Department of Human Genetics, Katholieke Universiteit Leuven, Herestraat 49, B-3000 Leuven, Belgium.
J Biol Chem. 2011 Dec 30;286(52):44669-78. doi: 10.1074/jbc.M111.285734. Epub 2011 Oct 27.
PDZ domains are well known protein-protein interaction modules that, as part of multidomain proteins, assemble molecular complexes. Some PDZ domains have been reported to interact with membrane lipids, in particular phosphatidylinositol phosphates, but few studies have been aimed at elucidating the prevalence or the molecular details of such interactions. We screened 46 Drosophila PDZ domains for phosphoinositide-dependent cellular localization and discovered that the second PDZ domain of polychaetoid (Pyd PDZ2) interacts with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) at the plasma membrane. Surface plasmon resonance binding experiments with recombinant protein established that Pyd PDZ2 interacts with phosphatidylinositol phosphates with apparent affinities in the micromolar range. Electrostatic interactions involving an extended positively charged surface of Pyd PDZ2 are crucial for the PtdIns(4,5)P(2)-dependent membrane interactions as shown by a combination of three-dimensional modeling, mutagenesis, binding, and localization studies. In vivo localization studies further suggested that both lipid and peptide binding contribute to membrane localization. We identified the transmembrane protein Crumbs as a Pyd PDZ2 ligand and probed the relation between peptide and PtdIns(4,5)P(2) binding. Contrary to the prevalent view on PDZ/peptide/lipid binding, we did not find competition between peptide and lipid ligands. Instead, preloading the protein with the 10-mer Crb3 peptide increased the apparent affinity of Pyd PDZ2 for PtdIns(4,5)P(2) 6-fold. Our results suggest that membrane localization of Pyd PDZ2 may be driven by a combination of peptide and PtdIns(4,5)P(2) binding, which raises the intriguing possibility that the domain may coordinate protein- and phospholipid-mediated signals.
PDZ 结构域是众所周知的蛋白质-蛋白质相互作用模块，作为多结构域蛋白的一部分，它们组装分子复合物。已经有报道称一些 PDZ 结构域与膜脂，特别是磷脂酰肌醇磷酸相互作用，但很少有研究旨在阐明这种相互作用的普遍性或分子细节。我们筛选了 46 种果蝇 PDZ 结构域的磷酸肌醇依赖性细胞定位，发现多毛状（Pyd PDZ2）的第二个 PDZ 结构域与质膜上的磷脂酰肌醇 4,5-二磷酸（PtdIns（4,5）P2）相互作用。用重组蛋白进行的表面等离子体共振结合实验表明，Pyd PDZ2 与磷脂酰肌醇磷酸以微摩尔级的表观亲和力相互作用。涉及 Pyd PDZ2 扩展正电荷表面的静电相互作用对于 PtdIns（4,5）P2 依赖性膜相互作用至关重要，这一点通过三维建模、突变、结合和定位研究的组合得到了证明。体内定位研究进一步表明，脂质和肽结合都有助于膜定位。我们确定跨膜蛋白 Crumbs 是 Pyd PDZ2 的配体，并探讨了肽和 PtdIns（4,5）P2 结合之间的关系。与 PDZ/肽/脂结合的普遍观点相反，我们没有发现肽和脂质配体之间的竞争。相反，用 10 肽 Crb3 预先加载蛋白会使 Pyd PDZ2 对 PtdIns（4,5）P2 的表观亲和力增加 6 倍。我们的结果表明，Pyd PDZ2 的膜定位可能是由肽和 PtdIns（4,5）P2 结合的组合驱动的，这提出了一个有趣的可能性，即该结构域可能协调蛋白质和磷脂介导的信号。
