The Disordered Spindly C-terminus Interacts with RZZ Subunits ROD-1 and ZWL-1 in the Kinetochore through the Same Sites in C. Elegans

Henen, Morkos A.; Myers, Walter; Schmitt, Lauren R.; Wade, Kristen J.; Born, Alexandra; Nichols, Parker J.; Vögeli, Beat

doi:10.1016/j.jmb.2021.166812

Cited by 11 publications

(11 citation statements)

References 49 publications

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“…The observation that binding of RZZ to Spindly was insufficient to unleash a conformational change compatible with DD binding motivated our investigation of the detailed mechanisms that hold Spindly in a closed conformation. A Spindly fragment encompassing residues 354-605 is sufficient to bind RZZ (Henen et al, 2021; Raisch et al, 2021), but the autoinhibited conformation requires a fragment of Spindly comprised between residues 276 and 309, and therefore positioned upstream of the minimal RZZ-binding region (Figure 7H). AF2 modeling (Evans et al, 2021; Jumper et al, 2021), which became available in the late phases of our work and proved to be instrumental as a validation tool, suggests that Spindly 1-275 adopts an open conformation that is closely reminiscent of that of BICD2 1-400 , whereas Spindly 1-309 may adopt a closed conformation.…”

Section: Discussionmentioning

confidence: 99%

Conformational transitions of the mitotic adaptor Spindly underlie its interaction with Dynein and Dynactin

d'Amico

Ahmad

Cmentowski

et al. 2022

Preprint

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Cytoplasmic Dynein 1, or Dynein, is a microtubule minus-end directed motor. Dynein motility requires Dynactin and a family of activating adaptors that stabilize the Dynein-Dynactin complex and promote regulated interactions with cargo in space and time. How activating adaptors limit Dynein activation to specialized subcellular locales is unclear. Here, we reveal that Spindly, a mitotic Dynein adaptor at the kinetochore corona, exists natively in a closed conformation that occludes binding of Dynein-Dynactin to its CC1 box and Spindly motif. A structure-based analysis identified various mutations promoting an open conformation of Spindly that binds Dynein-Dynactin. A region of Spindly downstream from the Spindly motif and not required for cargo binding faces the CC1 box and stabilizes the intramolecular closed conformation. This region is also required for robust kinetochore localization of Spindly, suggesting that kinetochores promote Spindly activation to recruit Dynein. This mechanism may be paradigmatic for Dynein activation by other adaptors at various cellular locales.

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Section: Discussionmentioning

confidence: 99%

Conformational transitions of the mitotic adaptor Spindly underlie its interaction with Dynein and Dynactin

d'Amico

Ahmad

Cmentowski

et al. 2022

Preprint

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“…How Spindly coordinates its interaction with RZZ with activation of DD motility and processivity remains poorly understood. Spindly binds directly to the RZZ complex through its C‐terminal region (forming the complex abbreviated as RZZS), and engaging an RZZ module comprising the ROD β‐propeller and Zwilch (Gama et al , 2017; Mosalaganti et al , 2017; Pereira et al , 2018; Sacristan et al , 2018; Henen et al , 2021). Furthermore, in humans and likely most other metazoans, Spindly is post‐translationally modified on Cys602 with farnesyl, an isoprenoid lipid.…”

Section: Introductionmentioning

confidence: 99%

Structure of the RZZ complex and molecular basis of Spindly‐driven corona assembly at human kinetochores

et al. 2022

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In metazoans, a ≈1 megadalton (MDa) multiprotein complex comprising the dynein–dynactin adaptor Spindly and the ROD–Zwilch–ZW10 (RZZ) complex is the building block of a fibrous biopolymer, the kinetochore fibrous corona. The corona assembles on mitotic kinetochores to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. We report here a high‐resolution cryo‐EM structure that captures the essential features of the RZZ complex, including a farnesyl‐binding site required for Spindly binding. Using a highly predictive in vitro assay, we demonstrate that the SAC kinase MPS1 is necessary and sufficient for corona assembly at supercritical concentrations of the RZZ–Spindly (RZZS) complex, and describe the molecular mechanism of phosphorylation‐dependent filament nucleation. We identify several structural requirements for RZZS polymerization in rings and sheets. Finally, we identify determinants of kinetochore localization and corona assembly of Spindly. Our results describe a framework for the long‐sought‐for molecular basis of corona assembly on metazoan kinetochores.

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“…Within the Nup358 sequence, there are many intrinsically disordered regions (IDRs), which are also commonly found in proteins involved in dynein-dependent transport ( Zhu et al, 2017 ; Celestino et al, 2019 ; Lee et al, 2020 ; Henen et al, 2021 ; Lee et al, 2018 ). Although IDRs and intrinsically disordered proteins (IDPs) make up ~30% of eukaryotic proteins and have important physiological functions ( Ward et al, 2004 ), they remain the most poorly characterized class of proteins in regards to their structure, dynamics, and interactions.…”

Section: Introductionmentioning

confidence: 99%

Coil-to-α-helix transition at the Nup358-BicD2 interface activates BicD2 for dynein recruitment

Gibson

Cui

Ali

et al. 2022

eLife

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Nup358, a protein of the nuclear pore complex, facilitates a nuclear positioning pathway that is essential for many biological processes, including neuromuscular and brain development. Nup358 interacts with the dynein adaptor Bicaudal D2 (BicD2), which in turn recruits the dynein machinery to position the nucleus. However, the molecular mechanisms of the Nup358/BicD2 interaction and the activation of transport remain poorly understood. Here for the first time, we show that a minimal Nup358 domain activates dynein/dynactin/BicD2 for processive motility on microtubules. Using nuclear magnetic resonance (NMR) titration and chemical exchange saturation transfer (CEST), mutagenesis and circular dichroism spectroscopy (CD), a Nup358 a-helix encompassing residues 2162-2184 was identified, which transitioned from a random coil to an a-helical conformation upon BicD2-binding and formed the core of the Nup358-BicD2 interface. Mutations in this region of Nup358 decreased the Nup358/BicD2 interaction, resulting in decreased dynein recruitment and impaired motility. BicD2 thus recognizes Nup358 though a 'cargo recognition a-helix', a structural feature that may stabilize BicD2 in its activated state and promote processive dynein motility.

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The Disordered Spindly C-terminus Interacts with RZZ Subunits ROD-1 and ZWL-1 in the Kinetochore through the Same Sites in C. Elegans

Cited by 11 publications

References 49 publications

Conformational transitions of the mitotic adaptor Spindly underlie its interaction with Dynein and Dynactin

Conformational transitions of the mitotic adaptor Spindly underlie its interaction with Dynein and Dynactin

Structure of the RZZ complex and molecular basis of Spindly‐driven corona assembly at human kinetochores

Coil-to-α-helix transition at the Nup358-BicD2 interface activates BicD2 for dynein recruitment

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