RESUMO
The Drosophila melanogaster Germ cell-expressed protein (GCE) is a paralog of the juvenile hormone (JH) receptor - Methoprene tolerant protein (MET). Both proteins mediate JH function, preventing precocious differentiation during D. melanogaster development. Despite that GCE and MET are often referred to as equivalent JH receptors, their functions are not fully redundant and show tissue specificity. Both proteins belong to the family of bHLH-PAS transcription factors. The similarity of their primary structure is limited to defined bHLH and PAS domains, while their long C-terminal fragments (GCEC, METC) show significant differences and are expected to determine differences in GCE and MET protein activities. In this paper we present the structural characterization of GCEC as a coil-like intrinsically disordered protein (IDP) with highly elongated and asymmetric conformation. In comparison to previously characterized METC, GCEC is less compacted, contains more molecular recognition elements (MoREs) and exhibits a higher propensity for induced folding. The NMR shifts perturbation experiment and pull-down assay clearly demonstrated that the GCEC fragment is sufficient to form an interaction interface with the ligand binding domain (LBD) of the nuclear receptor Fushi Tarazu factor-1 (FTZ-F1). Significantly, these interactions can force GCEC to adopt more fixed structure that can modulate the activity, structure and functions of the full-length receptor. The discussed relation of protein functionality with the structural data of inherently disordered GCEC fragment is a novel look at this protein and contributes to a better understanding of the molecular basis of the functions of the C-terminal fragments of the bHLH-PAS family. Video abstract.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Área Sob a Curva , Células COS , Chlorocebus aethiops , Simulação por Computador , Fluorescência , Hidrodinâmica , Espectroscopia de Ressonância Magnética , Ligação Proteica , Domínios Proteicos , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Multidrug resistance among Gram-negative bacteria is a major global public health threat. Metallo-ß-lactamases (MBLs) target the most widely used antibiotic class, the ß-lactams, including the most recent generation of carbapenems. Interspecies spread renders these enzymes a serious clinical threat, and there are no clinically available inhibitors. We present the crystal structures of IMP-13, a structurally uncharacterized MBL from the Gram-negative bacterium Pseudomonas aeruginosa found in clinical outbreaks globally, and characterize the binding using solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The crystal structures of apo IMP-13 and IMP-13 bound to four clinically relevant carbapenem antibiotics (doripenem, ertapenem, imipenem, and meropenem) are presented. Active-site plasticity and the active-site loop, where a tryptophan residue stabilizes the antibiotic core scaffold, are essential to the substrate-binding mechanism. The conserved carbapenem scaffold plays the most significant role in IMP-13 binding, explaining the broad substrate specificity. The observed plasticity and substrate-locking mechanism provide opportunities for rational drug design of novel metallo-ß-lactamase inhibitors, essential in the fight against antibiotic resistance.
Assuntos
beta-Lactamases , Antibacterianos/farmacologia , Inibidores de beta-Lactamases , beta-Lactamases/genética , beta-Lactamas , CarbapenêmicosRESUMO
Trypanosoma protists are pathogens leading to a spectrum of devastating infectious diseases. The range of available chemotherapeutics against Trypanosoma is limited, and the existing therapies are partially ineffective and cause serious adverse effects. Formation of the PEX14-PEX5 complex is essential for protein import into the parasites' glycosomes. This transport is critical for parasite metabolism and failure leads to mislocalization of glycosomal enzymes, with fatal consequences for the parasite. Hence, inhibiting the PEX14-PEX5 protein-protein interaction (PPI) is an attractive way to affect multiple metabolic pathways. Herein, we have used structure-guided computational screening and optimization to develop the first line of compounds that inhibit PEX14-PEX5 PPI. The optimization was driven by several X-ray structures, NMR binding data, and molecular dynamics simulations. Importantly, the developed compounds show significant cellular activity against Trypanosoma, including the human pathogen Trypanosoma brucei gambiense and Trypanosoma cruzi parasites.
