Science 2000 Apr 7;288(5463):88-95.

Vale RD, Milligan RA.

Abstract

The microtubule-based kinesin motors and actin-based myosin motors generate motions associated with intracellular trafficking, cell division, and muscle contraction. Early studies suggested that these molecular motors work by very different mechanisms. Recently, however, it has become clear that kinesin and myosin share a common core structure and convert energy from adenosine triphosphate into protein motion using a similar conformational change strategy. Many different types of mechanical amplifiers have evolved that operate in conjunction with the conserved core. This modular design has given rise to a remarkable diversity of kinesin and myosin motors whose motile properties are optimized for performing distinct biological functions.

Kinesin and Mysosin

In addition to operating on different polymers, kinesin's motor domain is less than one-half the size of myosin's, and initial sequence comparisons failed to reveal any important similarities between these two motors. Their motile properties also appeared to be quite different. Conventional kinesin was found to be a highly processive motor that could take several hundred steps on a microtubule without detaching , whereas muscle myosin was shown to execute a single “stroke” and then dissociate (Fig. 1).

Kinesins are involved in membrane transport, mitosis and meiosis, messenger RNA and protein transport, ciliary and flagellar genesis, signal transduction, and microtubule polymer dynamics

Although muscle myosin is a dimer, the two heads appear to act independently, and only one head attaches to actin at a given time (Fig. 1). In the last few years, this model has received increasing experimental support. A battery of biochemical, biophysical, and structural methods have shown that the converter and lever arm domains rotate relative to the catalytic core in a nucleotide-dependent manner. Thus, muscle myosin moves the actin filament by the angular rotation of its long, rigid lever arm. Because the motor is strongly attached to actin only during this brief motion-producing phase of its enzymatic cycle, a single muscle myosin molecule cannot move continuously along its track.

The different mechanical strategies of conventional kinesin and muscle myosin reflect their distinct biological roles. Conventional kinesin transports small membrane organelles or protein complexes, and processive motion along the polymer enables efficient long-range transport using one or a few motor proteins. In contrast, muscle myosin operates in the context of a large array of motors, where it is essential for each one to attach, produce motion, and then detach quickly so as not to impede the actions of other motors producing force on the same filament.

ATP binding causes the forward motion of kinesin's neck linker (power stroke) but causes myosin to dissociate from actin and recock its lever arm (recovery stroke). Conversely, release of phosphate after ATP hydrolysis causes myosin to bind tightly to actin and swing its lever arm forward while it weakens kinesin's grip on the microtubule and detaches the neck linker.

Kinesin Movement

In contrast to muscle myosin, conventional kinesin walks methodically along a microtubule protofilament, stepping from one tubulin subunit to the next (distance of 80 Å), similar to a person walking across a pond along a row of stepping stonesRice et alhave shown that unidirectional motion is produced by a pronounced conformational change in kinesin's “neck linker,” a 15–amino acid region that is COOH-terminal to the catalytic core. The neck linker is mobile when kinesin is bound to microtubules in its nucleotide-free and adenosine diphosphate (ADP)–bound states. However, when the microtubule-bound kinesin binds an ATP analog, the neck linker becomes docked on the catalytic core with its COOH-terminus pointing toward the microtubule plus end. Thus, the energy associated with ATP binding drives a forward motion of the neck linker and any object attached to its COOH-terminus.

In a truncated kinesin monomer whose neck linker is attached directly to a bead or slide surface, the docking of the neck linker on the core will deliver a plus-end–directed pull on its cargo.monomer-based motility is nonprocessive and slow relative to the kinesin dimer . In the native kinesin dimer, the neck linker is connected to a coiled-coil dimerization domain, and neck linker motion in one head is conveyed to its partner to enable processive motion.The tight binding of the partner head to its new tubulin site then locks the step in place and produces a force that pulls kinesin's cargo forward by 80 Å.

For motors to produce forward motion, the ATP hydrolysis cycle must be linked to a conformational change cycle....polymer binding or strain on the mechanical element can affect enzymatic rates.

