RESUMO
CBL is a RING type E3 ubiquitin ligase that functions as a negative regulator of tyrosine kinase signaling and loss of CBL E3 function is implicated in several forms of leukemia. The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL and are required for CBL-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling. Despite the established role of SLAP/SLAP2 in regulating CBL activity, the nature of the interaction and the mechanisms involved are not known. To understand the molecular basis of the interaction between SLAP/SLAP2 and CBL, we solved the crystal structure of CBL tyrosine kinase binding domain (TKBD) in complex with SLAP2. The carboxy-terminal region of SLAP2 adopts an α-helical structure which binds in a cleft between the 4H, EF-hand, and SH2 domains of the TKBD. This SLAP2 binding site is remote from the canonical TKBD phospho-tyrosine peptide binding site but overlaps with a region important for stabilizing CBL in its autoinhibited conformation. In addition, binding of SLAP2 to CBL in vitro activates the ubiquitin ligase function of autoinhibited CBL. Disruption of the CBL/SLAP2 interface through mutagenesis demonstrated a role for this protein-protein interaction in regulation of CBL E3 ligase activity in cells. Our results reveal that SLAP2 binding to a regulatory cleft of the TKBD provides an alternative mechanism for activation of CBL ubiquitin ligase function.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Proto-Oncogênicas c-cbl/química , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/química , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sítios de Ligação , Regulação para Baixo , Humanos , Conformação Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Receptores Proteína Tirosina Quinases/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Domínios de Homologia de srcRESUMO
The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL E3 ubiquitin ligase to downregulate antigen, cytokine and tyrosine kinase receptor signalling. In contrast to the phosphotyrosine-dependent binding of CBL substrates through its tyrosine kinase-binding domain (TKBD), CBL TKBD associates with the C-terminal tail of SLAP2 in a phospho-independent manner. To understand the distinct nature of this interaction, a purification protocol for SLAP2 in complex with CBL TKBD was established and the complex was crystallized. However, determination of the complex crystal structure was hindered by the apparent degradation of SLAP2 during the crystallization process, such that only the CBL TKBD residues could initially be modelled. Close examination of the CBL TKBD structure revealed a unique dimer interface that included two short segments of electron density of unknown origin. To elucidate which residues of SLAP2 to model into this unassigned density, a co-expression system was generated to test SLAP2 deletion mutants and define the minimal SLAP2 binding region. SLAP2 degradation products were also analysed by mass spectrometry. The model-building and map-generation features of the Phenix software package were employed, leading to successful modelling of the C-terminal tail of SLAP2 into the unassigned electron-density segments.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Proto-Oncogênicas c-cbl/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Elétrons , Humanos , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismoRESUMO
Serine hydroxymethyltransferase 2 (SHMT2) converts serine plus tetrahydrofolate (THF) into glycine plus methylene-THF and is upregulated at the protein level in lung and other cancers. In order to better understand the role of SHMT2 in cancer a model system of HeLa cells engineered for inducible over-expression or knock-down of SHMT2 was characterized for cell proliferation and changes in metabolites and proteome as a function of SHMT2. Ectopic over-expression of SHMT2 increased cell proliferation in vitro and tumor growth in vivo. Knockdown of SHMT2 expression in vitro caused a state of glycine auxotrophy and accumulation of phosphoribosylaminoimidazolecarboxamide (AICAR), an intermediate of folate/1-carbon-pathway-dependent de novo purine nucleotide synthesis. Decreased glycine in the HeLa cell-based xenograft tumors with knocked down SHMT2 was potentiated by administration of the anti-hyperglycinemia agent benzoate. However, tumor growth was not affected by SHMT2 knockdown with or without benzoate treatment. Benzoate inhibited cell proliferation in vitro, but this was independent of SHMT2 modulation. The abundance of proteins of mitochondrial respiration complexes 1 and 3 was inversely correlated with SHMT2 levels. Proximity biotinylation in vivo (BioID) identified 48 mostly mitochondrial proteins associated with SHMT2 including the mitochondrial enzymes Acyl-CoA thioesterase (ACOT2) and glutamate dehydrogenase (GLUD1) along with more than 20 proteins from mitochondrial respiration complexes 1 and 3. These data provide insights into possible mechanisms through which elevated SHMT2 in cancers may be linked to changes in metabolism and mitochondrial function.
Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Glicina Hidroximetiltransferase/metabolismo , Neoplasias Pulmonares/patologia , Metaboloma , Proteoma/análise , Serina/metabolismo , Animais , Antifúngicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Glicina Hidroximetiltransferase/antagonistas & inibidores , Glicina Hidroximetiltransferase/genética , Células HeLa , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Domínios e Motivos de Interação entre Proteínas , Benzoato de Sódio/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease. Historically, therapeutics targeting RTKs have been identified using in vitro kinase assays. Due to frequent development of drug resistance, however, there is a need to identify more diverse compounds that inhibit mutated but not wild-type RTKs. Here, we describe MaMTH-DS (mammalian membrane two-hybrid drug screening), a live-cell platform for high-throughput identification of small molecules targeting functional protein-protein interactions of RTKs. We applied MaMTH-DS to an oncogenic epidermal growth factor receptor (EGFR) mutant resistant to the latest generation of clinically approved tyrosine kinase inhibitors (TKIs). We identified four mutant-specific compounds, including two that would not have been detected by conventional in vitro kinase assays. One of these targets mutant EGFR via a new mechanism of action, distinct from classical TKI inhibition. Our results demonstrate how MaMTH-DS is a powerful complement to traditional drug screening approaches.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Inibidores de Proteínas Quinases/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular , Linhagem Celular Tumoral , DNA Nucleotidiltransferases/genética , Descoberta de Drogas , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Genes Reporter , Humanos , Luciferases/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação , Fosforilação/efeitos dos fármacos , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/farmacologia , Estaurosporina/análogos & derivados , Estaurosporina/farmacologiaRESUMO
Retinitis pigmentosa (RP) is a degenerative retinal disease, often caused by mutations in the G-protein-coupled receptor rhodopsin. The majority of pathogenic rhodopsin mutations cause rhodopsin to misfold, including P23H, disrupting its crucial ability to respond to light. Previous screens to discover pharmacological chaperones of rhodopsin have primarily been based on rescuing rhodopsin trafficking and localization to the plasma membrane. Here, we present methods utilizing a yeast-based assay to screen for compounds that rescue the ability of rhodopsin to activate an associated downstream G-protein signaling cascade. We engineered a yeast strain in which human rhodopsin variants were genomically integrated, and were able to demonstrate functional coupling to the yeast mating pathway, leading to fluorescent protein expression. We confirmed that a known pharmacological chaperone, 9-cis retinal, could partially rescue light-dependent activation of a disease-associated rhodopsin mutation (P23H) expressed in yeast. These novel yeast strains were used to perform a phenotypic screen of 4280 compounds from the LOPAC1280 library and a peptidomimetic library, to discover novel pharmacological chaperones of rhodopsin. The fluorescence-based assay was robust in a 96-well format, with a Z' factor of 0.65 and a signal-to-background ratio of above 14. One compound was selected for additional analysis, but it did not appear to rescue rhodopsin function in yeast. The methods presented here are amenable to future screens of small-molecule libraries, as they are robust and cost-effective. We also discuss how these methods could be further modified or adapted to perform screens of more compounds in the future.
