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Kinesin: switch I & II and the motor mechanism

F. Jon Kull^1,* and Sharyn A. Endow^2,

¹ Department of Biophysics, Max-Planck Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg Germany
² Department of Cell Biology, Duke University Medical Center, Durham, NC 27710 USA
^* Present address: Department of Chemistry, Dartmouth College, Hanover, NH 03755 USA

View larger version (29K): [in a new window]   Fig. 1. Structural comparison of kinesin and myosin. (A) Front view of kinesin (rat monomeric KHC, PDB 2KIN) (Sack et al., 1997) and myosin II (scallop S1-ADP·VO4, PDB 1DFL) (Houdusse et al., 2000). Core ß-strands with adjacent nucleotide-binding (P-loop) or switch regions are colored green (P-loop), purple (switch I) and cyan (switch II). The helix following switch II, the ‘relay’ helix, is red in the two motors. Other ß-strands and -helices common to kinesin and myosin are shown in dark blue-gray and unique areas are in light gray. The myosin converter and kinesin neck helix and neck linker are shown in pink-purple. The myosin lever arm is tan. (B) Back view, rotated 180° from the view in A, showing the filament-binding face of the motors. Switch II at the active site of myosin is connected to the relay helix, which interacts with the converter and lever arm at its other end - the converter can therefore convert changes at the active site into movements of the lever arm.

View larger version (48K): [in a new window]   Fig. 2. Comparison of the three observed structural conformations of myosin. The structures are weak-binding states, but are modeled as complexed with actin for illustration. Motor domains (gray) and converter domains/lever arms (colored) for the myosin detached state (red) (scallop S1-ADP, PDB 1B7T) (Houdusse et al., 1999), near-rigor state (green) (nucleotide-free scallop S1, PDB 1DFK) (Houdusse et al., 2000) and transition state (cyan) (scallop S1-ADP·VO4, PDB 1DFL) (Houdusse et al., 2000) are shown bound to a short actin filament of five molecules (yellow). In the detached state, the lever arm and associated light chains (not pictured) would collide with an extended actin filament. Motor domains were aligned using a least-squares alignment of 19 -carbon atoms, including residues 168-186.

View larger version (80K): [in a new window]   Fig. 3. Structural changes at the active site and the filament-binding region in kinesin and myosin. (A,B) Comparison of human kinesin (PDB 1BG2) (Kull et al., 1996) and rat kinesin (PDB 2KIN, monomer structure) (Sack et al., 1997) showing the nucleotide-binding site with bound ADP and movements of helices 4 and 5 (human, orange; rat, red). Helix 4 can be seen in (A) and is the upper red/orange helix pair in (B). The lower helix pair is 5. The superposed structures show little difference at the active site in the P-loop (green), switch I (purple), or switch II (cyan), but helices 4 and 5 are displaced relative to one another by a simple translation of 4-5Å. The kinesin neck linker, observed in the rat structure, is shown in magenta. (C,D) Superposed KIF1A-ADP (PDB 1I5S) and KIF1A-AMP·PCP (PDB 1I6I) (Kikkawa et al., 2001). The movement of helices 4 and 5 (KIF1A-ADP, orange; KIF1A-AMP·PCP, red) is more complex than in (B), and consists of a translation coupled with a rotation. There is also a substantial structural rearrangement of the switch I region (C) (KIF1A-ADP, pink-purple; KIF1A-AMP·PCP, yellow), which changes the short loop-helix-loop-helix (KIF1A-ADP) to a short pseudo-ß-hairpin (KIF1A-AMP·PCP). (E,F) Myosin-ADP·BeF3 CLOSED (F.J.K. & K.C. Holmes, unpublished) compared with myosin-ADP OPEN (PDB 1G8X) (Kliche et al., 2001). The relay helix (analogous to kinesin helix 4) in the CLOSED form (red) is translated along its axis 4.5Å toward the nucleotide-binding site with little rotational movement at its N-terminus. However, the pronounced bend in the middle of the relay helix in the CLOSED structure causes a 100° rotation of its C-terminal end, causing the position of the helix end in the OPEN (orange) and CLOSED (red) forms to differ by 15 Å. This movement is further amplified by the myosin converter domain, and is thought to drive the myosin power stroke. All of the comparisons and distances described in this paper are based on a least-squares alignment of 19 -carbon atoms in the structurally conserved P-loop, the preceding ß-strand, and the N-terminal end of the following -helix (residues 77-95 of human kinesin, 78-96 of rat kinesin, 89-107 of KIF1A, 466-484 of Kar3 and 171-189 of Dictyostelium myosin II).

