Commentary 15 Kinesin: switch I & II and the motor mechanism

F. Jon Kull1,* and Sharyn A. Endow2,‡ 1Department of Biophysics, Max-Planck Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg Germany 2Department of Cell Biology, Duke University Medical Center, Durham, NC 27710 USA *Present address: Department of Chemistry, Dartmouth College, Hanover, NH 03755 USA ‡Author for correspondence (e-mail: [email protected])

Journal of Cell Science 115, 15-23 (2002) © The Company of Biologists Ltd

Summary New crystal structures of the kinesin motors differ from transitions that occur in the kinesin motors during the ATP previously described motor-ADP atomic models, showing hydrolysis cycle. The movements of switch I residues striking changes both in the switch I region near the disrupt the water-mediated coordination of the bound nucleotide-binding cleft and in the switch II or ‘relay’ helix Mg2+, which could result in loss of Mg2+ and ADP, raising at the filament-binding face of the motor. The switch I the intriguing possibility that disruption of the switch I region, present as a short helix flanked by two loops in region leads to release of nucleotide by the kinesins. None previous motor-ADP structures, rearranges into a pseudo- of the new structures is a true motor-ATP state, however, β-hairpin or is completely disordered with melted helices probably because conformational changes at the active site to either side of the disordered switch I loop. The relay helix of the kinesins require interactions with microtubules to undergoes a rotational movement coupled to a translation stabilize the movements. that differs from the piston-like movement of the relay helix observed in myosin. The changes observed in the crystal Key words: Molecular motor, Structure/function, Conformational structures are interpreted to represent structural changes, Kinesin, Myosin

Introduction more steps of ATP binding or hydrolysis induces small Life inside the cell is an incessant frenzy of rapid back-and- conformational changes in the protein that, under load, create forth movements of myriad cellular components, including strain (Howard, 2001). The strain is relieved by further changes proteins, vesicles and organelles, driven by collisions of water in the motor that produce force and then amplify the force, and other molecules (see Howard, 2001). This Brownian resulting in movement of the motor along its filament. The motion allows molecules such as newly synthesized RNAs to structural elements that undergo strain are likely to have diffuse randomly from their site of entry into the cytoplasm but spring-like or elastic properties that allow them to extend or is far too slow to move molecules the relatively large distances rotate, and then recoil back into their original conformation required in eukaryotic cells and cannot localize them to specific (Howard, 2001). Movements of the motor catalytic core are regions of the cell (Wilkie and Davis, 2001). Localization to a further expected to involve the highly conserved switch regions site within the cell requires facilitated movement, usually aided (Kull et al., 1996; Sablin et al., 1996), switch I and switch II, by one of a battery of cytoskeletal motor proteins - the myosins, so-named because of their structural homology to regions of G kinesins or dyneins. proteins that move upon nucleotide hydrolysis and exchange (Sprang, 1997). Thus, an understanding of the motor mechanism is likely to come only after workers have identified Cytoskeletal motors the spring-like or elastic elements within the motor, together Cytoskeletal motor proteins are specially built to perform their with the force-producing structural changes in the motor and transport function in the cell: one region of the protein binds the steps in the hydrolysis cycle at which they occur. to an actin filament or microtubule, hydrolyzes ATP and moves The two best-studied cytoskeletal motors, myosin and along the filament, while another region attaches to cargo kinesin, are dimeric proteins that have two catalytic domains for transport to a specific site within the cell. Cytoskeletal joined by a coiled-coil rod or stalk. These two motor proteins motors are thus specialized ATPases that transport cargo by and other highly related proteins in their respective families coordinating the hydrolysis of ATP with binding to and contain a central core of structural elements that are remarkably movement along a filament. Remarkably, these molecular similar to one another (Kull et al., 1996) (Fig. 1). Despite this motors convert the chemical energy from ATP hydrolysis structural homology, however, there are indications that the directly into mechanical energy. How do motors capture the kinesin motors differ substantially from the myosins in their energy released by nucleotide hydrolysis and turn it into mechanism of function. A fundamental difference is the