Assuntos
Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Bibliotecas de Moléculas Pequenas , Leveduras/efeitos dos fármacos , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Mutação , Receptores Acoplados a Proteínas G/genética , Retinose Pigmentar/tratamento farmacológico , Retinose Pigmentar/etiologia , Rodopsina/genética , Transdução de Sinais/efeitos dos fármacos , Leveduras/genética , Leveduras/metabolismoRESUMO
The balance between comprehensively analyzing the proteome and using valuable mass spectrometry time is a genuine challenge in the field of proteomics. Multidimensional fractionation strategies have significantly increased proteome coverage, but often at the cost of increased mass analysis time, despite advances in mass spectrometer acquisition rates. Recently, the Evosep One liquid chromatography system was shown to analyze peptide samples in a high-throughput manner without sacrificing in-depth proteomics coverage. We demonstrate the incorporation of Evosep One technology into our multiplexing workflow for analysis of tandem mass tag (TMT)-labeled nonsmall cell lung carcinoma (NSCLC) patient-derived xenografts (PDXs). By the use of a 30 samples per day Evosep workflow, >12â¯000 proteins were identified in 48 h of mass spectrometry time, which is comparable to the number of proteins identified by our conventional concatenated EASY-nLC workflow in 60 h. Shorter Evosep gradient lengths reduced the number of protein identifications by 10% while decreasing the mass analysis time by 50%. This Evosep workflow will enable quantitative analysis of multiplexed samples in less time without conceding depth of proteome coverage.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/química , Cromatografia Líquida/métodos , Neoplasias Pulmonares/química , Proteínas de Neoplasias/isolamento & purificação , Peptídeos/isolamento & purificação , Proteoma/isolamento & purificação , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Cromatografia Líquida/instrumentação , Expressão Gênica , Xenoenxertos , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos SCID , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Peptídeos/química , Proteoma/química , Proteoma/genética , Proteoma/metabolismo , Coloração e Rotulagem/métodos , Espectrometria de Massas em Tandem , Fatores de Tempo , Fluxo de TrabalhoRESUMO
Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y(194) impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y(194) on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases.
Assuntos
Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Mieloide Aguda/enzimologia , Mieloma Múltiplo/enzimologia , Fosfotirosina/metabolismo , Proteômica/métodos , Quinases da Família src/química , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Sequência Conservada , Cristalografia por Raios X , Humanos , Modelos Moleculares , Estrutura Secundária de Proteína , Especificidade por Substrato , Domínios de Homologia de srcRESUMO
SLAP (Src like adaptor protein) contains adjacent Src homology 3 (SH3) and Src homology 2 (SH2) domains closely related in sequence to that of cytoplasmic Src family tyrosine kinases. Expressed most abundantly in the immune system, SLAP function has been predominantly studied in the context of lymphocyte signaling, where it functions in the Cbl dependent downregulation of antigen receptor signaling. However, accumulating evidence suggests that SLAP plays a role in the regulation of a broad range of membrane receptors including members of the receptor tyrosine kinase (RTK) family. In this review we highlight the role of SLAP in the ubiquitin dependent regulation of type III RTKs PDGFR, CSF-1R, KIT and Flt3, as well as Eph family RTKs. SLAP appears to bind activated type III and Eph RTKs via a conserved autophosphorylated juxtamembrane tyrosine motif in an SH2-dependent manner, suggesting that SLAP is important in regulating RTK signaling.
