RESUMO
Proteomics techniques can identify thousands of phosphorylation sites in a single experiment, the majority of which are new and lack precise information about function or molecular mechanism. Here we present a fast method to predict potential phosphorylation switches by mapping phosphorylation sites to protein-protein interactions of known structure and analysing the properties of the protein interface. We predict 1024 sites that could potentially enable or disable particular interactions. We tested a selection of these switches and showed that phosphomimetic mutations indeed affect interactions. We estimate that there are likely thousands of phosphorylation mediated switches yet to be uncovered, even among existing phosphorylation datasets. The results suggest that phosphorylation sites on globular, as distinct from disordered, parts of the proteome frequently function as switches, which might be one of the ancient roles for kinase phosphorylation.
Assuntos
Modelos Químicos , Fosfotransferases/química , Mapeamento de Interação de Proteínas/métodos , Proteoma/química , Análise de Sequência de Proteína/métodos , Sítios de Ligação , Simulação por Computador , Modelos Moleculares , Fosforilação , Fosfotransferases/ultraestrutura , Ligação Proteica , Conformação Proteica , Proteoma/ultraestrutura , Relação Estrutura-AtividadeRESUMO
Systematic interrogation of mutation or protein modification data is important to identify sites with functional consequences and to deduce global consequences from large data sets. Mechismo (mechismo.russellab.org) enables simultaneous consideration of thousands of 3D structures and biomolecular interactions to predict rapidly mechanistic consequences for mutations and modifications. As useful functional information often only comes from homologous proteins, we benchmarked the accuracy of predictions as a function of protein/structure sequence similarity, which permits the use of relatively weak sequence similarities with an appropriate confidence measure. For protein-protein, protein-nucleic acid and a subset of protein-chemical interactions, we also developed and benchmarked a measure of whether modifications are likely to enhance or diminish the interactions, which can assist the detection of modifications with specific effects. Analysis of high-throughput sequencing data shows that the approach can identify interesting differences between cancers, and application to proteomics data finds potential mechanistic insights for how post-translational modifications can alter biomolecular interactions.
Assuntos
Bases de Dados de Proteínas , Mutação , Proteínas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias/genética , Conformação Proteica , Mapeamento de Interação de Proteínas , ProteômicaRESUMO
The mitotic kinesin Eg5 (or KSP) is a crucial player in the development and function of the mitotic spindle. Inhibition of this protein leads to cell cycle arrest and apoptosis without interfering with other microtubule-dependent processes. Therefore, it is a potential target in cancer therapy. Here, we report the synthesis and biological evaluation of a small library of molecules based on the structure of the known Eg5 inhibitor HR22C16. One of these derivatives (compound trans-24) proved to be a potent and specific Eg5 inhibitor.
