RESUMEN
Reverse transcription of human immunodeficiency virus type 1 (HIV-1) initiates from the 3' end of human tRNALys3. The primer tRNALys3 is selectively packaged into the virus in the form of a complex with human lysyl-tRNA synthetase (LysRS). To facilitate reverse transcription initiation, part of the 5' leader (5'L) of HIV-1 genomic RNA (gRNA) evolves a tRNA anticodon-like element (TLE), which binds LysRS and releases tRNALys3 for primer annealing and reverse transcription initiation. Although TLE has been identified as a key element in 5'L responsible for LysRS binding, how the conformations and various hairpin structures of 5'L regulate 5'L-LysRS interaction is not fully understood. Here, these factors have been individually investigated using direct and competitive fluorescence anisotropy binding experiments. Our data showed that the conformation of 5'L significantly influences its binding affinity with LysRS. The 5'L conformation favoring gRNA dimerization and packaging exhibits much weaker binding affinity with LysRS compared to the alternative 5'L conformation that is not selected for packaging. Additionally, dimerization of 5'L impairs LysRS-5'L interaction. Furthermore, among various regions of 5'L, both the primer binding site/TLE domain and the stem-loop 3 are important for LysRS interaction, whereas the dimerization initiation site and the splicing donor plays a minor role. In contrast, the presence of the transacting responsive and the polyadenylation signal hairpins slightly inhibit LysRS binding. These findings reveal that the conformation and various regions of the 5'L of HIV-1 genome regulate its interaction with human LysRS and the reverse transcription primer release process.
Asunto(s)
Genoma Viral , VIH-1 , Lisina-ARNt Ligasa , Conformación de Ácido Nucleico , Transcripción Reversa , Lisina-ARNt Ligasa/metabolismo , Lisina-ARNt Ligasa/química , Lisina-ARNt Ligasa/genética , Humanos , VIH-1/genética , VIH-1/enzimología , ARN Viral/metabolismo , ARN Viral/química , ARN Viral/genética , Regiones no Traducidas 5' , Unión ProteicaRESUMEN
Aminoacyl-tRNA synthetases (aaRSs) play a central role in the translation of genetic code, serving as attractive drug targets. Within this family, the lysyl-tRNA synthetase (LysRS) constitutes a promising antimalarial target. ASP3026, an anaplastic lymphoma kinase (ALK) inhibitor was recently identified as a novel Plasmodium falciparum LysRS (PfLysRS) inhibitor. Here, based on cocrystal structures and biochemical experiments, we developed a series of ASP3026 analogues to improve the selectivity and potency of LysRS inhibition. The leading compound 36 showed a dissociation constant of 15.9 nM with PfLysRS. The inhibitory efficacy on PfLysRS and parasites has been enhanced. Covalent attachment of L-lysine to compound 36 resulted in compound 36K3, which exhibited further increased inhibitory activity against PfLysRS but significantly decreased activity against ALK. However, its inhibitory activity against parasites did not improve, suggesting potential future optimization directions. This study presents a new example of derivatization of kinase inhibitors repurposed to inhibit aaRS.
Asunto(s)
Quinasa de Linfoma Anaplásico , Antimaláricos , Lisina-ARNt Ligasa , Plasmodium falciparum , Inhibidores de Proteínas Quinasas , Plasmodium falciparum/enzimología , Plasmodium falciparum/efectos de los fármacos , Lisina-ARNt Ligasa/antagonistas & inhibidores , Lisina-ARNt Ligasa/metabolismo , Lisina-ARNt Ligasa/química , Lisina-ARNt Ligasa/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico/metabolismo , Quinasa de Linfoma Anaplásico/genética , Antimaláricos/farmacología , Antimaláricos/química , Relación Estructura-Actividad , Humanos , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genéticaRESUMEN
Tuberculosis is a major global cause of both mortality and financial burden mainly in low and middle-income countries. Given the significant and ongoing rise of drug-resistant strains of Mycobacterium tuberculosis within the clinical setting, there is an urgent need for the development of new, safe and effective treatments. Here the development of a drug-like series based on a fused dihydropyrrolidino-pyrimidine scaffold is described. The series has been developed against M. tuberculosis lysyl-tRNA synthetase (LysRS) and cellular studies support this mechanism of action. DDD02049209, the lead compound, is efficacious in mouse models of acute and chronic tuberculosis and has suitable physicochemical, pharmacokinetic properties and an in vitro safety profile that supports further development. Importantly, preliminary analysis using clinical resistant strains shows no pre-existing clinical resistance towards this scaffold.
