BACKGROUND The slit diaphragm is a specialized adhesion junction between opposing podocytes, establishing the final filtration barrier that prevents passage of proteins from the capillary lumen into the urinary space. Nephrin, the key structural and signaling adhesion molecule expressed in the slit diaphragm, contains an evolutionally conserved, atypical PDZ-binding motif (PBM) reported to bind to a variety of proteins in the slit diaphragm. Several mutations in NPHS1 (the gene encoding nephrin) that result in nephrin lacking an intact PBM are associated with glomerular diseases. However, the molecular basis of nephrin-PBM-mediated protein complexes is still unclear. METHODS Using a combination of biochemic, biophysic, and cell biologic approaches, we systematically investigated the interactions between nephrin-PBM and PDZ domain-containing proteins in the slit diaphragm. RESULTS We found that nephrin-PBM specifically binds to one member of the membrane-associated guanylate kinase family of scaffolding proteins, MAGI1, but not to another, MAGI2. The complex structure of MAGI1-PDZ3/nephrin-PBM reveals that the Gly at the -3 position of nephrin-PBM is the determining feature for MAGI1-PDZ3 recognition, which sharply contrasts with the typical PDZ/PBM binding mode. A single gain-of-function mutation within MAGI2 enabled nephrin-PBM binding. In addition, using our structural analysis, we developed a highly efficient inhibitory peptide capable of specifically blocking the nephrin/MAGI1 interaction. CONCLUSIONS MAGI1 interacts with nephrin-PBM with exquisite specificity. A newly developed, potent inhibitory peptide that blocks this interaction may be useful for future functional investigations in vivo. Our findings also provide possible explanations for the diseases caused by NPHS1 mutations.

中文翻译：

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狭缝隔膜组装和信号传导中 Nephrin 和 MAGI1 之间高度特异性相互作用的结构基础。

背景技术裂隙隔膜是相对足细胞之间的专门粘附连接，建立最终的过滤屏障，防止蛋白质从毛细血管腔进入尿腔。Nephrin 是狭缝隔膜中表达的关键结构和信号粘附分子，包含进化上保守的非典型 PDZ 结合基序 (PBM)，据报道可与狭缝隔膜中的多种蛋白质结合。NPHS1（编码 nephrin 的基因）中的几种突变导致 nephrin 缺乏完整的 PBM，这与肾小球疾病有关。然而，nephrin-PBM 介导的蛋白复合物的分子基础仍不清楚。方法 结合使用生物化学、生物物理学和细胞生物学方法，我们系统地研究了 nephrin-PBM 和裂隙隔膜中含有 PDZ 结构域的蛋白质之间的相互作用。结果 我们发现 nephrin-PBM 特异性结合支架蛋白 MAGI1 的膜相关鸟苷酸激酶家族的一个成员，但不结合另一个 MAGI2。MAGI1-PDZ3/nephrin-PBM 的复杂结构表明，nephrin-PBM -3 位的 Gly 是 MAGI1-PDZ3 识别的决定性特征，这与典型的 PDZ/PBM 结合模式形成鲜明对比。MAGI2 中的单个功能获得性突变使 nephrin-PBM 结合成为可能。此外，使用我们的结构分析，我们开发了一种能够特异性阻断 nephrin/MAGI1 相互作用的高效抑制肽。结论 MAGI1 与 nephrin-PBM 相互作用，具有极强的特异性。一个新开发的，阻断这种相互作用的强效抑制肽可能对未来的体内功能研究有用。我们的研究结果还为 NPHS1 突变引起的疾病提供了可能的解释。