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First published online November 7, 2007
doi: 10.1242/10.1242/jcs.016931

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A cool look at the structural changes in kinesin motor domains

¹ MRC Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK
² Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8562, Japan

View larger version (36K): [in this window] [in a new window]   Fig. 1. Tentative schemes for conformational changes during ATPase cycles. (A) Motor acting as a monomer to contribute, in this case, to minus-end-directed movement. The motor domain is shown in cyan with the switch II helix in orange, the coiled-coil neck in red. The second head, thought to move passively with the coiled-coil, is not shown. The nucleotide bound to the motor is indicated at each stage in the cycle. (1) ADP-bound motor domain waiting to make contact with tubulin (part of one protofilament in a microtubule is shown as green subunits). The coiled-coil neck (red) is docked on to the motor domain. (2) Contact is made, ADP is released and the empty motor domain binds strongly to tubulin. (3) ATP binds to the nucleotide pocket. The coiled-coil, no longer docked on to the ATP-filled motor domain, is free to swing towards the MT minus end and allow other motors on the same cargo to search for new sites while this one remains attached. In an alternative model (Yun et al., 2003), the coiled-coil would be released by the loss of ADP and be free to move at stage 2. (4) ATP is hydrolysed to ADP and phosphate (Pi). (5) The motor domain detaches as phosphate is lost. Whilst the motor is unattached, the neck docks back on to the ADP-bound motor domain. (B) Processive dimer, whose two heads take turns in stepping towards the plus end of the MT (Vale et al., 1996; Schnitzer and Block, 1997; Hancock and Howard, 1998; Young et al., 1998; Carter and Cross, 2005; Hackney, 2007). White letters (E, D, T, DP) indicate the nucleotide states of individual heads. (1) The tethered, ADP-bound lead head is free to find a new binding site. It needs to swing around the junction with the neck-linker (red line) to bind to the same protofilament as the rear head. (2) ADP is released on contact and the empty lead head binds strongly. It must wait for the trailing head to finish ATP hydrolysis, release phosphate and detach (Klumpp et al., 2004), then wait to bind fresh ATP itself. The presence of the tethered head on the top of the leading head may provide the signal that allows ATP to bind. (3) ATP binds and the neck-linker is able to dock on to a site (shown as a white line) on the motor domain that helps to bias the binding of the new lead head to a site in the plus direction. (4) ATP is hydrolysed, making detachment possible once the lead head is firmly bound. (5) The detached head parks on top of the lead head while this head waits for ATP to bind (see main text and Fig. 6). In moving from position 4 to 5 and back to 1, the tethered heads must pass the coiled-coil on different sides during alternate steps, to avoid building up a twist.

View larger version (113K): [in this window] [in a new window]   Fig. 2. Atomic structures of a C-terminal and an N-terminal kinesin family motor domain. (A,B) Crystal structures, represented in cartoon format, of the motor domain of Ncd, a minus-end-directed kinesin-14. In A, showing the conformation thought to predominate in the normal soluble ADP-filled state, the N-terminal neck helix (dark red) is docked against the edge of the central β sheet of the motor domain (Sablin et al., 1998; Kozielski et al., 1999); under the conditions used to crystallise the structure shown in B (Yun et al., 2003), the neck is not docked and has swung down around a hinge at its base. Yun et al. suggested that this conformation represents the empty state; alternatively, it may resemble an ATP-bound state, as suggested by Endres et al. (Endres et al., 2006). The crystal structure does not show any obvious changes that would indicate how the movement might be controlled, except that the adjacent loops are disordered. The switch II region (including L11, 4, L12 and 5) is coloured in gold, switch I in blue and loop L8 in green. The way these two conformations may produce movement along a MT is shown in Fig. 1A. (C) Simplified scheme of the polypeptide, with important loops and helices coloured as in A and B. (D,E) Crystal structures of the motor domain of kinesin-1. The C-terminal neck is not in a fixed position in the ADP state [D (Kull et al., 1996)]; however, the buffer conditions used for E (Sack et al., 1997; Sindelar et al., 2002) caused it to dock against the motor domain in a way that is thought to occur in the ATP-bound state (`ATP-like'). There are also some differences in the adjacent elements, loop L12, helix 5 and the switch II helix 4, that connect to the nucleotide-binding site. As indicated in Fig. 3C, crystal structures of Kif1a with ATP analogues bound show similar changes (Nitta et al., 2004). (F) F shows a schematic view of a plus-end-directed motor domain and its neck. PDB codes 1CZ7, 1N6M, 1BG2, 2KIN.

