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FEBS J. 2016 Dec;283(24):4528-4548. doi: 10.1111/febs.13943. Epub 2016 Nov 20.

Neighboring phosphoSer-Pro motifs in the undefined domain of IRAK1 impart bivalent advantage for Pin1 binding.

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Department of Molecular Biology & Genetics, Cornell University, Ithaca, NY, USA.
Department of Chemistry, University of Illinois at Urbana-Champaign, IL, USA.
Division of Translational Therapeutics, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.


The peptidyl prolyl isomerase Pin1 has two domains that are considered to be its binding (WW) and catalytic (PPIase) domains, both of which interact with phosphorylated Ser/Thr-Pro motifs. This shared specificity might influence substrate selection, as many known Pin1 substrates have multiple sequentially close phosphoSer/Thr-Pro motifs, including the protein interleukin-1 receptor-associated kinase-1 (IRAK1). The IRAK1 undefined domain (UD) contains two sets of such neighboring motifs (Ser131/Ser144 and Ser163/Ser173), suggesting possible bivalent interactions with Pin1. Using a series of NMR titrations with 15N-labeled full-length Pin1 (Pin1-FL), PPIase, or WW domain and phosphopeptides representing the Ser131/Ser144 and Ser163/Ser173 regions of IRAK1-UD, bivalent interactions were investigated. Binding studies using singly phosphorylated peptides showed that individual motifs displayed weak affinities (> 100 μm) for Pin1-FL and each isolated domain. Analysis of dually phosphorylated peptides binding to Pin1-FL showed that inclusion of bivalent states was necessary to fit the data. The resulting complex model and fitted parameters were applied to predict the impact of bivalent states at low micromolar concentrations, demonstrating significant affinity enhancement for both dually phosphorylated peptides (3.5 and 24 μm for peptides based on the Ser131/Ser144 and Ser163/Ser173 regions, respectively). The complementary technique biolayer interferometry confirmed the predicted affinity enhancement for a representative set of singly and dually phosphorylated Ser131/Ser144 peptides at low micromolar concentrations, validating model predictions. These studies provide novel insights regarding the complexity of interactions between Pin1 and activated IRAK1, and more broadly suggest that phosphorylation of neighboring Ser/Thr-Pro motifs in proteins might provide competitive advantage at cellular concentrations for engaging with Pin1.


NMR titration; Pin1; bivalent interaction; interleukin-1 receptor-associated kinase-1; multi-state equilibrium

[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

Dr. Lu declares that he is an inventor of Pin1 technology, which was licensed by BIDMC to Pinteon Therapeutics. Dr. Lu owns equity in, and consults for, Pinteon. His interests were reviewed and are managed by BIDMC in accordance with its conflict of interest policy.

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