work? Motor proteins are apparently capable of sensing and nucleotide-dependent interactions of the motors with their responding to the presence or absence of a γ-phosphate, and filament: myosin bound to ATP is weakly bound to or detached then transmitting this information along a pathway of from actin, whereas kinesin-ATP is strongly bound to increasingly larger conformational changes that culminates in microtubules. Conversely, myosin-ADP is strongly bound to a force-generating event. A prevailing idea is that one or actin, whereas kinesin-ADP is weakly bound to or detached 16 Journal of Cell Science 115 (1) from microtubules. A further basic difference between the motors have been elusive. Almost all of the crystal structures kinesins and myosins is that myosin hydrolyzes ATP while solved so far are complexed to ADP and are interpreted to detached from actin, whereas kinesin hydrolyses ATP while represent weak-binding or detached ADP states (reviewed by attached to the microtubule. But, for both motors, the rate- Sack et al., 1999). The kinesin-ADP models do differ from one limiting step in the hydrolysis cycle is accelerated by binding another, indicating that they may represent structural of the motor to its filament, which results in a characteristic transitions within the ADP state, which could help identify the actin- or microtubule-activated ATPase activity that underlies mechanical elements of the motor that undergo conformational the ability of the motor to move along its filament. changes during the hydrolysis cycle. For example, human kinesin (Kull et al., 1996) and rat kinesin (Kozielski et al., Myosin Work on myosin has identified three distinct conformations of the motor that are thought to correspond to different nucleotide states (Rayment et al., 1993a; Fisher et al., 1995; Dominguez et al., 1998; Houdusse et al., 1999; Houdusse et al., 2000). These have been classified as detached, near rigor, and transition forms (Houdusse et al., 2000) (Fig. 2), all of which bind weakly to actin. The nucleotide bound to the motor is not always an indication of the motor state, since the detached form can be crystallized with either ADP or ATP bound to the active site (Houdusse et al., 1999; Houdusse et al., 2000). The three forms show major differences in the position of the rod-like lever arm, as well as a striking change in the ‘converter’ region at the base of the lever arm. The converter, a rigid α/β subdomain of 67 residues that includes the first three turns of the helical rod (Fig. 1), is thought to convert movements at the nucleotide-binding cleft of the motor, which are transmitted by the adjacent switch II or ‘relay’ helix and the SH1 helix, into the swinging of the lever arm. The SH1 helix, which is also adjacent to the converter, is intact in the near-rigor and transition structures but disordered in the detached form, which has been interpreted to be a motor-ATP state (Houdusse et al., 1999). This mobility of the SH1 helix reflects its role in transmitting and directing movements of the relay helix to the converter domain and lever arm. Structural changes at the nucleotide-binding cleft of myosin are thus coupled to movements of the relay helix, SH1 helix, converter domain and the lever arm (Fig.1). The power stroke is thought to correspond to the swinging of the lever arm (Rayment et al., 1993a; Rayment et al., 1993b); however, the structural elements that undergo strain and the conformational changes in myosin that produce force have not yet been identified.

Kinesin motors By comparison with myosin, the structural states of the kinesin

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 β-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 β-strands and α-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. Kinesin: switch I & II and the motor mechanism 17 1997) show little change in the nucleotide-binding site (Fig. myosin) forms a hydrogen bond with an oxygen from the γ- 3A), but helices α4 and α5 are displaced in rat kinesin relative phosphate; (2) a conserved serine (or threonine) residue from to human kinesin (Fig. 3A,B). Similarly, the Kar3 motor shows switch I (SSRFG in myosin) forms a strong hydrogen bond a change in the length of helix α4 compared with human from its γ-oxygen to the Mg2+ ion and a weaker one to the γ- kinesin or Ncd - the helix is nine residues (two turns) longer phosphate of the nucleotide; and (3) the same serine or than in the other two motors (Gulick et al., 1998), indicating threonine forms a hydrogen bond from its amide nitrogen to that helix α4, the switch II (or relay) helix of the kinesins, can an oxygen on the γ-phosphate. Given that the KIF1A- change in length as well as orientation, and could undergo large AMP·PCP structure does not show any of these features, it changes in conformation during the hydrolysis cycle. Another probably represents a collision ATP complex - a first