View larger version (189K): [in this window] [in a new window]   Fig. 3. High-resolution EM maps of plus-end-directed motors bound to microtubules. (A) Surface representation showing kinesin-1 bound to a MT (Sindelar and Downing, 2007). The secondary structures (superimposed in cartoon form) from kinesin-ADP crystals (PDB code 1BG2) (Kull et al., 1996) dock rather precisely into the EM map. (B,C) Sections through EM maps (density represented as a grey net) of the Kif1a motor domain attached to tubulin in two nucleotide states (ADP-bound and AMPPNP-bound) with corresponding crystal structures superimposed (from Kikkawa and Hirokawa, 2006). The view from the MT plus end (B) compares two positions of helix 4 (yellow for the ADP-bound state, red for the AMPPNP-bound state) relative to the motor core. In the side view (C), changes in helix 6 (green) are indicated, as well as changes indicating the reduced mobility of the neck linker when AMPPNP is bound.

View larger version (272K): [in this window] [in a new window]   Fig. 4. High-resolution EM maps of Kar3 motor domains bound to microtubules. (A,C) Sections at two different levels through maps of the Kar3 motor domain in three different nucleotide states (ADP-bound, empty or AMPPNNP-bound) with the Kar3-ADP crystal structure (cyan -helices and β-strands; PDB code 1F9T) (Yun et al., 2001) docked into the EM density (represented by red and grey nets) (Hirose et al., 2006). The view is from the MT plus end and the positions of sections A and C within the 3D structure are indicated in panels B,D. Parts of - and β-tubulin are included, with the crystal structures (PDB code 1JFF) (Löwe et al., 2001) shown in gold and green. The red arrowhead in A indicates a feature in the EM density of the nucleotide-free Kar3 that is absent from either of the nucleotide-bound motors and may be loop L7 in a new position. Blue arrowheads in A indicate the point at the top of the motor domain that is retracted in the empty structure. A also shows a movement of helix 5, which is closer to tubulin in the empty and AMPPNP-bound states. A black arrowhead in C indicates a marked loss of density from the region occupied by the switch-II-helix 4 in the other structures. Orange arrowheads point to the L12 loop that also disappears in the empty structure. (E) Sections though the three maps viewed from the side. There are changes in the position of helix 6 relative to switch II helix 4 and helix H12 of -tubulin but, with only one Kar3 crystal structure available, we cannot be certain that the movement is the same as in Kif1a (Fig. 3C).

View larger version (32K): [in this window] [in a new window]   Fig. 5. Kar3 bound to tubulin. Binding orientation of Kar3 motor domain on β-tubulin, based on docking crystal structures into the EM maps (see Fig. 4). Different parts of Kar3 are coloured as in Fig. 2, including the switch II region in yellow. Loop L8 and helix 6 are also part of the interface with tubulin. Similar interactions for kinesin and Kif1a are apparent in Fig. 3A,C.

View larger version (78K): [in this window] [in a new window]   Fig. 6. Models of kinesin-1 dimers bound to tubulin. (A) Crystal structure of the rat kinesin-1 dimer (PDB code 3KIN) (Kozielski et al., 1997) with head 1 in the same orientation as the Kar3 head in Fig. 5. Note that the coiled-coil would clash with tubulin unless it moves from its position in the crystal structure. When MT were decorated with ADP-bound dimers (see C), a rotation of the whole motor domain apparently relieved the clash (Hirose et al., 1999). (B) The kinesin dimer crystal structure with the heads reoriented relative to each other to allow them both to dock into the EM map in D, with head 1 as the directly-bound head. The movement of head 2 is likely to have resulted from conformational changes in head 1 but the details are unknown. The coiled-coil (dark red) is shown as having shifted with head 2, though it was not detectable in D and was probably free to move anywhere. (C,D) Parts of the outer surfaces of low-resolution EM maps of kinesin-1 dimer bound in the ADP-bound and empty states to MTs (Hirose et al., 1999). Directly bound heads (1) and tethered heads (2) (both coloured here in cyan) can be identified. The MT is coloured green.