Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins.

Joseph Atherton,I-Mei Yu,Irene Farabella,Steven S Rosenfeld,Maya Topf,Carolyn A Moores,Anne Houdusse

doi:10.7554/elife.03680

Joseph Atherton, I-Mei Yu + Show 5 more

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https://doi.org/10.7554/elife.03680

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Abstract

Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles--including their nucleotide-free states--at ∼ 7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin-microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface.

Highlights

Kinesins are a large family of microtubule (MT)-based motors that play important roles in many cellular activities including mitosis, motility, and intracellular transport (Vale, 2003; Hirokawa and Noda, 2008; Hirokawa et al, 2010)
We show that relatively small structural transitions occur at the nucleotide-binding site on Mg-ATP binding, but that these lead to larger scale conformational changes and neck linker docking
The motor domain and nucleotide state, number of asymmetric units (AU) in the final reconstruction, the resolutions at a cut-off of 0.5 and 0.143 estimated by standard FSC (FSCt) and that corrected with the HRnoise substitution test (FSCtrue) (Chen et al, 2013) and by Rmeasure (Sousa and Grigorieff, 2007) and the EMDB accession number are given

Summary

Introduction

Kinesins are a large family of microtubule (MT)-based motors that play important roles in many cellular activities including mitosis, motility, and intracellular transport (Vale, 2003; Hirokawa and Noda, 2008; Hirokawa et al, 2010). A number of kinesins drive long distance transport of cellular cargo (Hirokawa et al, 2010; Soppina et al, 2014) with dimerisation allowing them to take multiple 8 nm ATP-driven steps toward MT plus ends (Svoboda et al, 1993). Their processivity depends on communication between the two motor domains, which is achieved via the neck linker that connects each motor domain to the dimer-forming coiled-coil (Hackney, 1994; Rice et al, 1999; Tomishige and Vale, 2000; Clancy et al, 2011).

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