Binding Kinetics between Membrane-Bound Kinesin Motors and Microtubules

Abstract

Microtubules (MTs) and their associated proteins are essential for many cellular processes, including maintenance of cellular structure, cell motility, cell division, and intracellular transport. Kinesin superfamily (KIFs) proteins are molecular motors that directionally transport organelles and cargos along MTs. As the first-discovered kinesins, the Kinesin-1 superfamily (KIF5s) is a group of highly processive motor proteins. Despite KIF5B's importance in cellular health, atomic level insight to the structure, dynamics, and microtubule interface are lacking. Magic Angle Spinning (MAS) NMR spectroscopy is well suited for structure and dynamics characterization of KIF5B motor domain in complex with MTs. We present an investigation into the atomic-resolution structure of KIF5B motor domain in complex with polymeric MTs by MAS NMR. We applied multidimensional (2D and 3D) homo-and heteronuclear experiments in U-13 C, 15 N-kinesin/MT complex for resonance assignments. All homo-and heteronuclear correlation spectra exhibited high resolution and revealed that more than 80% of the residues are present in the spectra. Chemical shift predictions were performed by ShiftX2 using the X-ray structure of KIF5B bound to a/b tubulin dimer. Based on the 2D/3D spectra, together with SHIFTX2 predictions, characteristic spin system assignments were identified and one third of residue specific assignments have been achieved. In addition, we applied homonuclear experiments in [1,6-Glu-13 C]-[U-15 N]-kinesin/MT complex and 1 H detection experiments under fast MAS in U-2 H, 13 C, 15 N-kinesin/MT complex to reduce spectral complexity and enhance resolution.