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<p>Microtubule (MT) branch nucleation requires Augmin and NEDD1 proteins, which recruit and activate the gamma-tubulin ring complex (γ-TuRC). Augmin is a fork-shaped assembly of eight coiled-coil subunits, while NEDD1 is a β-propeller protein bridging MTs, Augmin, and γ-TuRC. We reconstitute <i>Arabidopsis thaliana</i> Augmin assemblies and determine 3.7-7.3-Å cryo-EM structures of its V-junction and extended regions using crosslinking mass spectrometry. These structures reveal a complete plant Augmin model showing multi-coiled-coil interfaces stabilizing its 40-nm hetero-octameric fork architecture. The dual calponin homology (CH) domains at the V-junction terminus adopt open and closed conformations for MT binding. A 12-Å cryo-EM structure shows Augmin undergoes anti-parallel dimerization through conserved surfaces on its extended region. We determine the NEDD1 β-propeller structure with Augmin, revealing direct binding inside the V-junction that enhances dimerization. Direct coupling and evolutionary analyses identify co-varying residue pairs validating the eight-subunit model and NEDD1 interface. Cooperativity between dual CH domains and NEDD1 binding may regulate V-junction binding to MT lattices. This V-shaped dual binding anchors Augmin along MTs, creating platforms for γ-TuRC recruitment and branched MT nucleation.</p>

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Cryo-EM structures of plant Augmin reveal coiled-coil assembly, antiparallel dimerization, and NEDD1 binding

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月12日

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  • Md Ashaduzzaman,
  • Aryan Taheri,
  • Yuh-Ru Julie Lee,
  • Yuqi Tang,
  • Shubham Mittal,
  • Fei Guo,
  • Faruck Morcos,
  • Stephen D. Fried,
  • Bo Liu,
  • Jawdat Al-Bassam

摘要

Microtubule (MT) branch nucleation requires Augmin and NEDD1 proteins, which recruit and activate the gamma-tubulin ring complex (γ-TuRC). Augmin is a fork-shaped assembly of eight coiled-coil subunits, while NEDD1 is a β-propeller protein bridging MTs, Augmin, and γ-TuRC. We reconstitute Arabidopsis thaliana Augmin assemblies and determine 3.7-7.3-Å cryo-EM structures of its V-junction and extended regions using crosslinking mass spectrometry. These structures reveal a complete plant Augmin model showing multi-coiled-coil interfaces stabilizing its 40-nm hetero-octameric fork architecture. The dual calponin homology (CH) domains at the V-junction terminus adopt open and closed conformations for MT binding. A 12-Å cryo-EM structure shows Augmin undergoes anti-parallel dimerization through conserved surfaces on its extended region. We determine the NEDD1 β-propeller structure with Augmin, revealing direct binding inside the V-junction that enhances dimerization. Direct coupling and evolutionary analyses identify co-varying residue pairs validating the eight-subunit model and NEDD1 interface. Cooperativity between dual CH domains and NEDD1 binding may regulate V-junction binding to MT lattices. This V-shaped dual binding anchors Augmin along MTs, creating platforms for γ-TuRC recruitment and branched MT nucleation.