RESUMO
A key function of reversible protein phosphorylation is to regulate protein-protein interactions, many of which involve short linear motifs (3-12 amino acids). Motif-based interactions are difficult to capture because of their often low-to-moderate affinities. Here, we describe phosphomimetic proteomic peptide-phage display, a powerful method for simultaneously finding motif-based interaction and pinpointing phosphorylation switches. We computationally designed an oligonucleotide library encoding human C-terminal peptides containing known or predicted Ser/Thr phosphosites and phosphomimetic variants thereof. We incorporated these oligonucleotides into a phage library and screened the PDZ (PSD-95/Dlg/ZO-1) domains of Scribble and DLG1 for interactions potentially enabled or disabled by ligand phosphorylation. We identified known and novel binders and characterized selected interactions through microscale thermophoresis, isothermal titration calorimetry, and NMR We uncover site-specific phospho-regulation of PDZ domain interactions, provide a structural framework for how PDZ domains accomplish phosphopeptide binding, and discuss ligand phosphorylation as a switching mechanism of PDZ domain interactions. The approach is readily scalable and can be used to explore the potential phospho-regulation of motif-based interactions on a large scale.
Assuntos
Domínios PDZ/genética , Peptídeos/genética , Mapas de Interação de Proteínas/genética , Proteoma/genética , Sequência de Aminoácidos/genética , Sítios de Ligação , Proteína 4 Homóloga a Disks-Large/genética , Humanos , Ligantes , Oligonucleotídeos/genética , Biblioteca de Peptídeos , Fosforilação , Ligação Proteica/genética , Mapeamento de Interação de Proteínas , Proteína da Zônula de Oclusão-1/genéticaRESUMO
Denitrovibrio acetiphilus N2460(T) is one of the few members of the phylum Deferribacteres with a sequenced genome. N2460(T) was capable of growing with dimethyl sulfoxide, selenate, or arsenate provided as a terminal electron acceptor, and we identified 15 genes that could possibly encode respiratory reductases for these compounds. The protein encoded by one of these genes, YP_003504839, clustered with respiratory arsenate reductases on a phylogenetic tree. Transcription of the gene for YP_003504839, Dacet_2121, was highly induced when arsenate was provided as a terminal electron acceptor. Dacet_2121 exists in a possible operon that is distinct from the previously characterized respiratory arsenate reductase operon in Shewanella sp. ANA-3.
