Tetraspanins are eukaryotic membrane proteins that contribute to a variety of signaling processes by organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster partner receptors into tetraspanin-enriched microdomains is unknown. Here, we present crystal structures of the large extracellular loop of CD9 bound to nanobodies 4C8 and 4E8 and, the cryo-EM structure of 4C8-bound CD9 in complex with its partner EWI-F. CD9-EWI-F displays a tetrameric arrangement with two central EWI-F molecules, dimerized through their ectodomains, and two CD9 molecules, one bound to each EWI-F transmembrane helix through CD9-helices h3 and h4. In the crystal structures, nanobodies 4C8 and 4E8 bind CD9 at loops C and D, which is in agreement with the 4C8 conformation in the CD9-EWI-F complex. The complex varies from nearly twofold symmetric (with the two CD9 copies nearly anti-parallel) to ca. 50° bent arrangements. This flexible arrangement of CD9-EWI-F with potential CD9 homo-dimerization at either end provides a "concatenation model" for forming short linear or circular assemblies, which may explain the occurrence of tetraspanin-enriched microdomains.

中文翻译：

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基于具有EWI-F的CD9的结构，富含四跨素的微区组装的意义。

四跨膜蛋白是真核细胞膜蛋白，通过在质膜中组织伴侣受体分子来促进多种信号传导过程。四跨膜蛋白如何结合并聚集伴侣受体到富含四跨膜蛋白的微区中是未知的。在这里，我们介绍了与纳米抗体4C8和4E8结合的CD9大细胞外环的晶体结构，以及与它的伴侣EWI-F结合的4C8结合的CD9的冷冻电磁结构。CD9-EWI-F显示四聚体排列，其中有两个中央EWI-F分子通过其胞外域二聚，还有两个CD9分子，一个通过CD9-螺旋h3和h4与每个EWI-F跨膜螺旋结合。在晶体结构中，纳米抗体4C8和4E8在环C和D处结合CD9，这与CD9-EWI-F复合物中的4C8构象一致。复杂程度从几乎双重对称（两个CD9拷贝几乎反平行）到大约3个。50°弯曲的安排。CD9-EWI-F的这种灵活排列在任一端均具有潜在的CD9均二聚作用，为形成短的线性或圆形装配提供了“串联模型”，这可以解释富含四跨素的微区的发生。