Assuntos
Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-kit/química , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/química , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/química , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/química , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Tirosina Quinase 3 Semelhante a fms/química , Tirosina Quinase 3 Semelhante a fms/metabolismo , Domínios de Homologia de srcRESUMO
The protein kinase Rad53 is a key regulator of the DNA damage checkpoint in budding yeast. Its human ortholog, CHEK2, is mutated in familial breast cancer and mediates apoptosis in response to genotoxic stress. Autophosphorylation of Rad53 at residue Thr354 located in the kinase activation segment is essential for Rad53 activation. In this study, we assessed the requirement of kinase domain dimerization and the exchange of its activation segment during the Rad53 activation process. We solved the crystal structure of Rad53 in its dimeric form and found that disruption of the observed head-to-tail, face-to-face dimer structure decreased Rad53 autophosphorylation on Thr354 in vitro and impaired Rad53 function in vivo. Moreover, we provide critical functional evidence that Rad53 trans-autophosphorylation may involve the interkinase domain exchange of helix αEF via an invariant salt bridge. These findings suggest a mechanism of autophosphorylation that may be broadly applicable to other protein kinases.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/química , Quinase do Ponto de Checagem 2/genética , Cristalografia por Raios X , Dimerização , Ativação Enzimática , Humanos , Dados de Sequência Molecular , Mutação , Fosforilação , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , UltracentrifugaçãoRESUMO
The Src-like adaptor proteins (SLAP/SLAP2) are key components of Cbl-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling in hematopoietic cells. SLAP and SLAP2 consist of adjacent SH3 and SH2 domains that are most similar in sequence to Src family kinases (SFKs). Notably, the SH3-SH2 connector sequence is significantly shorter in SLAP/SLAP2 than in SFKs. To understand the structural implication of a short SH3-SH2 connector sequence, we solved the crystal structure of a protein encompassing the SH3 domain, SH3-SH2 connector, and SH2 domain of SLAP2 (SLAP2-32). While both domains adopt typical folds, the short SH3-SH2 connector places them in close association. Strand ße of the SH3 domain interacts with strand ßA of the SH2 domain, resulting in the formation of a continuous ß sheet that spans the length of the protein. Disruption of the SH3/SH2 interface through mutagenesis decreases SLAP-32 stability in vitro, consistent with inter-domain binding being an important component of SLAP2 structure and function. The canonical peptide binding pockets of the SH3 and SH2 domains are fully accessible, in contrast to other protein structures that display direct interaction between SH3 and SH2 domains, in which either peptide binding surface is obstructed by the interaction. Our results reveal potential sites of novel interaction for SH3 and SH2 domains, and illustrate the adaptability of SH2 and SH3 domains in mediating interactions. As well, our results suggest that the SH3 and SH2 domains of SLAP2 function interdependently, with implications on their mode of substrate binding.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Proto-Oncogênicas pp60(c-src)/química , Domínios de Homologia de src , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estabilidade Proteica , Estrutura Secundária de ProteínaRESUMO
PDZ (Post-synaptic density, 95 kDa, Discs large, Zona Occludens-1) domains are protein interaction domains that bind to the carboxy-terminal amino acids of binding partners, heterodimerize with other PDZ domains, and also bind phosphoinositides. PDZ domain containing proteins are frequently involved in the assembly of multi-protein complexes and clustering of transmembrane proteins. LNX1 (Ligand of Numb, protein X 1) is a RING (Really Interesting New Gene) domain-containing E3 ubiquitin ligase that also includes four PDZ domains suggesting it functions as a scaffold for a multi-protein complex. Here we use a human protein array to identify direct LNX1 PDZ domain binding partners. Screening of 8,000 human proteins with isolated PDZ domains identified 53 potential LNX1 binding partners. We combined this set with LNX1 interacting proteins identified by other methods to assemble a list of 220 LNX1 interacting proteins. Bioinformatic analysis of this protein list was used to select interactions of interest for future studies. Using this approach we identify and confirm six novel LNX1 binding partners: KCNA4, PAK6, PLEKHG5, PKC-alpha1, TYK2 and PBK, and suggest that LNX1 functions as a signalling scaffold.
Assuntos
Ubiquitina-Proteína Ligases/química , Biologia Computacional , Avaliação Pré-Clínica de Medicamentos , Humanos , Domínios PDZ , Ligação Proteica , Mapeamento de Interação de Proteínas , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/fisiologiaRESUMO
The AMP-activated protein kinase (AMPK) is a highly conserved trimeric protein complex that is responsible for energy homeostasis in eukaryotic cells. Here, a 1.9 A resolution crystal structure of the isolated kinase domain from the alpha2 subunit of human AMPK, the first from a multicellular organism, is presented. This human form adopts a catalytically inactive state with distorted ATP-binding and substrate-binding sites. The ATP site is affected by changes in the base of the activation loop, which has moved into an inhibited DFG-out conformation. The substrate-binding site is disturbed by changes within the AMPKalpha2 catalytic loop that further distort the enzyme from a catalytically active form. Similar structural rearrangements have been observed in a yeast AMPK homologue in response to the binding of its auto-inhibitory domain; restructuring of the kinase catalytic loop is therefore a conserved feature of the AMPK protein family and is likely to represent an inhibitory mechanism that is utilized during function.