Asunto(s)
Lisina-ARNt Ligasa , Mycobacterium tuberculosis , Tuberculosis , Animales , Lisina-ARNt Ligasa/química , Lisina-ARNt Ligasa/genética , Lisina-ARNt Ligasa/farmacología , Ratones , Mycobacterium tuberculosis/genética , Tuberculosis/tratamiento farmacológicoRESUMEN
There is a pressing need for new medicines to prevent and treat malaria. Most antimalarial drug discovery is reliant upon phenotypic screening. However, with the development of improved target validation strategies, target-focused approaches are now being utilized. Here, we describe the development of a toolkit to support the therapeutic exploitation of a promising target, lysyl tRNA synthetase (PfKRS). The toolkit includes resistant mutants to probe resistance mechanisms and on-target engagement for specific chemotypes; a hybrid KRS protein capable of producing crystals suitable for ligand soaking, thus providing high-resolution structural information to guide compound optimization; chemical probes to facilitate pulldown studies aimed at revealing the full range of specifically interacting proteins and thermal proteome profiling (TPP); as well as streamlined isothermal TPP methods to provide unbiased confirmation of on-target engagement within a biologically relevant milieu. This combination of tools and methodologies acts as a template for the development of future target-enabling packages.
Asunto(s)
Antimaláricos , Lisina-ARNt Ligasa , Malaria , Antimaláricos/química , Antimaláricos/farmacología , Descubrimiento de Drogas , Humanos , Lisina-ARNt Ligasa/química , Lisina-ARNt Ligasa/genética , Lisina-ARNt Ligasa/metabolismo , Plasmodium falciparum/metabolismoRESUMEN
Interactions between lysyl-tRNA synthetase (LysRS) and HIV-1 Gag facilitate selective packaging of the HIV-1 reverse transcription primer, tRNALys3. During HIV-1 infection, LysRS is phosphorylated at S207, released from a multi-aminoacyl-tRNA synthetase complex and packaged into progeny virions. LysRS is critical for proper targeting of tRNALys3 to the primer-binding site (PBS) by specifically binding a PBS-adjacent tRNA-like element (TLE), which promotes release of the tRNA proximal to the PBS. However, whether LysRS phosphorylation plays a role in this process remains unknown. Here, we used a combination of binding assays, RNA chemical probing, and small-angle X-ray scattering to show that both wild-type (WT) and a phosphomimetic S207D LysRS mutant bind similarly to the HIV-1 genomic RNA (gRNA) 5'UTR via direct interactions with the TLE and stem loop 1 (SL1) and have a modest preference for binding dimeric gRNA. Unlike WT, S207D LysRS bound in an open conformation and increased the dynamics of both the PBS region and SL1. A new working model is proposed wherein a dimeric phosphorylated LysRS/tRNA complex binds to a gRNA dimer to facilitate tRNA primer release and placement onto the PBS. Future anti-viral strategies that prevent this host factor-gRNA interaction are envisioned.