View larger version (88K): [in a new window]   Fig. 4. Coordination of the nucleotide and Mg2+ by essential switch elements in kinesin and myosin. (A) The nucleotide-binding site of KIF1A-ADP (PDB 1I5S) (Kikkawa et al., 2001). The nucleotide is primarily bound by the P-loop (green) and switch II (cyan). Switch I (magenta) does not interact with the nucleotide. The Mg2+ is shown as a purple sphere and is coordinated in an octahedral geometry by S104, an oxygen from the ß-phosphate of ADP, and four water molecules (cyan spheres). Dashed lines indicate hydrogen bonds. The conserved D248 from switch II (DLAGSE) is shown hydrogen bonded with one of the water molecules coordinating the Mg2+. The amide nitrogen of G251 from switch II is shown as a blue sphere. Side chains for the conserved S215 of switch I (SSRSH), as well as E253 and R216, which form the R-E salt bridge observed in some kinesin-ADP structures, are also shown. The salt bridge is not formed in this structure. The relay helix, 4, is shown in red. (B) A similar view for KIF1A-AMP·PCP (PDB 1I6I) (Kikkawa et al., 2001). Despite the structural rearrangement of switch I into a pseudo-ß-hairpin (magenta), the conserved S215 does not move enough to coordinate the Mg2+ ion. Likewise, the amide of G251, although 0.8 Å closer to the nucleotide than in the ADP structure, is unable to form a hydrogen bond to the -phosphate. The R-E salt bridge between R216 of switch I and E253 of switch II, thought to stabilize the CLOSED form of myosin, is not formed. In this conformation, the enzyme is unlikely to be capable of catalyzing ATP hydrolysis, and most likely represents a collision ATP state. (C) A view of myosin-ADP (PDB 1MMA) (Gulick et al., 1997), showing amino acid side chains analogous to those in KIF1A (A,B). The nucleotide and Mg2+ coordination are similar to that seen for KIF1A (A), with T186 of the P-loop replacing S104 in KIF1A and switch I (purple) much closer to the nucleotide, allowing S237 of switch I to coordinate the Mg2+ ion. D454 forms a hydrogen bond with a water coordinating the Mg2+ ion. In this OPEN conformation, the R-E salt bridge between R238 of switch I and E459 of switch II cannot form, as the two residues are too far apart. Likewise, the amide nitrogen of G457 of the P loop is not interacting with the nucleotide. (D) Myosin with bound ADP·BeF3 (F.J.K. & K.C. Holmes, unpublished). The structure, which is now CLOSED, likely represents a hydrolysis-competent state with BeF3 present instead of the -phosphate. S237 from switch I and an F from the BeF3 (instead of an O from the -phosphate if ATP were bound) are coordinating the Mg2+ ion, switch II has rotated about G457 and moved toward the nucleotide by 5.0 Å, the amide nitrogen from G457 forms a tight hydrogen bond with the BeF3 (representing the -phosphate), and the salt bridge between R238 and E459 has formed. The invariant hydrogen bond from the amide nitrogen of S237 to the -phosphate oxygen is not shown, for clarity. The relay helix of myosin (red) moves 4.5Å along its axis in the ADP·BeF3 state compared with the ADP state.

View larger version (44K): [in a new window]   Fig. 5. Comparison of wild-type and mutant R-E salt-bridge Kar3 motor domains. Wild-type Kar3 (PDB 1F9T) (Yun et al., 2001) superposed with (A) the E631A (EA) mutant structure (PDB 1F9W) (Yun et al., 2001) or (B) the R598A (RA) mutant structure (PDB 1F9V) (Yun et al., 2001). Blue regions represent areas of least change with <0.5 Å difference between main-chain atoms. Gray areas show differences of 0.5-1.0 Å, after which the color changes to orange (for wild type) or red (for the mutants), becoming solid at 2.0 Å structural difference. Yellow elements are present in the wild-type structure, but not the EA mutant. Green elements are observed in the RA mutant, but not wild-type Kar3. The ADP and Mg2+ from wild-type Kar3 are also shown. The arrow in (A) indicates the position of the switch I loop, which becomes disordered in the EA and RA mutants. The arrow in (B) indicates loop 11 and the N-terminus of helix 4, the relay helix, which become ordered upon mutation of R598. The -helix preceding switch I also undergoes a substantial movement in the RA mutant.