Magic-angle-spinning NMR structure of the kinesin-1 motor domain assembled with microtubules reveals the elusive neck linker orientation

Abstract

Microtubules (MTs) and their associated proteins play essential roles in maintaining cell structure, organelle transport, cell motility, and cell division. Two motors, kinesin and cytoplasmic dynein link the MT network to transported cargos using ATP for force generation. Here, we report an all-atom NMR structure of nucleotide-free kinesin-1 motor domain (apo-KIF5B) in complex with paclitaxel-stabilized microtubules using magic-angle-spinning (MAS) NMR spectroscopy. The structure reveals the position and orientation of the functionally important neck linker and how ADP induces structural and dynamic changes that ensue in the neck linker. These results demonstrate that the neck linker is in the undocked conformation and oriented in the direction opposite to the KIF5B movement. Chemical shift perturbations and intensity changes indicate that a significant portion of ADP-KIF5B is in the neck linker docked state. This study also highlights the unique capability of MAS NMR to provide atomic-level information on dynamic regions of biological assemblies.

Fig. 1. Characterization and MAS NMR spectra of KIF5B bound to MTs.

a TEM images of the NMR sample before (left, middle) and after (right) MAS NMR experiments. Similar results were reproduced in three independent experiments. b SDS-PAGE  of the KIF5B/MT complex. The co-sedimentation assay conditions are detailed in Methods. This experiment was replicated three times with similar results. c Characteristic Ala (top left), Ser/Thr (middle left), Gly (bottom left) and aromatic (right) regions of the CORD and NCACX spectra of U-¹³C,¹⁵N-KIF5B bound to MTs. d 2D ¹H-¹⁵N HETCOR spectra of U-²H,¹³C,¹⁵N-KIF5B bound to MTs. e Backbone walk for the A260-I265 stretch extracted from 3D NCACX (purple) and NCOCX (gray) spectra of U-¹³C,¹⁵N-KIF5B bound to MTs. Selected assignments are labeled in each spectrum. Source data are provided as a Source Data file.

Fig. 2. MAS NMR spectra and structure of KIF5B bound to MTs.

a Expansions of 2D CORD spectra (200 ms mixing time) of [1,6-¹³C-glucose,U-¹⁵N]-KIF5B/MT (gray) and [2-¹³C-glucose,U-¹⁵N]-KIF5B/MT (purple). Inter-residue correlations are labeled. b A matrix representation of assigned inter-residue contacts generated from the MAS NMR distance restraints. c Expansion of a single KIF5B (purple, with the neck linker colored in yellow) bound to a tubulin dimer (gray). d Side view of 22 KIF5B structures (purple) docked onto microtubules (gray).

Fig. 3. MAS NMR spectra and conformations of functionally important regions of KIF5B bound to MTs.

a Left: binding site of KIF5B (purple) in complex with MTs (cyan); Right: An expanded view of the KIF5B-tubulin dimer interface showing the interacting helices and loops. b Nucleotide-binding region in the apo-KIF5B structure. The T87 in P-loop is distant from E236, indicating the “open” state of nucleotide-binding pocket. Carbon, nitrogen, oxygen and sulfur atoms are colored with purple, cyan, orange and red, respectively. Hydrogen atoms are not shown. c Expansions of 2D CORD spectra of [1,6-¹³C-glucose,U-¹⁵N]-KIF5B/MT (cyan), [2-¹³C-glucose,U-¹⁵N]-KIF5B/MT (purple) and U-¹³C,¹⁵N-KIF5B/MT (gray) illustrating long-range correlations with the neck linker. Distance restraint network between neck linker and nearby residues within KIF5B mapped onto the structure is shown in black dashed lines. d Expansion of the superposition of 2D CORD spectra of apo-KIF5B/MT (gray) and ADP-KIF5B/MT (purple) illustrating chemical shift and peak intensity changes. Assigned peaks with pronounced changes are labeled in the spectra. e–g Regions that comprise residues for which chemical shift and/or intensity changes are observed in the CORD spectra, mapped onto MAS NMR structure of KIF5B bound to MTs. Undocked neck linker in apo-KIF5B e; Expected region for docked neck linker in ADP-KIF5B f; and nucleotide binding cleft g.