Asunto(s)
Seropositividad para VIH , VIH-1 , Lisina-ARNt Ligasa , Regiones no Traducidas 5' , Seropositividad para VIH/genética , VIH-1/genética , VIH-1/metabolismo , Humanos , Lisina-ARNt Ligasa/química , Lisina-ARNt Ligasa/genética , Conformación de Ácido Nucleico , ARN Guía de Kinetoplastida , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , ARN Viral/genética , ARN Viral/metabolismoRESUMEN
Malaria is a parasitic illness caused by the genus Plasmodium from the apicomplexan phylum. Five plasmodial species of P. falciparum (Pf), P. knowlesi, P. malariae, P. ovale, and P. vivax (Pv) are responsible for causing malaria in humans. According to the World Malaria Report 2020, there were 229 million cases and ~ 0.04 million deaths of which 67% were in children below 5 years of age. While more than 3 billion people are at risk of malaria infection globally, antimalarial drugs are their only option for treatment. Antimalarial drug resistance keeps arising periodically and thus threatens the main line of malaria treatment, emphasizing the need to find new alternatives. The availability of whole genomes of P. falciparum and P. vivax has allowed targeting their unexplored plasmodial enzymes for inhibitor development with a focus on multistage targets that are crucial for parasite viability in both the blood and liver stages. Over the past decades, aminoacyl-tRNA synthetases (aaRSs) have been explored as anti-bacterial and anti-fungal drug targets, and more recently (since 2009) aaRSs are also the focus of antimalarial drug targeting. Here, we dissect the structure-based knowledge of the most advanced three aaRSs-lysyl- (KRS), prolyl- (PRS), and phenylalanyl- (FRS) synthetases in terms of development of antimalarial drugs. These examples showcase the promising potential of this family of enzymes to provide druggable targets that stall protein synthesis upon inhibition and thereby kill malaria parasites selectively.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Antimaláricos/química , Inhibidores Enzimáticos/química , Lisina-ARNt Ligasa/química , Fenilalanina-ARNt Ligasa/química , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/química , Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Antimaláricos/farmacología , Dominio Catalítico , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Expresión Génica , Humanos , Lisina-ARNt Ligasa/antagonistas & inhibidores , Lisina-ARNt Ligasa/genética , Lisina-ARNt Ligasa/metabolismo , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Modelos Moleculares , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Fenilalanina-ARNt Ligasa/genética , Fenilalanina-ARNt Ligasa/metabolismo , Plasmodium falciparum/química , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Aminoacyl-tRNA synthetases (AARSs) catalyze the ligation of amino acids to their cognate tRNAs and therefore play an essential role in protein biosynthesis in all living cells. The KARS gene in human encodes both cytosolic and mitochondrial lysyl-tRNA synthetase (LysRS). A recent study identified a missense mutation in KARS gene (c.517T > C) that caused autosomal recessive nonsyndromic hearing loss. This mutation led to a tyrosine to histidine (YH) substitution in both cytosolic and mitochondrial LysRS proteins, and decreased their aminoacylation activity to different levels. Here, we report the crystal structure of LysRS YH mutant at a resolution of 2.5 Å. We found that the mutation did not interfere with the active center, nor did it cause any significant conformational changes in the protein. The loops involved in tetramer interface and tRNA anticodon binding site showed relatively bigger variations between the mutant and wild type proteins. Considering the differences between the cytosolic and mitochondrial tRNAlyss, we suggest that the mutation triggered subtle changes in the tRNA anticodon binding region, and the interferences were further amplified by the different D and T loops in mitochondrial tRNAlys, and led to a complete loss of the aminoacylation of mitochondrial tRNAlys.
Asunto(s)
Sordera/enzimología , Lisina-ARNt Ligasa/química , Mutación , Aminoacilación , Anticodón , Cristalografía por Rayos X , Sordera/genética , Sordera/metabolismo , Sordera/patología , Predisposición Genética a la Enfermedad , Humanos , Lisina-ARNt Ligasa/genética , Lisina-ARNt Ligasa/aislamiento & purificación , Lisina-ARNt Ligasa/metabolismo , Mitocondrias/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/aislamiento & purificación , Proteínas Mutantes/metabolismo , Biosíntesis de Proteínas , Elementos Estructurales de las Proteínas , ARN de Transferencia/química , ARN de Transferencia/genética , ARN de Transferencia/metabolismoRESUMEN
Replication of human immunodeficiency virus type 1 (HIV-1) requires the packaging of tRNALys,3 from the host cell into the new viral particles. The GagPol viral polyprotein precursor associates with mitochondrial lysyl-tRNA synthetase (mLysRS) in a complex with tRNALys, an essential step to initiate reverse transcription in the virions. The C-terminal integrase moiety of GagPol is essential for its association with mLysRS. We show that integrases from HIV-1 and HIV-2 bind mLysRS with the same efficiency. In this work, we have undertaken to probe the three-dimensional (3D) architecture of the complex of integrase with mLysRS. We first established that the C-terminal domain (CTD) of integrase is the major interacting domain with mLysRS. Using the pBpa-photo crosslinking approach, inter-protein cross-links were observed involving amino acid residues located at the surface of the catalytic domain of mLysRS and of the CTD of integrase. In parallel, using molecular docking simulation, a single structural model of complex was found to outscore other alternative conformations. Consistent with crosslinking experiments, this structural model was further probed experimentally. Five compensatory mutations in the two partners were successfully designed which supports the validity of the model. The complex highlights that binding of integrase could stabilize the tRNALys:mLysRS interaction.