step in difference in the kinesin-ADP models is the structure of the ATP binding that does not produce major conformational neck linker, the region that joins the coiled-coil stalk to the changes (Cooke, 1986) - rather than a catalytically active state catalytic core: the neck linker is disordered in human kinesin similar to Ras-GMP·PNP (PDB 5P21) (Pai et al., 1990) or (Kull et al., 1996), but it is visible in rat kinesin, forming β- myosin-VO4 (PDB 1VOM) (Smith and Rayment, 1996). strands that interact with the β-sheet of the motor core Crystal structures of kinesin mutants designed to destabilize (Kozielski et al., 1997; Sack et al., 1997). The transition from the ADP state, stabilizing conformations that may normally be a disordered to a visible state indicates that the neck linker is unstable or transient, reveal extensive changes in the inherently mobile and could therefore play a critical role in the conformation of switch I and switch II (Yun et al., 2001). The motor mechanism. kinesin mutants affect highly conserved or invariant arginine The kinesin-ADP structures have thus yielded insights into and glutamic acid residues of switch I (SSRSH) and switch II the mechanical movements of the motors, but do not substitute (DLAGSE), respectively, and disrupt a salt bridge between the for crystal structures of other nucleotide states. Why has an two residues that is observed in some, but not all, kinesin-ADP ADP-Pi state or a true ATP state not yet been seen in the kinesin structures (Sack et al., 1999; Yun et al., 2001). The R-E salt crystals, even though both crystal states have been observed for bridge is also observed in myosin (Fig. 4D), where it has been myosin? The reason is probably that the structural changes at the nucleotide-binding cleft of the kinesins are more dependent on filament binding than those of myosin - restructuring the kinesin active site may require stabilization by interactions between the motor and microtubules, with the microtubule performing the role of the upper 50 KDa domain of myosin, which is largely missing in the kinesin motors (Fig. 1). Crystals of kinesin proteins complexed with ATP analogues have been grown, but the structures of these crystals closely resemble the ADP-bound forms of the motors. One of the structures has recently been reported to be an ‘ATP-like’ form of the KIF1A motor (Kikkawa et al., 2001). The new KIF1A structure is complexed with AMP·PCP, a slowly hydrolysable ATP analogue, and shows two striking changes compared with KIF1A bound to ADP and other ADP-bound kinesin motors: (1) switch I has a substantially different conformation, assuming a pseudo-β-hairpin structure rather than a short loop-helix-loop-helix (as in KIF1A-ADP) or a short helix flanked by two loops (as in other kinesin- ADP structures) (Fig. 3A,C); and (2) helix α4, the relay helix, is tilted by 20° and the first two turns of the helix are unwound (Fig. 3D). The neck linker is also docked against the catalytic core of the motor rather than disordered and absent from the model as in the human kinesin-ADP structure (Kull et al., 1996). The neck linker conformation of the KIF1A- AMP·PCP structure does not differ, however, from that Fig. 2. Comparison of the three observed structural conformations of myosin. of the rat kinesin atomic models (Kozielski et al., 1997; The structures are weak-binding states, but are modeled as complexed with Sack et al., 1997). Furthermore, the KIF1A-AMP·PCP actin for illustration. Motor domains (gray) and converter domains/lever arms structure lacks several important interactions that are a (colored) for the myosin detached state (red) (scallop S1-ADP, PDB 1B7T) prerequisite for a hydrolysis-competent state (Fig. 4). (Houdusse et al., 1999), near-rigor state (green) (nucleotide-free scallop S1, PDB 1DFK) (Houdusse et al., 2000) and transition state (cyan) (scallop S1- In both G proteins and myosin, the NTP-bound and ADP·VO4, PDB 1DFL) (Houdusse et al., 2000) are shown bound to a short transition states show three invariant interactions actin filament of five molecules (yellow). In the detached state, the lever arm between conserved switch I and switch II residues and and associated light chains (not pictured) would collide with an extended the nucleotide (Fig. 4D): (1) the amide nitrogen from actin filament. Motor domains were aligned using a least-squares alignment the conserved glycine residue in switch II (DIXGFE in of 19 α-carbon atoms, including residues 168-186. 