Assuntos
Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Biocatálise , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Alinhamento de SequênciaRESUMO
Eph receptor tyrosine kinases regulate many important biological processes. In the present study, we explored the substrate specificity of the EphA4 receptor tyrosine kinase using peptide arrays. We define a consensus substrate motif for EphA4 and go on to identify and test a number of potential EphA4 substrates and map their putative site(s) of phosphorylation. Cotransfection studies validate two of the predicted substrates: Nck2 and Dok1. Our findings identify several potential EphA4 substrates and demonstrate the general utility of using peptide arrays to rapidly identify and map protein kinase phosphorylation sites.
Assuntos
Peptídeos/metabolismo , Análise Serial de Proteínas , Receptor EphA4/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Peptídeos/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptor EphA4/genética , Reprodutibilidade dos Testes , Especificidade por Substrato , Tirosina/metabolismoRESUMO
Eph receptor tyrosine kinases (RTKs) mediate numerous developmental processes. Their activity is regulated by auto-phosphorylation on two tyrosines within the juxtamembrane segment (JMS) immediately N-terminal to the kinase domain (KD). Here, we probe the molecular details of Eph kinase activation through mutational analysis, X-ray crystallography and NMR spectroscopy on auto-inhibited and active EphB2 and EphA4 fragments. We show that a Tyr750Ala gain-of-function mutation in the KD and JMS phosphorylation independently induce disorder of the JMS and its dissociation from the KD. Our X-ray analyses demonstrate that this occurs without major conformational changes to the KD and with only partial ordering of the KD activation segment. However, conformational exchange for helix alphaC in the N-terminal KD lobe and for the activation segment, coupled with increased inter-lobe dynamics, is observed upon kinase activation in our NMR analyses. Overall, our results suggest that a change in inter-lobe dynamics and the sampling of catalytically competent conformations for helix alphaC and the activation segment rather than a transition to a static active conformation underlies Eph RTK activation.
Assuntos
Receptor EphA4/química , Receptor EphA4/metabolismo , Receptor EphB2/química , Receptor EphB2/metabolismo , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Cristalografia por Raios X , Análise Mutacional de DNA , Ativação Enzimática , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Tirosina/metabolismoRESUMO
The rise of antibiotic resistance as a public health concern has led to increased interest in studying the ways in which bacteria avoid the effects of antibiotics. Enzymatic inactivation by several families of enzymes has been observed to be the predominant mechanism of resistance to aminoglycoside antibiotics such as kanamycin and gentamicin. Despite the importance of acetyltransferases in bacterial resistance to aminoglycoside antibiotics, relatively little is known about their structure and mechanism. Here we report the three-dimensional atomic structure of the aminoglycoside acetyltransferase AAC(6')-Ii in complex with coenzyme A (CoA). This structure unambiguously identifies the physiologically relevant AAC(6')-Ii dimer species, and reveals that the enzyme structure is similar in the AcCoA and CoA bound forms. AAC(6')-Ii is a member of the GCN5-related N-acetyltransferase (GNAT) superfamily of acetyltransferases, a diverse group of enzymes that possess a conserved structural motif, despite low sequence homology. AAC(6')-Ii is also a member of a subset of enzymes in the GNAT superfamily that form multimeric complexes. The dimer arrangements within the multimeric GNAT superfamily members are compared, revealing that AAC(6')-Ii forms a dimer assembly that is different from that observed in the other multimeric GNAT superfamily members. This different assembly may provide insight into the evolutionary processes governing dimer formation.