nuclear division and formation of intracellular structures was interrupted. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites.
Assuntos
Merozoítos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Humanos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Ubiquitina/genéticaRESUMO
Pyridoxal 5'-phosphate (PLP) is an essential cofactor for neurotransmitter metabolism. Pyridoxal phosphatase (PDXP) deficiency in mice increases PLP and γ-aminobutyric acid levels in the brain, yet how PDXP is regulated is unclear. Here, we identify the Ca2+ - and integrin-binding protein 1 (CIB1) as a PDXP interactor by yeast two-hybrid screening and find a calmodulin (CaM)-binding motif that overlaps with the PDXP-CIB1 interaction site. Pulldown and crosslinking assays with purified proteins demonstrate that PDXP directly binds to CIB1 or CaM. CIB1 or CaM does not alter PDXP phosphatase activity. However, elevated Ca2+ concentrations promote CaM binding and, thereby, diminish CIB1 binding to PDXP, as both interactors bind in a mutually exclusive way. Hence, the PDXP-CIB1 complex may functionally differ from the PDXP-Ca2+ -CaM complex.
RESUMO
The absence of fluorine from most biomolecules renders it an excellent probe for NMR spectroscopy to monitor inhibitor-protein interactions. However, predicting the binding mode of a fluorinated ligand from a chemical shift (or vice versa) has been challenging due to the high electron density of the fluorine atom. Nonetheless, reliable 19 F chemical-shift predictions to deduce ligand-binding modes hold great potential for inâ silico drug design. Herein, we present a systematic QM/MM study to predict the 19 Fâ NMR chemical shifts of a covalently bound fluorinated inhibitor to the essential oxidoreductase tryparedoxin (Tpx) from African trypanosomes, the causative agent of African sleeping sickness. We include many protein-inhibitor conformations as well as monomeric and dimeric inhibitor-protein complexes, thus rendering it the largest computational study on chemical shifts of 19 F nuclei in a biological context to date. Our predicted shifts agree well with those obtained experimentally and pave the way for future work in this area.
Assuntos
Inibidores Enzimáticos/química , Ressonância Magnética Nuclear Biomolecular/métodos , Pirimidinonas/química , Tiofenos/química , Tiorredoxinas/química , Tripanossomicidas/química , Inibidores Enzimáticos/metabolismo , Flúor/química , Mutação , Ligação Proteica , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Pirimidinonas/metabolismo , Tiofenos/metabolismo , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Tripanossomicidas/metabolismo , Trypanosoma brucei brucei/enzimologiaRESUMO
In recent years, three-dimensional density maps reconstructed from single particle images obtained by electron cryo-microscopy (cryo-EM) have reached unprecedented resolution. However, map interpretation can be challenging, in particular if the constituting structures require de-novo model building or are very mobile. Herein, we demonstrate the potential of convolutional neural networks for the annotation of cryo-EM maps: our network Haruspex has been trained on a carefully curated set of 293â experimentally derived reconstruction maps to automatically annotate RNA/DNA as well as protein secondary structure elements. It can be straightforwardly applied to newly reconstructed maps in order to support domain placement or as a starting point for main-chain placement. Due to its high recall and precision rates of 95.1 % and 80.3 %, respectively, on an independent test set of 122â maps, it can also be used for validation during model building. The trained network will be available as part of the CCP-EM suite.
Assuntos
Microscopia Crioeletrônica/métodos , DNA/química , Redes Neurais de Computação , Oligonucleotídeos/metabolismo , RNA/química , Humanos , Modelos Moleculares , Estrutura Secundária de ProteínaRESUMO
Targeting multiprotein receptor complexes, rather than receptors directly, is a promising concept in drug discovery. This is particularly relevant to the GABAB receptor complex, which plays a prominent role in many brain functions and diseases. Here, we provide the first studies targeting a key protein-protein interaction of the GABAB receptor complex-the interaction with KCTD proteins. By employing the µSPOT technology, we first defined the GABAB receptor-binding epitope mediating the KCTD interaction. Subsequently, we developed a highly potent peptide-based inhibitor that interferes with the KCTD/GABAB receptor complex and efficiently isolates endogenous KCTD proteins from mouse brain lysates. X-ray crystallography and SEC-MALS revealed inhibitor induced oligomerization of KCTD16 into a distinct hexameric structure. Thus, we provide a template for modulating the GABAB receptor complex, revealing a fundamentally novel approach for targeting GABAB receptor-associated neuropsychiatric disorders.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/metabolismo , Receptores de GABA-B/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Encéfalo/metabolismo , Cristalografia por Raios X , Polarização de Fluorescência , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Peptídeos/química , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores de GABA-B/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Glycine transporter 2 (GlyT2) mutations across the entire sequence have been shown to represent the presynaptic component of the neurological disease hyperekplexia. Dominant, recessive and compound heterozygous mutations have been identified, most of them leading to impaired glycine uptake. Here, we identified a novel loss of function mutation of the GlyT2 resulting from an amino acid exchange of proline 429 to leucine in a family with both parents being heterozygous carriers. A homozygous child suffered from severe neuromotor deficits. We characterised the GlyT2P429L variant at the molecular, cellular and protein level. Functionality was determined by glycine uptake assays. Homology modelling revealed that the mutation localises to α-helix 5, presumably disrupting the integrity of this α-helix. GlyT2P429L shows protein trafficking through various intracellular compartments to the cellular surface. However, the protein expression at the whole cell level was significantly reduced. Although present at the cellular surface, GlyT2P429L demonstrated a loss of protein function. Coexpression of the mutant with the wild-type protein, reflecting the situation in the parents, did not affect transporter function, thus explaining their non-symptomatic phenotype. Nevertheless, when the mutant was expressed in excess compared with the wild-type protein, glycine uptake was significantly reduced. Thus, these data demonstrate that the proline residue at position 429 is structurally important for the correct formation of α-helix 5. The failure in functionality of the mutated GlyT2 is most probably due to structural changes localised in close proximity to the sodium-binding site of the transporter.