18 Journal of Cell Science 115 (1) proposed to stabilize the CLOSED form of the motor, in which become mobile and the helices on either side to partially melt the switch I and switch II regions close in around the bound (Fig. 5). The mutant conformations have been interpreted to nucleotide, a conformation thought to be essential for ATP represent new ADP crystal forms that differ from those hydrolysis (Geeves and Holmes, 1999). In contrast, the OPEN previously reported (Sack et al., 1999) or intermediates conformation of myosin, in which the R-E salt bridge is not between the kinesin-ADP and kinesin-ATP states (Yun et al., formed (Geeves and Holmes, 1999; Kliche et al., 2001), is 2001). catalytically incompetent (Furch et al., 1999). The kinesin salt- The structural transitions between KIF1A-ADP and KIF1A- bridge mutants show striking changes in the switch I and AMP·PCP, together with those observed in the two Kar3 salt- switch II regions that include transitions from a disordered to bridge mutants, shed important new light on the mechanism of a visible switch II loop and from ordered to disordered switch the kinesin motors. They show that the switch I region can I loop and two flanking helices (Yun et al., 2001) (Fig. 5). The dramatically alter its conformation, changing from a small mutants thus stabilize the switch II region, resulting in a longer helix or helices [as in human KHC (Kull et al., 1996), rat KHC switch II helix as well as a visible switch II loop, and (Kozielski et al., 1997) and KIF1A-ADP (Kikkawa et al., destabilize the switch I region, causing the switch I loop to 2001)] to a pseudo-β-hairpin [as in KIF1A-AMP·PCP

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 α4 and α5 (human, orange; rat, red). Helix α4 can be seen in (A) and is the upper red/orange helix pair in (B). The lower helix pair is α5. The superposed structures show little difference at the active site in the P-loop (green), switch I (purple), or switch II (cyan), but helices α4 and α5 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 α4 and α5 (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-β- 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 α4) 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 α-carbon atoms in the structurally conserved P-loop, the preceding β-strand, and the N-terminal end of the following α-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). Kinesin: switch I & II and the motor mechanism 19

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 β- 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, α4, 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-β-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 γ-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 γ-phosphate. S237 from switch I and an 2+ F from the BeF3 (instead of an O from the γ-phosphate if ATP were bound) are coordinating the Mg 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 γ- phosphate), and the salt bridge between R238 and E459 has formed. The invariant hydrogen bond from the amide nitrogen of S237 to the γ- 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.

(Kikkawa et al., 2001)] to a completely disordered structure [as microscopy (cryoEM) density maps of KIF1A bound to in the Kar3 salt-bridge mutants (Yun et al., 2001)]. The switch microtubules in the presence of ADP or the ATP analogue I region can almost certainly adopt a conformation not yet AMP·PNP (Kikkawa et al., 2001) have been interpreted to observed in the kinesin motors in which conserved serine show that the long axis of the motor is offset by ~20° in the residues (SSRSH) interact with the Mg·ATP at the active site. AMP·PNP state compared with the ADP state, suggesting that The intriguing possibility that arises from structural analysis of the motor undergoes a directional rotation between these two the Kar3 mutants (Yun et al., 2001) is that disruption of the states. However, the difference in motor orientation between switch I region may be responsible for the mechanism of the two states is slight, and this, together with the low nucleotide release in the kinesins. A disruption of the switch I resolution of the ADP density map (22 Å) and the absence of region following nucleotide hydrolysis could interfere with the structural features in the central region of the motor that could coordination of Mg2+, causing Mg2+ and, subsequently, ADP be used as landmarks to orient the motor, makes it difficult to to be released. demonstrate unequivocally that such a rotation occurs. The KIF1A-ADP and KIF1A-AMP·PCP crystal structures have also been fitted into the cryoEM density maps - but the crystal Motor-microtubule interactions structures do not fit precisely into the density maps, and What do the new structures tell us about interactions between structural elements can be seen to protrude from the electron the kinesin motors and microtubules? Cryoelectron density even though the fittings have been optimized to 20 Journal of Cell Science 115 (1) minimize these effects. This indicates that the positions or which evolved into the kinesin and myosin motor mechanisms. conformations of at least some of the structural elements in the This could explain the non-processive, plus-end-directed microtubule-bound motors differ from the atomic models. This motility of neck-mutated