The nucleotide site: Swinging loops act as triggers

To change conformation between ATP- and ADP-bound states, motor proteins must sense the presence or absence of a single phosphate group. The identity of the “γ-phosphate sensor” became evident when myosin structures with and without bound ATP analogs were compared. The sensor consists of two loops, called switch I and switch II, which form hydrogen bonds with the γ-phosphate and also position a catalytic water and important side chains for cleavage of the β- to γ-phosphate bond. To accomplish these actions, the switch II loop operates like a spring-loaded gate that swings in by several angstroms to interact with the γ-phosphate and swings out when the γ-phosphate is released. The “ATP”-bound state of the sensor is also stabilized by a salt bridge that forms between the switch I and II loops. Kinesins contain switch I and II loops that are almost identical to those in myosinasimilar “γ-phosphate sensor” operates in the G protein superfamily as well indicating that the switch loops are ancient and predate the appearance of molecular motors.

Motor Functional Elements

The relay helix is the key structural element in the communication pathway linking the catalytic site, the polymer binding site, and the mechanical element in both kinesin and myosin. Myosin's relay helix undergoes a nucleotide-dependent conformational change that approximates the motion of a piston the motion of the switch II loop toward the γ-phosphate tilts and translates the relay helix along its axis toward the nucleotideConversely, without a nucleotide in the active site, switch II swings away and the relay helix moves to a “downstroke” position.

The relay helices in both myosins and kinesins undergo similar conformational changes that resemble the motions of a piston During the normal enzymatic cycle, the upstroke motion of the relay helix is a consequence of the inward motion of switch II toward the γ-phosphate, whereas the downstroke is initiated by phosphate release. Because the relay helix is long but incompressible, it is a perfect device for transmitting information from the nucleotide site to distant polymer binding and mechanical elements.

Binding

In kinesin, a 12–amino acid loop serves as the main microtubule binding element, whereas in myosin this loop is attached to a ∼140–amino acid actin binding domain.

The polymer binding site. Although the polymer binding interfaces of kinesin and myosin are different, they appear to communicate with the relay helix in a similar way. In kinesin, the microtubule binding loop begins COOH-terminal to the relay helix, doubles back, and makes extensive contacts with the relay helix (green loop in Fig. 2). Myosin has a comparable loop that follows a similar path, but, unlike kinesin, it does not contact actin directly. Instead, this loop plays an intermediate role by linking the relay helix to the actin binding elements.

These loop motions very likely affect polymer binding affinity in both motor proteins, but in opposite ways. Myosin in its ATP/ADP-Pi–bound state binds polymer more weakly than in its ADP-bound state, whereas in kinesin the opposite is true.



