Mechanistic insights into the interactions of dynein regulator Ndel1 with neuronal ankyrins and implications in polarity maintenance

Abstract

Ankyrin-G (AnkG), a highly enriched scaffold protein in the axon initial segment (AIS) of neurons, functions to maintain axonal polarity and the integrity of the AIS. At the AIS, AnkG regulates selective intracellular cargo trafficking between soma and axons via interaction with the dynein regulator protein Ndel1, but the molecular mechanism underlying this binding remains elusive. Here we report that Ndel1’s C-terminal coiled-coil region (CT-CC) binds to giant neuron-specific insertion regions present in both AnkG and AnkB with 2:1 stoichiometry. The high-resolution crystal structure of AnkB in complex with Ndel1 CT-CC revealed the detailed molecular basis governing the AnkB/Ndel1 complex formation. Mechanistically, AnkB binds with Ndel1 by forming a stable 5-helix bundle dominated by hydrophobic interactions spread across 6 distinct interaction layers. Moreover, we found that AnkG is essential for Ndel1 accumulation at the AIS. Finally, we found that cargo sorting at the AIS can be disrupted by blocking the AnkG/Ndel1 complex formation using a peptide designed based on our structural data. Collectively, the atomic structure of the AnkB/Ndel1 complex together with studies of cargo sorting through the AIS establish the mechanistic basis for AnkG/Ndel1 complex formation and for the maintenance of axonal polarity. Our study will also be valuable for future studies of the interaction between AnkB and Ndel1 perhaps at distal axonal cargo transport.

Cytoskeletal organization of axons in vertebrates and invertebrates

“…Furthermore, the dynein regulator Ndel1 binds to the AnkG tail (asterisks in Fig. 3 B), and this interaction is essential for AIS filter functions (Ye et al, 2020). In agreement with these functional studies, EM analyses suggest that a fraction of MTs are in contact with the undercoat ( Fig.…”

Cargo hold and delivery: Ankyrins, spectrins, and their functional patterning of neurons

“…and ankyrin-G with 2:1 stoichiometry (Ye et al, 2020). The crystal structure of the ankyrin-B-NDEL1's CT-CC complex revealed a stable 5-helix bundle dominated by hydrophobic interactions spread across six distinct interaction layers (Ye et al, 2020).…”

“…Ankyrin‐G is necessary for NDEL1 localization to the AIS, where it promotes the polarized distribution of dendritic cargo by reversing the direction of transport of somatodendritic vesicles at the AIS (discussed below; Kuijpers et al, ). A recent report showed that NDEL1's C‐terminal coiled‐coil region (CT‐CC) binds a similar ~70‐amino acids stretch within the inserted region of both ankyrin‐B and ankyrin‐G with 2:1 stoichiometry (Ye et al, ). The crystal structure of the ankyrin‐B‐NDEL1's CT‐CC complex revealed a stable 5‐helix bundle dominated by hydrophobic interactions spread across six distinct interaction layers (Ye et al, ).…”

“…A recent report showed that NDEL1's C‐terminal coiled‐coil region (CT‐CC) binds a similar ~70‐amino acids stretch within the inserted region of both ankyrin‐B and ankyrin‐G with 2:1 stoichiometry (Ye et al, ). The crystal structure of the ankyrin‐B‐NDEL1's CT‐CC complex revealed a stable 5‐helix bundle dominated by hydrophobic interactions spread across six distinct interaction layers (Ye et al, ). However, while blocking of the NDEL1‐binding region in ankyrin‐G impaired NDEL1 accumulation at the AIS (Ye et al, ), the significance of the ankyrin‐B‐NDEL1 interaction remains unknown.…”

“…The crystal structure of the ankyrin‐B‐NDEL1's CT‐CC complex revealed a stable 5‐helix bundle dominated by hydrophobic interactions spread across six distinct interaction layers (Ye et al, ). However, while blocking of the NDEL1‐binding region in ankyrin‐G impaired NDEL1 accumulation at the AIS (Ye et al, ), the significance of the ankyrin‐B‐NDEL1 interaction remains unknown. Similarly, 480 kDa ankyrin‐G interacts with GABARAP, a MAP member of the ubiquitin‐like LC3 family at extrasynaptic somatodendritic plasma membranes of pyramidal neurons (Tseng et al, ).…”