Ncd-kinesin and Ncd constructs is not unexpected, since the crystal structures of the motor are (Endow and Waligora, 1998; Case et al., 2000) that appears to not complexed with tubulin and are highly unlikely to be in the be intrinsic to the kinesin motor domain. Amplification by the same states as the motor bound to microtubules and imaged by neck and/or neck linker of such an initial strain-generating cryoEM. The authors interpret their fittings of the atomic movement could direct motor movement towards the structures into the cryoEM maps to show that the rotational microtubule plus or minus end. movement they observe is centered around helix α4, the relay The salt-bridge mutants provide further information about helix, which rotates 20° between the ADP and AMP·PCP the changes that occur in the kinesin motors in states atomic models. They propose that, when the motor is bound to subsequent to the ADP state. Biochemical analysis of the the microtubule, the motor itself rotates around the helix, mutant motors demonstrates that they can hydrolyze ATP and which remains fixed in place (Kikkawa et al., 2001). bind to microtubules but show no microtubule-activated Kikkawa et al. propose that the rotational movement of the ATPase activity (Yun et al., 2001), which is essential for catalytic core drives docking of the neck linker onto the motor movement of the motor along the microtubule. Activation of core and is accompanied by a displacement of the motor the motor ATPase is thought to occur in the kinesin motors by towards the plus end of the microtubule, thus accounting for accelerating the rate-limiting step, under nonsaturating the plus-end-directed movement of KIF1A and other kinesin microtubule concentrations, of ADP release (Hackney, 1988). motors (Kikkawa et al., 2001). They further propose that a Because the mutants can bind to microtubules but binding to similar rotation of the catalytic core of Ncd, a kinesin-related microtubules fails to activate their ATPase, the mutants motor that has the opposite directionality, disrupts an ‘decouple’ (Ruppel and Spudich, 1996) microtubule- and interaction between the neck and the motor core that drives nucleotide-binding by the motor (Song and Endow, 1998). The movement of Ncd towards the minus end. These interpretations decoupling of these two essential motor activities is interpreted of the static crystal structures provide models for motor to be due to a block in communication between the function that require confirmation by further experimental microtubule- and nucleotide-binding regions of the motor (Yun work. Several aspects of the proposed mechanisms lend et al., 2001). Together with a previous mutant in which themselves to testing - for example, by biochemical analysis of microtubule-activated ATPase activity is blocked (Song and mutants or spectrophotometric analysis of motors containing Endow, 1998), the salt-bridge mutants define a structural fluorescent probes at specific sites (Xing et al., 2000) to determine whether the proposed rotation occurs during force production by the motor and is therefore relevant to motor function. It is also of interest to determine whether the rotation, if it occurs, is coupled to directional movement of the motor. Such a rotation could represent a basic force-producing movement of the motor with the relay helix acting like a spring or elastic element that undergoes strain and enables the motor to produce force. If so, the movement would differ from the piston-like movement of the relay helix thought to occur in myosin (Dominguez et al., 1998) (Fig. 3E,F) and proposed to drive the swinging of the lever arm (Vale and Milligan, 2000). Although the Fig. 5. Comparison of wild-type and mutant R-E salt-bridge Kar3 motor domains. Wild-type Kar3 relay helix could serve as the (PDB 1F9T) (Yun et al., 2001) superposed with (A) the E631A (EA) mutant structure (PDB 1F9W) spring or elastic element involved (Yun et al., 2001) or (B) the R598A (RA) mutant structure (PDB 1F9V) (Yun et al., 2001). Blue in force generation in both kinesin regions represent areas of least change with <0.5 Å difference between main-chain atoms. Gray and myosin, the mechanistic 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 details of force generation would wild-type structure, but not the EA mutant. Green elements are observed in the RA mutant, but not differ. The rotational movement wild-type Kar3. The ADP and Mg2+ from wild-type Kar3 are also shown. The arrow in (A) indicates thought to occur in KIF1A the position of the switch I loop, which becomes disordered in the EA and RA mutants. The arrow in could represent an ancient (B) indicates loop 11 and the N-terminus of helix α4, the relay helix, which become ordered upon conformational movement mutation of R598. The α-helix preceding switch I also undergoes a substantial movement in the RA inherited from the G proteins, mutant. Kinesin: