Rogals Monique J, Greenwood Alexander I, Kwon Jeahoo, Lu Kun Ping, Nicholson Linda K
Department of Molecular Biology & Genetics, Cornell University, Ithaca, NY, USA.
Department of Chemistry, University of Illinois at Urbana-Champaign, IL, USA.
FEBS J. 2016 Dec;283(24):4528-4548. doi: 10.1111/febs.13943. Epub 2016 Nov 20.
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.
肽基脯氨酰异构酶Pin1有两个结构域，分别被认为是其结合结构域（WW）和催化结构域（PPIase），二者均与磷酸化的丝氨酸/苏氨酸 - 脯氨酸基序相互作用。这种共同的特异性可能会影响底物选择，因为许多已知的Pin1底物都有多个连续靠近的磷酸化丝氨酸/苏氨酸 - 脯氨酸基序，包括蛋白质白细胞介素 - 1受体相关激酶 - 1（IRAK1）。IRAK1的未定义结构域（UD）包含两组这样相邻的基序（Ser131/Ser144和Ser163/Ser173），提示可能与Pin1存在二价相互作用。使用一系列用15N标记的全长Pin1（Pin1-FL）、PPIase或WW结构域以及代表IRAK1-UD的Ser131/Ser144和Ser163/Ser173区域的磷酸肽进行的核磁共振滴定，研究了二价相互作用。使用单磷酸化肽的结合研究表明，单个基序对Pin1-FL和每个分离的结构域显示出较弱的亲和力（>100μM）。对与Pin1-FL结合的双磷酸化肽的分析表明，纳入二价状态对于拟合数据是必要的。所得的复合物模型和拟合参数被用于预测低微摩尔浓度下二价状态的影响，证明了对两种双磷酸化肽都有显著的亲和力增强（基于Ser131/Ser144和Ser163/Ser173区域的肽分别为3.5和24μM）。互补技术生物层干涉术证实了在低微摩尔浓度下对一组代表性的单磷酸化和双磷酸化Ser131/Ser144肽预测的亲和力增强，验证了模型预测。这些研究为Pin1与活化的IRAK1之间相互作用的复杂性提供了新的见解，并且更广泛地表明，蛋白质中相邻的丝氨酸/苏氨酸 - 脯氨酸基序的磷酸化可能在细胞浓度下为与Pin1结合提供竞争优势。
