Structure of an Enzyme-Derived Phosphoprotein Recognition Domain

Membrane Associated Guanylate Kinases (MAGUKs) contain a protein interaction domain (GKdom) derived from the enzyme Guanylate Kinase (GKenz). Here we show that GKdom from the MAGUK Discs large (Dlg) is a phosphoprotein recognition domain, specifically recognizing the phosphorylated form of the mitotic spindle orientation protein Partner of Inscuteable (Pins). We determined the structure of the Dlg-Pins complex to understand the dramatic transition from nucleotide kinase to phosphoprotein recognition domain. The structure reveals that the region of the GKdom that once served as the GMP binding domain (GBD) has been co-opted for protein interaction. Pins makes significantly more contact with the GBD than does GMP, but primarily with residues that are conserved between enzyme and domain revealing the versatility of the GBD as a platform for nucleotide and protein interactions. Mutational analysis reveals that the GBD is also used to bind the GK ligand MAP1a, suggesting that this is a common mode of MAGUK complex assembly. The GKenz undergoes a dramatic closing reaction upon GMP binding but the protein-bound GKdom remains in the ‘open’ conformation indicating that the dramatic conformational change has been lost in the conversion from nucleotide kinase to phosphoprotein recognition domain.

膜相关鸟苷酸激酶（Membrane Associated Guanylate Kinases，MAGUKs）包含一个源自鸟苷酸激酶（Guanylate Kinase，GKenz）的蛋白质相互作用结构域（GKdom）。本研究发现，来自MAGUK家族成员盘状大蛋白（Discs large，Dlg）的GKdom是一种磷酸化蛋白质识别结构域，可特异性识别有丝分裂纺锤体定向蛋白Inscuteable结合蛋白（Partner of Inscuteable，Pins）的磷酸化形式。为阐明其从核苷酸激酶向磷酸化蛋白质识别结构域的显著功能转变，我们解析了Dlg-Pins复合物的三维结构。结构分析显示，原本作为GMP结合结构域（GMP binding domain，GBD）的GKdom区域，已被重新用于蛋白质相互作用。与GMP相比，Pins与GBD的相互作用界面显著更大，且二者的结合主要依赖于酶与该结构域间保守的氨基酸残基，这揭示了GBD作为核苷酸与蛋白质相互作用平台的多功能性。突变分析表明，GBD还可结合鸟苷酸激酶配体MAP1a，提示这是MAGUK复合物组装的通用模式。GKenz在结合GMP时会发生显著的构象闭合反应，但结合蛋白质的GKdom则始终维持“开放”构象，这表明从核苷酸激酶向磷酸化蛋白质识别结构域的转化过程中，该剧烈构象变化已丢失。