switch I & II and the motor mechanism 21 signaling pathway within the motor for ATPase activation by 3E,F and Fig. 4C,D). In the new kinesin structures, switch I microtubules. This pathway extends from helix α4, the relay moves towards the bound nucleotide when the motor is helix, at the microtubule-binding region of the motor to the R- complexed with AMP·PCP (Kikkawa et al., 2001) or when the E salt bridge between switch I and switch II to the active site. salt bridge between switch I and switch II is disrupted (Yun et The mutants further show that direct interactions between al., 2001), whereas switch II remains unchanged in position. But switch I and switch II are required for activation of the motor the movement of the invariant switch I serine residues (SSRSH) ATPase by microtubules. in the KIF1A-AMP·PCP model and the salt-bridge mutants is The salt-bridge mutants are trapped in states that alter the small (<1.0 Å) and does not result in additional interactions ability of the motor to bind to microtubules: one of the mutants between switch I and the nucleotide. This means that we have (RA) binds weakly to microtubules compared with the wild not yet seen the critical transition in kinesin corresponding to type, whereas the other (EA) binds tightly (Yun et al., 2001). the CLOSED conformation of myosin in which the switch II The regions of the motors that are altered in the crystal glycine flips in to hydrogen bond with the γ-phosphate. When structures identify structural elements that are likely to be kinesin does this, there is likely to be a movement of switch II involved in weak and strong binding of the motor to toward the nucleotide, although perhaps not so large as that in microtubules. The structures of the switch II loop and helix myosin, since switch II of kinesin is already intermediate in differ between the RA and EA atomic models, and this may be position between that of myosin in its OPEN and CLOSED responsible for differences in the interactions of the motors forms. It also seems likely that this conformation will be seen with microtubules - the switch II helix is longer by one turn in only when the motor is complexed with microtubules (as noted the RA mutant compared with one of the two available wild- above) or in a mutant that somehow mimics the binding of the type structures, and the switch II loop is visible in the RA motor to microtubules. The requirement for stabilization of mutant rather than disordered, as in the EA mutant. motor movements by the microtubule is a crucial point for the Stabilization of the switch II loop and the N-terminus of the kinesin motors - it not only explains why a true ATP state has switch II helix might interfere sterically with microtubule not yet been seen in the crystal structures but is important for binding, causing the weak binding of the RA mutant to the interpretation of available structures. microtubules. Loop L8 is displaced slightly in the EA mutant Mutants that disrupt the salt bridge between switch I and relative to its position in the wild-type motor. This difference, switch II inhibit or block the filament-activated ATPase activity although small, may reflect an inherent difference in loop of both myosin and the kinesin motors (Onishi et al., 1998; mobility that causes the mutant motor to bind more tightly to Furch et al., 1999; Yun et al., 2001). This indicates that direct microtubules than the wild type. Previous studies implicate the interactions between switch I and switch II are needed for switch II loop/helix and loop L8 in binding to tubulin (Woehlke activation of the myosin and kinesin motor ATPase. Disruption et al., 1997; Alonso et al., 1998; Hirose et al., 1999; Kikkawa of the salt bridge in myosin appears to interfere with the et al., 2000), supporting these interpretations. formation of the catalytically active CLOSED conformation The atomic structures of the salt-bridge mutants further (Kliche et al., 2001), shifting the mutants toward the switch II show that changes in the water-mediated coordination of the OPEN conformation, whereas it causes the kinesins to move nucleotide at the active site occur when the salt bridge between towards a switch I OPEN conformation. The disrupted salt switch I and switch II is disrupted (Yun et al., 2001), as noted bridge of both myosin and the kinesins thus appears to permit above. The movements of switch I residues toward the movements of the switch I and switch II regions that are nucleotide, although <1 Å, disrupt coordination of the Mg2+ prevented when the salt bridge is formed (Geeves and Holmes, ion by water molecules. This could result in loss of Mg2+, 1999; Yun et al., 2001). This could prevent the mutants from which would be followed immediately by ADP release. Thus, populating a state essential for ATPase activation in the wild- the rate-limiting step in nucleotide hydrolysis would be type