Asunto(s)
Integrasa de VIH/química , Lisina-ARNt Ligasa/química , Mitocondrias/enzimología , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Dominios Proteicos , Técnicas del Sistema de Dos HíbridosRESUMEN
Aminoacyl-tRNA synthetases are attractive targets for the development of antibacterial, antifungal, antiparasitic agents and for the treatment of other human diseases. Lysyl-tRNA synthetase (LysRS) from this family has been validated as a promising target for the development of antimalarial drugs. Here, we developed a high-throughput compatible assay and screened 1215 bioactive compounds to identify Plasmodium falciparum cytoplasmic LysRS (PfLysRS) inhibitor. ASP3026, an anaplastic lymphoma kinase inhibitor that was used in clinical trials for the treatment of B-cell lymphoma and solid tumors, was identified as a novel PfLysRS inhibitor. ASP3026 suppresses the enzymatic activity of PfLysRS at nanomolar potency, which is >380-fold more effective than inhibition of the human counterpart. In addition, the compound suppressed blood-stage P. falciparum growth. To understand the molecular mechanism of inhibition by ASP3026, we further solved the cocrystal structure of PfLysRS-ASP3026 at a resolution of 2.49 Å, providing clues for further optimization of the compound. Finally, primary structure-activity relationship analyses indicated that the inhibition of PfLysRS by ASP3026 is highly structure specific. This work not only provides a new chemical scaffold with good druggability for antimalarial development but also highlights the potential for repurposing kinase-inhibiting drugs to tRNA synthetase inhibitors to treat human diseases.
Asunto(s)
Antimaláricos/farmacología , Inhibidores Enzimáticos/farmacología , Lisina-ARNt Ligasa/antagonistas & inhibidores , Plasmodium falciparum/enzimología , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Animales , Antimaláricos/química , Inhibidores Enzimáticos/química , Humanos , Lisina-ARNt Ligasa/química , Modelos Moleculares , Plasmodium falciparum/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Conejos , Relación Estructura-Actividad , Sulfonas/química , Sulfonas/farmacología , Triazinas/química , Triazinas/farmacologíaRESUMEN
Human lysyl-tRNA synthetase (hLysRS) is essential for aminoacylation of tRNALys Higher eukaryotic LysRSs possess an N-terminal extension (Nterm) previously shown to facilitate high-affinity tRNA binding and aminoacylation. This eukaryote-specific appended domain also plays a critical role in hLysRS nuclear localization, thus facilitating noncanonical functions of hLysRS. The structure is intrinsically disordered and therefore remains poorly characterized. Findings of previous studies are consistent with the Nterm domain undergoing a conformational transition to an ordered structure upon nucleic acid binding. In this study, we used NMR to investigate how the type of RNA, as well as the presence of the adjacent anticodon-binding domain (ACB), influences the Nterm conformation. To explore the latter, we used sortase A ligation to produce a segmentally labeled tandem-domain protein, Nterm-ACB. In the absence of RNA, Nterm remained disordered regardless of ACB attachment. Both alone and when attached to ACB, Nterm structure remained unaffected by titration with single-stranded RNAs. The central region of the Nterm domain adopted α-helical structure upon titration of Nterm and Nterm-ACB with RNA hairpins containing double-stranded regions. Nterm binding to the RNA hairpins resulted in CD spectral shifts consistent with an induced helical structure. NMR and fluorescence anisotropy revealed that Nterm binding to hairpin RNAs is weak but that the binding affinity increases significantly upon covalent attachment to ACB. We conclude that the ACB domain facilitates induced-fit conformational changes and confers high-affinity RNA hairpin binding, which may be advantageous for functional interactions of LysRS with a variety of different binding partners.
