RESUMO
To decipher the molecular basis for RET kinase activation and oncogenic deregulation, we defined the temporal sequence of RET autophosphorylation by label-free quantitative mass spectrometry. Early autophosphorylation sites map to regions flanking the kinase domain core, while sites within the activation loop only form at later time points. Comparison with oncogenic RET kinase revealed that late autophosphorylation sites become phosphorylated much earlier than wild-type RET, which is due to a combination of an enhanced enzymatic activity, increased ATP affinity, and surprisingly, by providing a better intermolecular substrate. Structural analysis of oncogenic M918T and wild-type RET kinase domains reveal a cis-inhibitory mechanism involving tethering contacts between the glycine-rich loop, activation loop, and αC-helix. Tether mutations only affected substrate presentation but perturbed the autophosphorylation trajectory similar to oncogenic mutations. This study reveals an unappreciated role for oncogenic RET kinase mutations in promoting intermolecular autophosphorylation by enhancing substrate presentation.
Assuntos
Regulação Enzimológica da Expressão Gênica , Mutação , Proteínas Proto-Oncogênicas c-ret/química , Proteínas Proto-Oncogênicas c-ret/genética , Homologia de Sequência de Aminoácidos , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Humanos , Insetos , Ligantes , Espectrometria de Massas , Dados de Sequência Molecular , Fosforilação , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Especificidade por Substrato , Fatores de Tempo , Tirosina/químicaRESUMO
We have previously developed a rapid microplate-based approach for measuring the denaturation curves by intrinsic tryptophan fluorescence for simple monomeric and two-state unfolding proteins. Here we demonstrate that it can accurately resolve the multiple conformational transitions that occur during the denaturation of a complex multimeric and cofactor associated protein. We have also analyzed the effect of two active-site mutations, D381A and Y440A upon the denaturation pathway of transketolase using intrinsic fluorescence measurements, and we compare the results from classical and microplate-based instrumentation. This work shows that the rapid assay is able to identify changes in the denaturation pathway, due to mutations or removal of cofactors, which affect the stability of the native and intermediate states. This would be of significant benefit for the directed evolution of protein stability, optimizing enzyme stability under biocatalytic process conditions, and also for engineering specific transitions in protein unfolding pathways.
Assuntos
Escherichia coli/enzimologia , Análise Serial de Proteínas/métodos , Transdução de Sinais/fisiologia , Transcetolase/química , Transcetolase/metabolismo , Estabilidade Enzimática , Desnaturação ProteicaRESUMO
Receptor tyrosine kinases exhibit a variety of activation mechanisms despite highly homologous catalytic domains. Such diversity arises through coupling of extracellular ligand-binding portions with highly variable intracellular sequences flanking the tyrosine kinase domain and specific patterns of autophosphorylation sites. Here, we show that the juxtamembrane (JM) segment enhances RET catalytic domain activity through Y687. This phospho-site is also required by the JM region to rescue an otherwise catalytically deficient RET activation-loop mutant lacking tyrosines. Structure-function analyses identified interactions between the JM hinge, αC helix, and an unconventional activation-loop serine phosphorylation site that engages the HRD motif and promotes phospho-tyrosine conformational accessibility and regulatory spine assembly. We demonstrate that this phospho-S909 arises from an intrinsic RET dual-specificity kinase activity and show that an equivalent serine is required for RET signaling in Drosophila. Our findings reveal dual-specificity and allosteric components for the mechanism of RET activation and signaling with direct implications for drug discovery.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas Proto-Oncogênicas c-ret/química , Proteínas Proto-Oncogênicas c-ret/metabolismo , Receptores Proteína Tirosina Quinases/química , Relação Estrutura-Atividade , Regulação Alostérica/genética , Sequência de Aminoácidos/genética , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Ativação Enzimática/genética , Fosforilação , Proteínas Proto-Oncogênicas c-ret/genética , Receptores Proteína Tirosina Quinases/genética , Serina/metabolismo , Transdução de Sinais/genéticaRESUMO
UNLABELLED: During the last decade the use of transaminases for the production of pharmaceutical and fine chemical intermediates has attracted a great deal of attention. Transaminases are versatile biocatalysts for the efficient production of amine intermediates and many have (S)-enantiospecificity. Transaminases with (R)-specificity are needed to expand the applications of these enzymes in biocatalysis. In this work we have identified a fungal putative (R)-specific transaminase from the Eurotiomycetes Nectria haematococca, cloned a synthetic version of this gene, demonstrated (R)-selective deamination of several substrates including (R)-α-methylbenzylamine, as well as production of (R)-amines, and determined its crystal structure. The crystal structures of the holoenzyme and the complex with an inhibitor gabaculine offer the first detailed insight into the structural basis for substrate specificity and enantioselectivity of the industrially important class of (R)-selective amine : pyruvate transaminases. DATABASE: The atomic coordinates and structure factors for the Nectria TAm in holoenzyme and gabaculine-bound forms have been deposited in the PDB as entries 4cmd and 4cmf respectively.
Assuntos
Nectria/enzimologia , Transaminases/metabolismo , Sequência de Aminoácidos , Biocatálise , Clonagem Molecular , Genes Fúngicos , Modelos Moleculares , Dados de Sequência Molecular , Nectria/genética , Conformação Proteica , Estereoisomerismo , Especificidade por Substrato , Transaminases/química , Transaminases/genéticaRESUMO
We have previously shown that the denaturation of TK with urea follows a non-aggregating though irreversible denaturation pathway in which the cofactor binding appears to become altered but without dissociating, then followed at higher urea by partial denaturation of the homodimer prior to any further unfolding or dissociation of the two monomers. Urea is not typically present during biocatalysis, whereas access to TK enzymes that retain activity at increased temperature and extreme pH would be useful for operation under conditions that increase substrate and product stability or solubility. To provide further insight into the underlying causes of its deactivation in process conditions, we have characterised the effects of temperature and pH on the structure, stability, aggregation and activity of Escherichia coli transketolase. The activity of TK was initially found to progressively improve after pre-incubation at increasing temperatures. Loss of activity at higher temperature and low pH resulted primarily from protein denaturation and subsequent irreversible aggregation. By contrast, high pH resulted in the formation of a native-like state that was only partially inactive. The apo-TK enzyme structure content also increased at pH 9 to converge on that of the holo-TK. While cofactor dissociation was previously proposed for high pH deactivation, the observed structural changes in apo-TK but not holo-TK indicate a more complex mechanism.