motors. overcome (Hackney, 1988), explaining the activation of the Although it is unclear how far the mechanistic similarities motor ATPase by microtubules. between kinesin and myosin extend, the following movements are expected to occur during the kinesin ATPase cycle on the basis of the structural changes observed in the myosins and G Kinesin and myosin motor mechanisms – a proteins. As noted above, the new kinesin structures show a comparison conformation predicted to occur in myosin that we refer to as Although motor-filament interactions of myosin and the kinesin switch I OPEN, switch II OPEN (F.J.K. and K.C. Holmes, motors during the hydrolysis cycle differ, the structural unpublished). Nucleotide hydrolysis in both myosin and G homology of their catalytic cores suggests that movements of proteins requires both switch elements to be CLOSED. When conserved structural elements parallel one another. For kinesin is not bound to microtubules, the microtubule-binding example, analogous movements of the switch I and switch II region and switch II appear to be uncoupled from one another regions, which are highly conserved in myosin and the kinesin (see below), and it seems likely that the link between these motors, are expected to occur during nucleotide hydrolysis and regions is established upon microtubule binding, perhaps exchange in the two classes of motors. Such movements of the through the ordering of loop 11 by interactions with the switch regions have now been observed in crystal structures of microtubule. If binding to the microtubule causes switch II to both motors. In myosin, the movements change the structure of become even more OPEN, this, in combination with the the nucleotide-binding cleft, as in the G proteins (Sprang, already OPEN switch I, could result in destabilization and 1997): conversion of the OPEN form of myosin to the CLOSED release of the bound Mg·ADP. Subsequent binding of ATP form is accompanied by movement of switch II into the active might then pull switch II into a CLOSED position, in which a site (Fisher et al., 1995; Geeves and Holmes, 1999) (see Fig. hydrogen bond forms between the invariant switch II glycine 22 Journal of Cell Science 115 (1) (DLAGSE) and the γ-phosphate, thereby causing the switch II noted that the KIF1A structures solved were those of a chimera helix at the microtubule-binding site to change in conformation consisting of the neck linker from conventional kinesin fused and the neck linker to dock against (or undock from) the motor to the KIF1A catalytic core (Kikkawa et al., 2001). It is core (Schief and Howard, 2001). In myosin, the conserved salt therefore still uncertain exactly how the native KIF1A protein bridge between switch I and switch II stabilizes the CLOSED behaves, especially given its unusual nature. KIF1A has been conformation, which is necessary for ATP hydrolysis. The role reported to move in a biased diffusional manner along the of the salt bridge in the kinesins is less certain - the salt bridge microtubule (Okada and Hirokawa, 1999), rather than by is observed in some, but not all, motor-ADP crystal structures alternative binding of two heads like conventional kinesin and, in cases in which it forms, its geometry is imperfect. It is (Howard, 2001), and it is the alternative binding of the kinesin not clear whether a perfect salt bridge forms when the motor heads that the neck linker docking and undocking are thought is in a hydrolysis-competent state, since the salt bridge is not to regulate. Whether KIF1A is regulated by nucleotide binding formed in the closest structure yet to an ATP state, KIF1A- in the same way as conventional kinesin is unclear, given its AMP·PCP, or in the Kar3 salt-bridge mutants, which can be atypical mechanism of motility. interpreted to be transitions towards an ATP-bound state. If the Moreover, the structural changes observed in helices α4 and switch I and switch II elements in the hydrolysis-competent α5 of KIF1A-AMP·PCP are uncoupled to changes in the conformation of kinesin assume the same conformation as they nucleotide-binding site. In myosin, the C-terminal end of the do in myosin, the salt bridge would form with perfect relay helix is linked to the converter domain (Fig. 1) by strong geometry, leading to the formation of a hydrolysis-competent hydrophobic interactions and the two move as a rigid body - CLOSED conformation similar to that of myosin and the G when the relay helix moves, so does the converter, and a tightly proteins. However, the switch I region of kinesin has never coupled pathway of conformational change connects the relay been observed in a conformation that resembles switch I helix/converter to the nucleotide-binding site. If γ-phosphate is CLOSED of myosin and could function in a different manner, present at the active site, the relay helix is pulled up and in, the reflecting differences that exist between myosin and