Asunto(s)
Lisina-ARNt Ligasa/química , Modelos Moleculares , Pliegue del ARN , ARN de Transferencia/química , Humanos , Espectroscopía de Resonancia Magnética , Dominios ProteicosRESUMEN
High-quality antibody (Ab) production depends on the availability of immunologically relevant antigens. We present a potentially universal platform for generating soluble antigens from bacterial hosts, tailored to immunized animals for Ab production. A novel RNA-dependent chaperone, in which the target antigen is genetically fused with an RNA-interacting domain (RID) docking tag derived from the immunized host, promotes the solubility and robust folding of the target antigen. We selected the N-terminal tRNA-binding domain of lysyl-tRNA synthetase (LysRS) as the RID for fusion with viral proteins and demonstrated the expression of the RID fusion proteins in their soluble and native conformations; immunization predominantly elicited Ab responses to the target antigen, whereas the "self" RID tag remained nonimmunogenic. Differential immunogenicity of the fusion proteins greatly enriched and simplified the screening of hybridoma clones of monoclonal antibodies (mAbs), enabling specific and sensitive serodiagnosis of MERS-CoV infection. Moreover, mAbs against the consensus influenza hemagglutinin stalk domain enabled a novel assay for trivalent seasonal influenza vaccines. The Fc-mediated effector function was demonstrated, which could be harnessed for the design of next-generation "universal" influenza vaccines. The nonimmunogenic built-in antigen folding module tailored to a repertoire of immunized animal hosts will drive immunochemical diagnostics, therapeutics, and designer vaccines.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Antivirales/biosíntesis , Antígenos Virales/química , Infecciones por Coronavirus/diagnóstico , Hibridomas/inmunología , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Chaperonas Moleculares , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Antígenos Virales/genética , Antígenos Virales/inmunología , Infecciones por Coronavirus/sangre , Ensayo de Inmunoadsorción Enzimática , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Inmunización , Vacunas contra la Influenza , Lisina-ARNt Ligasa/química , Lisina-ARNt Ligasa/genética , Ratones , Ratones Endogámicos BALB C , Conformación Proteica , Dominios Proteicos , Pliegue de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Pruebas Serológicas , SolubilidadRESUMEN
Aminoacyl-tRNA synthetases, the essential enzyme family for protein translation, are attractive targets for developing antibacterial, antifungal, and antiparasitic agents and for treating other human diseases. The antimalarial natural product cladosporin was discovered recently as a novel lysyl-tRNA synthetase (LysRS) specific inhibitor. Here, we report a thorough analysis of cladosporin derivatives using chemical synthesis, biophysical, and biochemical experiments. A series of isocoumarin derivatives with only one nonhydrogen atom/bond change per compound was synthesized. These changes include replacements of methyltetrahydropyran moiety by methylcyclohexane or cyclohexane, lactone by lactam, hydroxyl groups by methoxyl groups, and dismission of the chiral center at C3 with a Δ3,4 double bond. We evaluated these compounds by thermal shift assays and enzymatic experiments and further studied their molecular recognition by the Plasmodium falciparum LysRS through total five high-resolution crystal structures. Our results showed that the methyltetrahydropyran moiety of cladosporin could be replaced by a more stable methylcyclohexane without reducing binding ability. Removing the methyl group from the methylcyclohexane moiety slightly decreased the interaction with LysRS. Besides, the replacement with a lactam group or a conjugated Δ3,4 double bond within the scaffold could be two more options to optimize the compound. Lastly, the two phenolic hydroxyl groups were critical for the compounds to bind LysRS. The detailed analyses at atomic resolution in this study provide a foundation for the further development of new antibiotics from cladosporin derivatives.