Assuntos
Proteínas de Membrana/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Piridinas/síntese química , Piridinas/farmacologia , Tripanossomicidas/síntese química , Tripanossomicidas/farmacologia , Animais , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/biossíntese , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mioblastos/efeitos dos fármacos , Mioblastos/parasitologia , Proteínas de Protozoários/biossíntese , Ratos , Relação Estrutura-Atividade , Trypanosoma brucei gambiense/efeitos dos fármacos , Trypanosoma brucei gambiense/metabolismo , Trypanosoma brucei rhodesiense/efeitos dos fármacosRESUMO
The bHLH-PAS (basic helix-loop-helix/ Period-ARNT-Single minded) proteins are a family of transcriptional regulators commonly occurring in living organisms. bHLH-PAS members act as intracellular and extracellular "signals" sensors, initiating response to endo- and exogenous signals, including toxins, redox potential, and light. The activity of these proteins as transcription factors depends on nucleocytoplasmic shuttling: the signal received in the cytoplasm has to be transduced, via translocation, to the nucleus. It leads to the activation of transcription of particular genes and determines the cell response to different stimuli. In this review, we aim to present the current state of knowledge concerning signals that affect shuttling of bHLH-PAS transcription factors. We summarize experimentally verified and published nuclear localization signals/nuclear export signals (NLSs/NESs) in the context of performed in silico predictions. We have used most of the available NLS/NES predictors. Importantly, all our results confirm the existence of a complex system responsible for protein localization regulation that involves many localization signals, which activity has to be precisely controlled. We conclude that the current stage of knowledge in this area is still not complete and for most of bHLH-PAS proteins an experimental verification of the activity of further NLS/NES is needed.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Transdução de Sinais , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Transporte , Regulação da Expressão Gênica , Humanos , Espaço Intracelular/metabolismo , Família Multigênica , Sinais de Localização Nuclear , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Relação Estrutura-AtividadeRESUMO
The basic helix-loop-helix/Per-ARNT-SIM (bHLH-PAS) proteins are a class of transcriptional regulators, commonly occurring in living organisms and highly conserved among vertebrates and invertebrates. These proteins exhibit a relatively well-conserved domain structure: the bHLH domain located at the N-terminus, followed by PAS-A and PAS-B domains. In contrast, their C-terminal fragments present significant variability in their primary structure and are unique for individual proteins. C-termini were shown to be responsible for the specific modulation of protein action. In this review, we present the current state of knowledge, based on NMR and X-ray analysis, concerning the structural properties of bHLH-PAS proteins. It is worth noting that all determined structures comprise only selected domains (bHLH and/or PAS). At the same time, substantial parts of proteins, comprising their long C-termini, have not been structurally characterized to date. Interestingly, these regions appear to be intrinsically disordered (IDRs) and are still a challenge to research. We aim to emphasize the significance of IDRs for the flexibility and function of bHLH-PAS proteins. Finally, we propose modern NMR methods for the structural characterization of the IDRs of bHLH-PAS proteins.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Biologia Computacional/métodos , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Anatômicos , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Relação Estrutura-AtividadeRESUMO
The thermodynamic acid dissociation constants (pKa1 and pKa2) of 16 anthracycline antibiotics, including doxorubicin (DOX) and daunorubicin (DAU), their epimers, epidoxorubicin (EDOX) and epidaunorubicin (EDAU), as well as novel anthracycline derivatives containing piperidine (FPIP), morpholine (FMOR) and hexamethylenoimine (FHEX) rings in the formamidine group of the daunosamine moiety were determined by analysis of the dependence between measured electrophoretic mobility and the pH of the buffer using the capillary zone electrophoresis method. The results obtained confirmed the ampholytic character of anthracyclines with at least two ionization states. The determined values were in the range of 8.36-9.28 and 9.38-11.48 for pKa1 and pKa2 arising from ionization of amino and phenolic groups, respectively. Structural modifications in the daunosamine moiety of the studied anthracyclines affected their pharmacological properties, such as antiproliferative activity.
Assuntos
Antraciclinas/química , Antibióticos Antineoplásicos/química , Eletroforese Capilar/métodos , Hexosaminas/química , Amidinas , Antraciclinas/farmacologia , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Hexosaminas/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Dinâmica não Linear , TermodinâmicaRESUMO
Methoprene tolerant protein (Met) has recently been confirmed as the long-sought juvenile hormone (JH) receptor. This protein plays a significant role in the cross-talk of the 20-hydroxyecdysone (20E) and JH signalling pathways, which are important for control of insect development and maturation. Met belongs to the basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) family of transcription factors. In these proteins, bHLH domains are typically responsible for DNA binding and dimerization, whereas the PAS domains are crucial for the choice of dimerization partner and the specificity of target gene activation. The C-terminal region is usually responsible for the regulation of protein complex activity. The sequence of the Met C-terminal region (MetC) is not homologous to any sequence deposited in the Protein Data Bank (PDB) and has not been structurally characterized to date. In this study, we show that the MetC exhibits properties typical for an intrinsically disordered protein (IDP). The final averaged structure obtained with small angle X-ray scattering (SAXS) experiments indicates that intrinsically disordered MetC exists in an extended conformation. This extended shape and the long unfolded regions characterise proteins with high flexibility and dynamics. Therefore, we suggest that the multiplicity of conformations adopted by the disordered MetC is crucial for its activity as a biological switch modulating the cross-talk of different signalling pathways in insects.