letters 844 nature structural biology • volume 9 number 11 • november 2002 Two conformations in the human kinesin power stroke defined by X-ray crystallography and EPR spectroscopy Charles V. Sindelar 1 , Mary Jane Budny 1 , Sarah Rice 2 , Nariman Naber 1 , Robert Fletterick 1 and Roger Cooke 1 1 Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143, USA. 2 Department of Biochemistry, Stanford University, Palo Alto, California 94305, USA. Published online 7 October 2002; doi:10.1038/nsb852 Crystal structures of the molecular motor kinesin show con- formational variability in a structural element called the neck linker. Conformational change in the neck linker, initi- ated by ATP exchange, is thought to drive the movement of kinesin along the microtubule track. We use site-specific EPR measurements to show that when microtubules are absent, the neck linker exists in equilibrium between two structural states (disordered and ‘docked’). The active site nucleotide does not control the position taken by the neck linker. However, we find that sulfate can specifically bind near the nucleotide site and stabilize the docked neck linker confor- mation, which we confirmed by solving a new crystal struc- ture. Comparing the crystal structures of our construct with the docked or undocked neck linker reveals how microtubule binding may activate the nucleotide-sensing mechanism of kinesin, allowing neck linker transitions to power motility. The molecular motor protein kinesin is essential in organisms that contain microtubules 1,2 . Powered by ATP, dimers of kinesin step along the protofilament tracks of microtubules, generating mechanical work through alternating binding and release steps of their monomer catalytic domains 3–5 . Many essential details of this walking mechanism are not yet known. The highly conserved neck linker domain of kinesin has been implicated in the movement and force production of kinesin 6–9 . This short (∼15 amino acid) segment connects the C-terminus of the catalytic core of conventional kinesin (Fig. 1) to the coiled-coil stalk, which stabilizes the dimer and leads to a globu- lar tail domain used for binding cellular cargo. On microtubules, the neck linker of kinesin was found to make an ATP-triggered zippering transition 6,7,9 , suggesting a ‘power stroke’ model of motility. The ‘disordered’ and ‘docked’ conformations of the neck linker at the beginning and end of this power stroke, respectively, are thought to correspond to the two distinct con- a b c Fig. 1 Two crystallized conformations of human kinesin. a, Regions of conformational change in the new crystal structure of the monomeric human kinesin construct K349 (with a docked neck linker), with respect to the published form 10 that has a disordered neck linker. The new K349 structure is colored by residue movement relative to a least-squares alignment. The ADP active site nucleotide is represented as a space-filled model, as are two specifically bound sulfate anions found in the docked K349 structure to the rear and right of the molecule from this viewing angle. The three positions on the neck linker used for site-labeled EPR probes are indicated by red stars. b, The new K349 crystal structure com- pared with the crystal structure of monomeric rat kinesin. Two sulfate ions (space filled) are found in the rat structure at the same positions as in the docked K349 structure. c, Uncoupling in the switch II domain of kinesin. In the alignment of human kinesin crystal structures, the con- served switch II nucleotide sensor moves <0.7 Å, whereas the switch II cluster shifts downward and to the right (docked structure, red) relative to the undocked (gray) structure. The switch II cluster definition here does not include the nucleotide-sensing segment or L11, differing slightly from the original definition proposed by Kikkawa et al. 17 Molecular drawings were generated by the Swiss-Pdb Viewer 32 and rendered using MegaPOV (www.povray.org). © 2002 Nature Publishing Group http://www.nature.com/naturestructuralbiology