Pedersen Søren W, Albertsen Louise, Moran Griffin E, Levesque Brié, Pedersen Stine B, Bartels Lina, Wapenaar Hannah, Ye Fei, Zhang Mingjie, Bowen Mark E, Strømgaard Kristian
Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, University of Copenhagen , Universitetsparken 2, DK-2100 Copenhagen, Denmark.
Department of Physiology & Biophysics, Stony Brook University , Stony Brook, New York 11794, United States.
ACS Chem Biol. 2017 Sep 15;12(9):2313-2323. doi: 10.1021/acschembio.7b00361. Epub 2017 Aug 1.
The postsynaptic density protein of 95 kDa (PSD-95) is a key scaffolding protein that controls signaling at synapses in the brain through interactions of its PDZ domains with the C-termini of receptors, ion channels, and enzymes. PSD-95 is highly regulated by phosphorylation. To explore the effect of phosphorylation on PSD-95, we used semisynthetic strategies to introduce phosphorylated amino acids at four positions within the PDZ domains and examined the effects on interactions with a large set of binding partners. We observed complex effects on affinity. Most notably, phosphorylation at Y397 induced a significant increase in affinity for stargazin, as confirmed by NMR and single molecule FRET. Additionally, we compared the effects of phosphorylation to phosphomimetic mutations, which revealed that phosphomimetics are ineffective substitutes for tyrosine phosphorylation. Our strategy to generate site-specifically phosphorylated PDZ domains provides a detailed understanding of the role of phosphorylation in the regulation of PSD-95 interactions.
95千道尔顿的突触后致密蛋白（PSD - 95）是一种关键的支架蛋白，它通过其PDZ结构域与受体、离子通道和酶的C末端相互作用来控制大脑突触处的信号传导。PSD - 95受到磷酸化的高度调控。为了探究磷酸化对PSD - 95的影响，我们采用半合成策略在PDZ结构域内的四个位置引入磷酸化氨基酸，并研究了其对与大量结合伴侣相互作用的影响。我们观察到对亲和力的复杂影响。最显著的是，Y397位点的磷酸化导致对stargazin的亲和力显著增加，这通过核磁共振（NMR）和单分子荧光共振能量转移（FRET）得到证实。此外，我们比较了磷酸化与模拟磷酸化突变的影响，结果表明模拟磷酸化不是酪氨酸磷酸化的有效替代物。我们生成位点特异性磷酸化PDZ结构域的策略为详细了解磷酸化在PSD - 95相互作用调控中的作用提供了依据。