the helix bends, the converter rotates, and the lever arm swings. kinesins in the motor-filament state during the hydrolysis step. These movements occur even in detached motors not bound to Formation of the salt bridge, even with imperfect geometry, actin, in which they function to reprime the myosin head to when the motor is detached from the filament may stabilize the position it for binding to the next actin site. In the kinesins, unbound motor, whereas the absence of the salt bridge may be there is a short loop (L12) between helix α4, the relay helix, needed to permit changes in the active site when the motor is and helix α5 that differs from the large actin-binding domain bound to its filament. inserted at this site in myosin. The movement of the two helices appears to be coupled in the KIF1A-AMP·PCP structure (Kikkawa et al., 2001), but the link between the nucleotide- Further uncertainties binding site and the two helices, which form part of the motor In addition to the uncertainties regarding the rotation of the interface with the microtubule (Woehlke et al., 1997; Alonso relay helix that has been inferred from the KIF1A atomic et al., 1998; Hirose et al., 1999; Kikkawa et al., 2000), is weak structures and cryoEM analysis (Kikkawa et al., 2001), in motors not bound to microtubules. That is, the changes in questions regarding other aspects of the kinesin mechanism helices α4 and α5 at the microtubule-binding site of KIF1A remain. One of these concerns the docking of the neck linker appear to be unlinked to structural changes at the active site, onto the motor catalytic core. Several crystal structures now at least in the absence of microtubules. The Kar3 uncoupling show a visible neck linker docked onto the motor core forming and salt-bridge mutants (Yun et al., 2001) show that a pathway β-sheet structure with other β-strands of the catalytic core, of structural changes from helix α4 to the active site does exist rather than disordered and not visible in the model. But it is in the kinesins, but the structural details of this pathway are not still uncertain whether the docking occurs in the ADP or ATP evident from the KIF1A-AMP·PCP crystal structure. The state, since the neck linker has been observed docked onto the conformational changes described by Kikkawa et al. could motor core in motors bound either to ADP (Kozielski et al., therefore be a result of crystal contacts affecting the position 1997; Sack et al., 1997) or the ATP analogue, AMP·PCP of the helices, rather than the nucleotide state inducing a (Kikkawa et al., 2001). Kikkawa and co-workers interpret the conformational change (Kikkawa et al., 2001). This is also a previously reported kinesin-ADP structures with a docked neck likely explanation for the differences in the positions of helix linker (Kozielski et al., 1997; Sack et al., 1997) to be motor- α4 and α5 observed in the rat and human kinesin structures ATP structures. But the distinction between the two states is (Sack et al., 1999). Variable regions in different crystal not clear if the ADP and ATP conformations differ only slightly structures can be indicative of movements that occur in vivo, and if the states do not depend on the bound nucleotide because however. The observed movements of helices α4 and α5 could the energy transition between them is low, as is seen in myosin therefore approximate actual conformational changes that (Urbanke and Wray, 2001). occur during the kinesin cycle, although they may also differ Another way of phrasing this question is what is the significantly in detail and magnitude. The true ATP state of structure of the neck linker of kinesin in a conformationally kinesin remains to be visualized. distinct ATP state? A recent model proposes that the neck linker docks against the catalytic core when the motor binds to ATP and microtubules, directing the motor towards the plus Future directions end (Rice et al., 1999). However, evidence supporting this A major question that has not yet been answered either for model is controversial (see Schief and Howard, 2001), and myosin or the kinesins is what are the changes in conformation more work is needed to determine its validity. It should also be that occur in the strongly bound states of the motor? High- Kinesin: switch I & II and the motor mechanism 23 resolution atomic structures that reveal the changes in position Kliche, W., Fujita-Becker, S., Kollmar, M., Manstein, D. J. and Kull, F. J. and conformation of structural elements both at the motor (2001). Structure of a genetically engineered molecular motor. EMBO J. 20, filament-binding interface and the nucleotide-binding cleft are 40-46. Kozielski, F., Sack, S., Marx, A., Thormählen, M., Schönbrunn, E., Biou, needed to understand the motor mechanism. The fitting of V., Thompson, A., Mandelkow, E.-M. and Mandelkow, E. (1997). 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