Asunto(s)
Antimaláricos/química , Inhibidores Enzimáticos/química , Isocumarinas/química , Lisina-ARNt Ligasa/antagonistas & inhibidores , Antimaláricos/síntesis química , Antimaláricos/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Isocumarinas/síntesis química , Isocumarinas/metabolismo , Lisina-ARNt Ligasa/química , Lisina-ARNt Ligasa/metabolismo , Plasmodium falciparum/enzimología , Unión ProteicaRESUMEN
Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections and has highly developed systems for acquiring resistance against numerous antibiotics. The gene (lysS) encoding P. aeruginosa lysyl-tRNA synthetase (LysRS) was cloned and overexpressed, and the resulting protein was purified to 98% homogeneity. LysRS was kinetically evaluated, and the Km values for the interaction with lysine, adenosine triphosphate (ATP), and tRNALys were determined to be 45.5, 627, and 3.3 µM, respectively. The kcatobs values were calculated to be 13, 22.8, and 0.35 s-1, resulting in kcatobs/KM values of 0.29, 0.036, and 0.11 s-1µM-1, respectively. Using scintillation proximity assay technology, natural product and synthetic compound libraries were screened to identify inhibitors of function of the enzyme. Three compounds (BM01D09, BT06F11, and BT08F04) were identified with inhibitory activity against LysRS. The IC50 values were 17, 30, and 27 µM for each compound, respectively. The minimum inhibitory concentrations were determined against a panel of clinically important pathogens. All three compounds were observed to inhibit the growth of gram-positive organisms with a bacteriostatic mode of action. However, two compounds (BT06F11 and BT08F04) were bactericidal against cultures of gram-negative bacteria. When tested against human cell cultures, BT06F11 was not toxic at any concentration tested, and BM01D09 was toxic only at elevated levels. However, BT08F04 displayed a CC50 of 61 µg/mL. In studies of the mechanism of inhibition, BM01D09 inhibited LysRS activity by competing with ATP for binding, and BT08F04 was competitive with ATP and uncompetitive with the amino acid. BT06F11 inhibited LysRS activity by a mechanism other than substrate competition.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Lisina-ARNt Ligasa/antagonistas & inhibidores , Lisina-ARNt Ligasa/química , Pseudomonas aeruginosa/enzimología , Relación Dosis-Respuesta a Droga , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Estructura Molecular , Pseudomonas aeruginosa/efectos de los fármacos , Bibliotecas de Moléculas PequeñasRESUMEN
Intrinsic disorders are a common feature of hub proteins in eukaryotic interactomes controlling the signaling pathways. The intrinsically disordered proteins (IDPs) are prone to misfolding, and maintaining their functional stability remains a major challenge in validating their therapeutic potentials. Considering that IDPs are highly enriched in RNA-binding proteins (RBPs), here we reasoned and confirmed that IDPs could be stabilized by fusion to RBPs. Dickkopf2 (DKK2), Wnt antagonist and a prototype IDP, was fused with lysyl-tRNA synthetase (LysRS), with or without the fragment crystallizable (Fc) domain of an immunoglobulin and expressed predominantly as a soluble form from a bacterial host. The functional competence was confirmed by in vitro Wnt signaling reporter and tube formation in human umbilical vein endothelial cells (HUVECs) and in vivo Matrigel plug assay. The removal of LysRS by site-specific protease cleavage prompted the insoluble aggregation, confirming that the linkage to RBP chaperones the functional competence of IDPs. While addressing to DKK2 as a key modulator for cancer and ischemic vascular diseases, our results suggest the use of RBPs as stabilizers of disordered proteinaceous materials for acquiring and maintaining the structural stability and functional competence, which would impact the druggability of a variety of IDPs from human proteome.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Lisina-ARNt Ligasa/química , Lisina-ARNt Ligasa/genética , Lisina-ARNt Ligasa/metabolismo , Motivos de Unión al ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Vía de Señalización Wnt/genética , Vía de Señalización Wnt/fisiologíaRESUMEN
