Structural basis for interaction between the conserved cell polarity proteins Inscuteable and Leu-Gly-Asn repeat-enriched protein (LGN)
Structural basis for interaction between the conserved cell polarity proteins Inscuteable and Leu-Gly-Asn repeat-enriched protein (LGN)
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DOI：
10.1073/pnas.1110951108
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发表时间：
2011-11
期刊：
影响因子：
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通讯作者：
S. Yuzawa;S. Kamakura;Yuko Iwakiri;Junya Hayase;H. Sumimoto
中科院分区：
文献类型：
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作者：
S. Yuzawa;S. Kamakura;Yuko Iwakiri;Junya Hayase;H. Sumimoto
哺乳动物细胞极性蛋白 mInsc（Inscuteable 的哺乳动物同源物）和 Leu-Gly-Asn 重复序列富集蛋白 (LGN) 之间的相互作用，以及它们各自的果蝇同源物 Inscuteable 和 Inscuteable 伴侣 (Pins) 之间的相互作用，在有丝分裂纺锤体方向（导致不对称细胞分裂的过程）中发挥着至关重要的作用。在这里，我们以 2.6-Å 的分辨率报道了人 mInsc 的 LGN 结合域 (LBD) 与人 LGN 的 N 端四肽重复 (TPR) 基序复合的晶体结构。在复合物中，mInsc-LBD采用具有三个结合模块的细长结构——α螺旋、延伸区域和与环连接的β折叠——沿着TPR形成的超螺旋的凹面与LGN反向平行。结构分析和基于结构的诱变定义了对 mInsc-LGN 关联至关重要的残基，并揭示了 G 蛋白信号传导 3 (AGS3) 结合蛋白 Frmpd1 [4.1/ezrin/radixin/moesin (FERM) 和 PSD-95/Dlg/ZO-1 (PDZ) 结构域含有蛋白 1] 及其相关 Frmpd4 通过一个区域与 LGN 相互作用的激活剂 与 mInsc-LBD 的一部分同源，而核有丝分裂装置蛋白 (NuMA) 和 LGN 的 C 末端以与 mInsc 不同的方式识别 TPR 结构域。 mInsc 以最高亲和力 (KD ≈ 2.4 nM) 与 LGN 结合，并有效取代 Frmpd 蛋白、NuMA 和 LGN C 末端，表明 mInsc 优先与 LGN 结合。我们还使用突变蛋白证明，mInsc-LGN 相互作用对于 LGN 的稳定和 mInsc 的细胞内定位至关重要。
Interaction between the mammalian cell polarity proteins mInsc (mammalian homologue of Inscuteable) and Leu-Gly-Asn repeat-enriched protein (LGN), as well as that between their respective Drosophila homologues Inscuteable and Partner of Inscuteable (Pins), plays crucial roles in mitotic spindle orientation, a process contributing to asymmetric cell division. Here, we report a crystal structure of the LGN-binding domain (LBD) of human mInsc complexed with the N-terminal tetratricopeptide repeat (TPR) motifs of human LGN at 2.6-Å resolution. In the complex, mInsc-LBD adopts an elongated structure with three binding modules—an α-helix, an extended region, and a β-sheet connected with a loop—that runs antiparallel to LGN along the concave surface of the superhelix formed by the TPRs. Structural analysis and structure-based mutagenesis define residues that are critical for mInsc–LGN association, and reveal that the activator of G-protein signaling 3 (AGS3)-binding protein Frmpd1 [4.1/ezrin/radixin/moesin (FERM) and PSD-95/Dlg/ZO-1 (PDZ) domain-containing protein 1] and its relative Frmpd4 interact with LGN via a region homologous to a part of mInsc-LBD, whereas nuclear mitotic apparatus protein (NuMA) and the C terminus of LGN recognize the TPR domain in a manner different from that by mInsc. mInsc binds to LGN with the highest affinity (KD ≈ 2.4 nM) and effectively replaces the Frmpd proteins, NuMA, and the LGN C terminus, suggesting the priority of mInsc in binding to LGN. We also demonstrate, using mutant proteins, that mInsc–LGN interaction is vital for stabilization of LGN and for intracellular localization of mInsc.