external image moz-screenshot.pngexternal image moz-screenshot-1.png
Figure 1 Models for the motility cycles of muscle myosin and conventional kinesin [see animation (23)]. (A) Muscle myosin. Frame 1: Muscle myosin is a dimer of two identical motor heads (catalytic cores are blue; lever arms in the prestroke ADP-Pi state are yellow), which are anchored to the thick filament (top) by a coiled coil (gray rod extending to the upper right). In the ADP-Pi–bound state, the catalytic core binds weakly to actin. Frame 2: One head docks properly onto an actin binding site (green). The two myosin heads act independently, and only one attaches to actin at a time. Frame 3: Actin docking causes phosphate release from the active site. The lever arm then swings to the poststroke, ADP-bound state (red), which moves the actin filament by ∼100 Å. Frame 4: After completing the stroke, ADP dissociates and ATP binds to the active site, which rapidly reverts the catalytic core to its weak-binding actin state. The lever arm will then recock back to its prestroke state (i.e., back to frame 1). (B) Conventional kinesin. Unlike myosin, the two heads of the kinesin dimer work in a coordinated manner to move processively along the track. The coiled coil (gray) extends toward the top and leads up to the kinesin cargo. Frame 1: Each catalytic core (blue) is bound to a tubulin heterodimer (green, β subunit; white, α subunit) along a microtubule protofilament (the cylindrical microtubule is composed of 13 protofilament tracks). To adopt this position, the neck linker points forward on the trailing head (orange; neck linker next to but not tightly docked to the core) and rearward on the leading head (red). ATP binding to the leading head will initiate neck linker docking. Frame 2: Neck linker docking is completed by the leading head (yellow), which throws the partner head forward by 160 Å (arrow) toward the next tubulin binding site. Frame 3: After a random diffusional search, the new leading head docks tightly onto the binding site, which completes the 80 Å motion of the attached cargo. Polymer binding also accelerates ADP release, and during this time, the trailing head hydrolyzes ATP to ADP-Pi. Frame 4: After ADP dissociates, an ATP binds to the leading head and the neck linker begins to zipper onto the core (partially docked neck indicated by the orange color). The trailing head, which has released its Pi and detached its neck linker (red) from the core, is in the process of being thrown forward. The surface features of the motors and filaments were rendered by G. Johnson (fiVth media: www.fiVth.com) using the programs MolView, Strata Studio Pro, and Cinema 4D (also for Figs. 4 and 5). Protein Data Bank (PDB) files used throughout the figures are as follows: ADP-AlF4 − smooth muscle myosin [prestroke, yellow: 1BR2 (16)], nucleotide-free chicken skeletal myosin [poststroke, red: 2MYS (14)], human conventional kinesin [prestroke, red: 1BG2 (6)], and rat conventional kinesin [poststroke, yellow: 2KIN (40)]. Scale bars, 60 Å (A) and 40 Å (B).
Figure 1 Models for the motility cycles of muscle myosin and conventional kinesin [see animation (23)]. (A) Muscle myosin. Frame 1: Muscle myosin is a dimer of two identical motor heads (catalytic cores are blue; lever arms in the prestroke ADP-Pi state are yellow), which are anchored to the thick filament (top) by a coiled coil (gray rod extending to the upper right). In the ADP-Pi–bound state, the catalytic core binds weakly to actin. Frame 2: One head docks properly onto an actin binding site (green). The two myosin heads act independently, and only one attaches to actin at a time. Frame 3: Actin docking causes phosphate release from the active site. The lever arm then swings to the poststroke, ADP-bound state (red), which moves the actin filament by ∼100 Å. Frame 4: After completing the stroke, ADP dissociates and ATP binds to the active site, which rapidly reverts the catalytic core to its weak-binding actin state. The lever arm will then recock back to its prestroke state (i.e., back to frame 1). (B) Conventional kinesin. Unlike myosin, the two heads of the kinesin dimer work in a coordinated manner to move processively along the track. The coiled coil (gray) extends toward the top and leads up to the kinesin cargo. Frame 1: Each catalytic core (blue) is bound to a tubulin heterodimer (green, β subunit; white, α subunit) along a microtubule protofilament (the cylindrical microtubule is composed of 13 protofilament tracks). To adopt this position, the neck linker points forward on the trailing head (orange; neck linker next to but not tightly docked to the core) and rearward on the leading head (red). ATP binding to the leading head will initiate neck linker docking. Frame 2: Neck linker docking is completed by the leading head (yellow), which throws the partner head forward by 160 Å (arrow) toward the next tubulin binding site. Frame 3: After a random diffusional search, the new leading head docks tightly onto the binding site, which completes the 80 Å motion of the attached cargo. Polymer binding also accelerates ADP release, and during this time, the trailing head hydrolyzes ATP to ADP-Pi. Frame 4: After ADP dissociates, an ATP binds to the leading head and the neck linker begins to zipper onto the core (partially docked neck indicated by the orange color). The trailing head, which has released its Pi and detached its neck linker (red) from the core, is in the process of being thrown forward. The surface features of the motors and filaments were rendered by G. Johnson (fiVth media: www.fiVth.com) using the programs MolView, Strata Studio Pro, and Cinema 4D (also for Figs. 4 and 5). Protein Data Bank (PDB) files used throughout the figures are as follows: ADP-AlF4 − smooth muscle myosin [prestroke, yellow: 1BR2 (16)], nucleotide-free chicken skeletal myosin [poststroke, red: 2MYS (14)], human conventional kinesin [prestroke, red: 1BG2 (6)], and rat conventional kinesin [poststroke, yellow: 2KIN (40)]. Scale bars, 60 Å (A) and 40 Å (B).
Figure 2 Atomic structures of the myosin and kinesin motor domains and conformational changes triggered by the relay helix. The motor domains of smooth muscle myosin (ADP-AlF4 −) and rat conventional kinesin are shown in the upper panels (both structures are proposed in this article to represent an “ATP/ADP-Pi” conformation). The common structural elements in the catalytic cores are highlighted in blue, the relay helices and polymer loops are dark green, and the mechanical elements [neck linker for kinesin; the converter and lever arm domains for myosin (17)] are yellow, and nucleotide is shown as an off-white space-filling model. The complete lever arm was not obtained in the smooth muscle myosin crystal structure, but was added here by atomic modeling for purposes of illustration. The kinesin and myosin structures are shown in the same orientation (by superimposing their P-loops) and are displayed as viewed from the polymer surface (∼90° clockwise view of the motors shown in Fig. 4). The similar positions of the relay helices and the mechanical elements in kinesin and myosin can be seen in relation to their common cores. The details and similarity of the conformational changes elicited by the relay helix are shown in the lower panels. For both myosin and kinesin, the relay helix undergoes similar motions during the transition from the “ATP/ADP-Pi–bound” state (dark green; upstroke) to the “ADP/nucleotide-free state” (light green; downstroke). A loop following the relay helix (in greens), which likely controls polymer affinity, and the mechanical elements [“ATP/ADP-Pi” (yellow) and “ADP/nucleotide-free” states (red)] both shift their positions in response to the relay helix motion. With the relay helix in an upstroke position, kinesin Ile325 (orange space-filling residue) in the neck linker inserts into a pocket on the catalytic core [see (48)]. This event is proposed to trigger the docking of the rest of the neck linker onto the catalytic core. In the downstroke position, the relay helix (light green) occludes the pocket, which pushes Ile325 out, and the neck linker becomes disordered (red dots). Nucleotide-free chicken skeletal myosin (red, light green) and ADP-AlF4 − smooth muscle myosin (yellow, dark green) are shown on the left, and human conventional kinesin (red, light green) and rat conventional kinesin (yellow, dark green) are shown on the right.
Figure 2 Atomic structures of the myosin and kinesin motor domains and conformational changes triggered by the relay helix. The motor domains of smooth muscle myosin (ADP-AlF4 −) and rat conventional kinesin are shown in the upper panels (both structures are proposed in this article to represent an “ATP/ADP-Pi” conformation). The common structural elements in the catalytic cores are highlighted in blue, the relay helices and polymer loops are dark green, and the mechanical elements [neck linker for kinesin; the converter and lever arm domains for myosin (17)] are yellow, and nucleotide is shown as an off-white space-filling model. The complete lever arm was not obtained in the smooth muscle myosin crystal structure, but was added here by atomic modeling for purposes of illustration. The kinesin and myosin structures are shown in the same orientation (by superimposing their P-loops) and are displayed as viewed from the polymer surface (∼90° clockwise view of the motors shown in Fig. 4). The similar positions of the relay helices and the mechanical elements in kinesin and myosin can be seen in relation to their common cores. The details and similarity of the conformational changes elicited by the relay helix are shown in the lower panels. For both myosin and kinesin, the relay helix undergoes similar motions during the transition from the “ATP/ADP-Pi–bound” state (dark green; upstroke) to the “ADP/nucleotide-free state” (light green; downstroke). A loop following the relay helix (in greens), which likely controls polymer affinity, and the mechanical elements [“ATP/ADP-Pi” (yellow) and “ADP/nucleotide-free” states (red)] both shift their positions in response to the relay helix motion. With the relay helix in an upstroke position, kinesin Ile325 (orange space-filling residue) in the neck linker inserts into a pocket on the catalytic core [see (48)]. This event is proposed to trigger the docking of the rest of the neck linker onto the catalytic core. In the downstroke position, the relay helix (light green) occludes the pocket, which pushes Ile325 out, and the neck linker becomes disordered (red dots). Nucleotide-free chicken skeletal myosin (red, light green) and ADP-AlF4 − smooth muscle myosin (yellow, dark green) are shown on the left, and human conventional kinesin (red, light green) and rat conventional kinesin (yellow, dark green) are shown on the right.