Mutations in genes encoding aminoacyl-tRNA synthetases have been reported in several neurological disorders. KARS is a dual localized lysyl-tRNA synthetase and its cytosolic isoform belongs to the multiple aminoacyl-tRNA synthetase complex (MSC). Biallelic mutations in the KARS gene were described in a wide phenotypic spectrum ranging from nonsyndromic deafness to complex impairments. Here, we report on a patient with severe neurological and neurosensory disease investigated by whole-exome sequencing and found to carry biallelic mutations c.683C>T (p.Pro228Leu) and c.871T>G (p.Phe291Val), the second one being novel, in the KARS gene. The patient presented with an atypical clinical presentation with an optic neuropathy not previously reported. At the cellular level, we show that cytoplasmic KARS was expressed at a lower level in patient cells and displayed decreased interaction with MSC. In vitro, these two KARS variants have a decreased aminoacylation activity compared with wild-type KARS, the p.Pro228Leu being the most affected. Our data suggest that dysfunction of cytoplasmic KARS resulted in a decreased level of translation of the nuclear-encoded lysine-rich proteins belonging to the respiratory chain complex, thus impairing mitochondria functions.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Lisina-ARNt Ligasa/genética , Mutación , Enfermedades del Sistema Nervioso/complicaciones , Enfermedades del Sistema Nervioso/genética , Enfermedades del Nervio Óptico/complicaciones , Trastornos de la Sensación/complicaciones , Trastornos de la Sensación/genética , Alelos , Secuencia de Aminoácidos , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/metabolismo , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Fibroblastos/metabolismo , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Lisina-ARNt Ligasa/química , Lisina-ARNt Ligasa/metabolismo , Imagen por Resonancia Magnética , Modelos Moleculares , Enfermedades del Sistema Nervioso/diagnóstico , Enfermedades del Nervio Óptico/diagnóstico , Linaje , Unión Proteica , Conformación Proteica , Trastornos de la Sensación/diagnóstico , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Cladosporin (CLD) is a fungal metabolite that kills the malaria parasite via inhibiting its cytoplasmic lysyl-tRNA synthetase (KRS) and abrogating protein translation. Here we provide structural and drug selectivity analyses on CLD interacting residues in apo and holo KRSs from Plasmodium falciparum, Homo sapiens, Cryptosporidium parvum, and Mycobacterium ulcerans. We show that both gross and subtle alterations in protein backbone and sidechains drive the active site structural plasticity that allows integration of CLD in KRSs. The ligand-induced fit of CLD in PfKRS is marked by closure and stabilization of three disordered loops and one alpha helix. However, these structural rearragements are not evident in KRS-CLD complexes from H. sapiens, C. parvum, or M. ulcerans. Strikingly, CLD fits into the MuKRS active site due to a remarkable rotameric alteration in its clash-prone methionine residue that provides accommodation for the methyl moiety in CLD. Although the high concentrations of drugs used for Hs, Cp, and MuKRS-CLD complexes in co-crystallization studies enable elucidation of a structural framework for understanding drug binding in KRSs, we propose that these data should be concurrently assessed via biochemical studies of potency and drug selectivity given the poor cell-based activity of CLD against human and bacterial cells. Our comprehensive analyses of KRS-CLD interactions, therefore, highlight vital issues in structure-based drug discovery studies.
Asunto(s)
Isocumarinas/metabolismo , Lisina-ARNt Ligasa/metabolismo , Plasmodium falciparum/enzimología , Cryptosporidium parvum/enzimología , Isocumarinas/química , Lisina-ARNt Ligasa/química , Mycobacterium ulcerans/enzimología , Unión ProteicaRESUMEN
Eukaryotic lysyl-tRNA synthetases (LysRS) have an N-terminal appended tRNA-interaction domain (RID) that is absent in their prokaryotic counterparts. This domain is intrinsically disordered and lacks stable structures. The disorder-to-order transition is induced by tRNA binding and has implications on folding and subsequent assembly into multi-tRNA synthetase complexes. Here, we expressed and purified RID from human LysRS (hRID) in Escherichia coli and performed a detailed mutagenesis of the appended domain. hRID was co-purified with nucleic acids during Ni-affinity purification, and cumulative mutations on critical amino acid residues abolished RNA binding. Furthermore, we identified a structural ensemble between disordered and helical structures in non-RNA-binding mutants and an equilibrium shift for wild-type into the helical conformation upon RNA binding. Since mutations that disrupted RNA binding led to an increase in non-functional soluble aggregates, a stabilized RNA-mediated structural transition of the N-terminal appended domain may have implications on the functional organization of human LysRS and multi-tRNA synthetase complexes in vivo.
