Ashaduzzaman Md, Taheri Aryan, Lee Yuh-Ru Julie, Tang Yuqi, Guo Fei, Fried Stephen D, Liu Bo, Al-Bassam Jawdat
Department of Molecular Cellular Biology, University of California, Davis, CA, USA.
Present address: Molecular Cell Biology Department, University of California, Berkeley, CA, USA.
bioRxiv. 2025 Feb 27:2025.02.25.640204. doi: 10.1101/2025.02.25.640204.
Microtubule (MT) branch nucleation is fundamental for building parallel MT networks in eukaryotic cells. In plants and metazoans, MT branch nucleation requires Augmin and NEDD1 proteins which bind along MTs and then recruit and activate the gamma-tubulin ring complex (γ-TuRC). Augmin is a fork-shaped assembly composed of eight coiled-coil subunits, while NEDD1 is a WD40 β-propellor protein that bridges across MTs, Augmin, and γ-TuRC during MT branch nucleation. Here, we reconstitute hetero-tetrameric and hetero-octameric Arabidopsis thaliana Augmin assemblies, resolve their subunit interactions using crosslinking mass spectrometry and determine 3.7 to 7.3-Å cryo-EM structures for the V-junction and extended regions of Augmin. These structures allowed us to generate a complete de novo plant Augmin model that reveals the long-range multi coiled-coil interfaces that stabilize its 40-nm hetero-octameric fork-shaped organization. We discovered the dual calponin homology (CH) domain forming its MT binding site at the end of its V-junction undertake open and closed conformations. We determined a 12-Å dimeric Augmin cryo-EM structure revealing Augmin undergoes anti-parallel dimerization through two conserved surfaces along Augmin's extended region. We reconstituted the NEDD1 WD40 β-propellor with Augmin revealing it directly binds on top its V-junction and enhances Augmin dimerization. Our studies suggest that cooperativity between the Augmin dual CH domains and NEDD1 WD40 binding site may regulate Augmin V-junction dual binding to MT lattices. This unique V-shaped dual binding and organization anchors Augmins along MTs generating a platform to recruit γ-TuRC and activate branched MT nucleation.
微管（MT）分支成核对于在真核细胞中构建平行MT网络至关重要。在植物和后生动物中，MT分支成核需要Augmin和NEDD1蛋白，它们沿着MT结合，然后招募并激活γ-微管蛋白环复合物（γ-TuRC）。Augmin是一种由八个卷曲螺旋亚基组成的叉形组装体，而NEDD1是一种WD40β-螺旋桨蛋白，在MT分支成核过程中横跨MT、Augmin和γ-TuRC。在这里，我们重组了拟南芥Augmin的异源四聚体和异源八聚体组装体，使用交联质谱解析了它们的亚基相互作用，并确定了Augmin V形连接和延伸区域的3.7至7.3埃冷冻电镜结构。这些结构使我们能够生成一个完整的从头植物Augmin模型，该模型揭示了稳定其40纳米异源八聚体叉形结构的远程多卷曲螺旋界面。我们发现，在其V形连接末端形成其MT结合位点的双钙调蛋白同源（CH）结构域呈现开放和闭合构象。我们确定了一个12埃的二聚体Augmin冷冻电镜结构，揭示Augmin通过沿着Augmin延伸区域的两个保守表面进行反平行二聚化。我们将NEDD1 WD40β-螺旋桨与Augmin重组，发现它直接结合在其V形连接的顶部并增强Augmin二聚化。我们的研究表明，Augmin双CH结构域与NEDD1 WD40结合位点之间的协同作用可能调节Augmin V形连接与MT晶格的双重结合。这种独特的V形双重结合和组织将Augmins锚定在MT上，形成一个招募γ-TuRC并激活分支MT成核的平台。