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Figure 4  A model for the “power strokes” of myosin and kinesin motors complexed with their polymer tracks. In myosin, a ∼100 Å motion of the lever arm domain is generated when the motor undergoes a transition from an ADP-Pi–bound state to an ADP/nucleotide-free conformation (78). This figure was generated by superimposing the structures of smooth muscle myosin (ADP-AlF4 −) and the nucleotide-free chicken skeletal myosin. Shown are the converter/lever arm positions in ADP-Pi (yellow) and nucleotide-free (red) states, the similar catalytic cores (blue), and the actin filament (gray; “pointed end” toward the top). In the motility cycle of a kinesin dimer (only one head shown here) along a microtubule (a single protofilament is shown in gray; “plus end” toward the top), the neck linker swings from a rearward-pointing position (ADP/nucleotide-free; red) to a forward-pointing position (ATP/ADP-Pi; yellow). The “ATP/ADP-Pi” state is rat conventional kinesin, whereas the “ADP/nucleotide-free” position of the neck linker was modeled on the basis of cryo-electron microscopy from Rice et al. (26). Myosin and kinesin structures were superimposed using their P-loops, showing that they bind in similar orientations to their tracks. (Note: The actin filament runs parallel to the plane of the image, but the microtubule is tilted ∼20° with respect to the plane of the image.) Although the mechanical elements are similarly positioned in kinesin and myosin, the power strokes occur in opposite directions (arrows) because of the different polymer binding cycles of the two motors (see text for details). Scale bar, 80 Å.
Figure 4 A model for the “power strokes” of myosin and kinesin motors complexed with their polymer tracks. In myosin, a ∼100 Å motion of the lever arm domain is generated when the motor undergoes a transition from an ADP-Pi–bound state to an ADP/nucleotide-free conformation (78). This figure was generated by superimposing the structures of smooth muscle myosin (ADP-AlF4 −) and the nucleotide-free chicken skeletal myosin. Shown are the converter/lever arm positions in ADP-Pi (yellow) and nucleotide-free (red) states, the similar catalytic cores (blue), and the actin filament (gray; “pointed end” toward the top). In the motility cycle of a kinesin dimer (only one head shown here) along a microtubule (a single protofilament is shown in gray; “plus end” toward the top), the neck linker swings from a rearward-pointing position (ADP/nucleotide-free; red) to a forward-pointing position (ATP/ADP-Pi; yellow). The “ATP/ADP-Pi” state is rat conventional kinesin, whereas the “ADP/nucleotide-free” position of the neck linker was modeled on the basis of cryo-electron microscopy from Rice et al. (26). Myosin and kinesin structures were superimposed using their P-loops, showing that they bind in similar orientations to their tracks. (Note: The actin filament runs parallel to the plane of the image, but the microtubule is tilted ∼20° with respect to the plane of the image.) Although the mechanical elements are similarly positioned in kinesin and myosin, the power strokes occur in opposite directions (arrows) because of the different polymer binding cycles of the two motors (see text for details). Scale bar, 80 Å.