Asunto(s)
Lisina-ARNt Ligasa/química , Lisina-ARNt Ligasa/metabolismo , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Humanos , Mutación , Unión Proteica , Estructura Secundaria de Proteína , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Relación Estructura-ActividadRESUMEN
The dependence of drug potency on diastereomeric configurations is a key facet. Using a novel general divergent synthetic route for a three-chiral center antimalarial natural product cladosporin, we built its complete library of stereoisomers (cladologs) and assessed their inhibitory potential using parasite-, enzyme-, and structure-based assays. We show that potency is manifest via tetrahyropyran ring conformations that are housed in the ribose binding pocket of parasite lysyl tRNA synthetase (KRS). Strikingly, drug potency between top and worst enantiomers varied 500-fold, and structures of KRS-cladolog complexes reveal that alterations at C3 and C10 are detrimental to drug potency whereas changes at C3 are sensed by rotameric flipping of glutamate 332. Given that scores of antimalarial and anti-infective drugs contain chiral centers, this work provides a new foundation for focusing on inhibitor stereochemistry as a facet of antimicrobial drug development.
Asunto(s)
Antimaláricos/química , Antimaláricos/farmacología , Isocumarinas/química , Isocumarinas/farmacología , Plasmodium falciparum/efectos de los fármacos , Antimaláricos/metabolismo , Evaluación Preclínica de Medicamentos , Isocumarinas/metabolismo , Lisina-ARNt Ligasa/química , Lisina-ARNt Ligasa/metabolismo , Modelos Moleculares , Plasmodium falciparum/enzimología , Conformación Proteica , EstereoisomerismoRESUMEN
Aminoacyl-tRNA synthetases (aaRSs) are enzymes that function at the first step of translation, catalyzing the conjugation of amino acids to their cognate tRNAs for protein synthesis. While preserving this essential role, higher eukaryotic aaRSs, such as human cytoplasmic aaRSs, have developed other functions during evolution, including angiogenesis, inflammation, development, tumorigenesis, etc. These translational and nontranslational functions of aaRSs are attractive targets for developing antibacterial, antifungal, anticancer agents and for treating other human diseases. Structural characterization of aaRS functions in both categories has deepened our understanding and provided insightful platform for further structure-based drug design. The convergence of the mechanism of action, together with their divergent functions, offers a possible protocol for studying these features of aaRSs in general. To guide this objective in future, we provide here a review on the methods used in structural analysis, which may be applied to study this special group of housekeeping proteins.
Asunto(s)
Lisina-ARNt Ligasa/química , Lisina/química , Procesamiento Proteico-Postraduccional , ARN de Transferencia de Lisina/química , Línea Celular Tumoral , Clonación Molecular , Cristalografía por Rayos X/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Linfocitos/química , Linfocitos/citología , Linfocitos/enzimología , Lisina/metabolismo , Lisina-ARNt Ligasa/genética , Lisina-ARNt Ligasa/metabolismo , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , ARN de Transferencia de Lisina/genética , ARN de Transferencia de Lisina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Lysyl-tRNA synthetase (KRS) is an enzyme that conjugates lysine to its cognate tRNAs in the process of protein synthesis. In addition to its catalytic function, KRS binds to the 67-kDa laminin receptor (LR) on the cell membrane and facilitates cell migration and metastasis. Modulation of this interaction by small-molecule inhibitors can be exploited to suppress cancer metastasis. In this study, we present fragment-based methods for the identification of inhibitors and monitoring protein-protein interactions between KRS and LR. First, we identified the amino acid residues, located on the KRS anticodon-binding domain, which interact with the C-terminal extension of the LR. One-dimensional (1D) relaxation-edited nuclear magnetic resonance spectroscopy (NMR) and competition experiments were designed and optimized to screen the fragment library. For screening using two-dimensional (2D) NMR, we identified the indicative signals in the KRS anticodon-binding domain and selected inhibitors that bind to KRS and compete with LR at the KRS-LR binding interface. These methods may offer an efficient approach for the discovery of anti-metastatic drugs.