Figure 5 A critical two-head–bound intermediate for processive movement requires mechanical elements of very different sizes in myosin V and kinesin. Because of the helical arrangement of subunits in an actin filament and the linear arrangement of subunits in a microtubule protofilament, equivalent motor binding sites (green subunits) occur every 360 Å along actin (“pointed end” toward the top) and every 80 Å along the microtubule protofilament (“plus end” toward the top). The direction of motion is shown by the arrows next to the coiled-coil domains (gray). The two heads of myosin V and conventional kinesin can span the distance between these binding sites only if the trailing head is in a poststroke state [“ADP/nucleotide-free” for myosin V (red); “ATP/ADP-Pi” for kinesin (yellow)] and the forward head is in a prestroke state [“ATP/ADP-Pi” for myosin V (yellow); “ADP/nucleotide-free” for kinesin (red)]. Myosin V is modeled here by extending the lever arm crystal structures of chicken skeletal muscle (upper head; red lever arm) and chicken smooth muscle myosin (ADP-AlF4 −; lower head, yellow lever arm) to include six light chains. The two myosin catalytic cores (blue) are docked in same orientation (by aligning their P-loops), but appear slightly different because of structural changes in the core associated with these two nucleotide states. Scale bar, 40 Å.
Figure 5 A critical two-head–bound intermediate for processive movement requires mechanical elements of very different sizes in myosin V and kinesin. Because of the helical arrangement of subunits in an actin filament and the linear arrangement of subunits in a microtubule protofilament, equivalent motor binding sites (green subunits) occur every 360 Å along actin (“pointed end” toward the top) and every 80 Å along the microtubule protofilament (“plus end” toward the top). The direction of motion is shown by the arrows next to the coiled-coil domains (gray). The two heads of myosin V and conventional kinesin can span the distance between these binding sites only if the trailing head is in a poststroke state [“ADP/nucleotide-free” for myosin V (red); “ATP/ADP-Pi” for kinesin (yellow)] and the forward head is in a prestroke state [“ATP/ADP-Pi” for myosin V (yellow); “ADP/nucleotide-free” for kinesin (red)]. Myosin V is modeled here by extending the lever arm crystal structures of chicken skeletal muscle (upper head; red lever arm) and chicken smooth muscle myosin (ADP-AlF4 −; lower head, yellow lever arm) to include six light chains. The two myosin catalytic cores (blue) are docked in same orientation (by aligning their P-loops), but appear slightly different because of structural changes in the core associated with these two nucleotide states. Scale bar, 40 Å.