CHAPTER 36
Motor Proteins
M olecular motors use adenosine triphosphate (ATP)
hydrolysis to power movements of subcellular com
ponents, such as organelles and chromosomes, along
the two polarized cytoskeletal fibers: actin filaments
and microtubules. No motors are known to move on
intermediate filaments. Motor proteins also produce
force locally within the network of cytoskeletal poly
mers, which transmits these forces to determine
the shape of each cell and, ultimately, the architecture
of tissues and whole organisms. Chapters 37 to 39
and 44 illustrate how motors move cells and their
internal parts.
Just three families of motor proteinsmyosin,
kinesin, and dyneinpower most eukaryotic cellular
movements (Fig. 36.1 and Table 36.1). During evolution,
myosin, kinesin, and Ras family guanosine triphospha
tases (GTPases) appear to have shared a common ances
tor (Fig. 36.1), whereas dynein is a member of the AAA
adenosine triphosphatase (ATPase) family (Box
36.1). Although the ancestral genes appeared in prokary
otes, and prokaryotes have homologs of both actin and
tubulin, none of these motor proteins has been found
in prokaryotes. Over time, gene duplication and diver
gence in eukaryotes gave rise to multiple genes for
myosin, dynein, and kinesin, each encoding proteins
Primodial NTPase
Primodial GTPase Primodial myosin
Many Many
contemporary contemporary
GTPases myosins
FIGURE 36.1 Evolution of myosin, kinesin, and dynein adenosine triphosphatase (ATPase) motors from genes that encoded two primordial
proteins that bound and hydrolyzed nucleoside triphosphates. Gene duplication and divergence created genes for many contemporary motors.
with specialized functions. Even the slimmed down
genome of budding yeast includes genes for five myosins,
six kinesins, and one dynein. Table 36.1 lists other
protein machines that produce molecular movements
during protein and nucleic acid synthesis, proton
pumping, and bacterial motility.
Motor proteins have two parts: a motor domain that
uses ATP hydrolysis to produce movements and a tail
that allows the motors to self-associate and/or to bind
particular cargo. Within the three families, the tails are
more diverse than the motor domains, allowing for spe
cialized functions of each motor isoform.
All motor proteins are enzymes that convert chemical
energy stored in ATP into molecular motion to produce
force upon an associated cytoskeletal polymer (Fig.
36.2). If the motor is anchored, the polymer may move.
If the polymer is anchored, the motor and any attached
cargo may move. If both are anchored, the force
stretches elastic elements in the molecules transiently,
but nothing moves, and the energy is lost as heat. Cells
use all these options (see Chapters 37 to 39).
Biochemists originally discovered and purified these
motors using enzyme (eg, ATP hydrolysis) or in vitro
motility assays (Fig. 36.11). With the prototype motors
identified, investigators found further examples and
Primodial kinesin Primodial AAA ATPase
Many Dyneins Other AAA-
contemporary ATPases
kinesins
623
624 SECTION IX n Cytoskeleton and Cellular Motility

TABLE 36.1 Mechanochemical Enzymes and Other Proteins That Produce Movements
Families Track Direction Cargo Energy
ATPases
Myosins
Muscle myosin Actin Barbed end Myosin filament ATP
Myosin II Actin Barbed end Myosin, actin ATP
Myosin I Actin Barbed end Membranes ATP
Myosin V Actin Barbed end Organelles ATP
Myosin VI Actin Pointed end Endocytic vesicles ATP
Dyneins
Axonemal Microtubule Minus end Microtubules ATP
Cytoplasmic Microtubule Minus end Membranes, chromosomes ATP
Kinesins
Kinesin-1 Microtubule Plus end Membranes, intermediate filaments ATP
Kinesin-14 Microtubule Minus end ? Microtubules ATP
Other Mechanochemical Systems
Polymerases and Helicases
Ribosome mRNA 5 to 3 None GTP
DNA polymerase DNA 5 to 3 None ATP
RNA polymerase DNA 5 to 3 None ATP
CMG DNA helicase DNA DNA ATP
RNA helicases RNA RNA ATP
Conformational System
Spasmin/centrin None None Cell, basal body Ca2+
Polymerizing Systems
Actin filaments None Barbed end Membranes ATP
Microtubules None Plus end Chromosomes GTP
Worm sperm MSP None Not polar Cytoskeleton
Rotary Motors
Bacterial flagella None Bidirectional Cell H+ or Na+ gradient
F-type ATPase None Bidirectional None H+ or ATP
V-type ATPase pump None None ATP

ATP, adenosine triphosphate; ATPase, adenosine triphosphatase; GTP, guanosine triphosphate; mRNA, messenger RNA; MSP, major sperm protein. The
terms barbed and pointed end refer to the appearance of actin filaments decorated with a myosin fragment (Fig. 33.8).

variant isoforms of each by purifying proteins, cloning
complementary DNAs (cDNAs), sequencing genomes, or
genetic screening.
Myosins
Myosins are the only motors that are known to use actin
filaments as tracks. Members of the diverse myosin
superfamily arose from a common ancestor and share a
motor unit called a myosin head that produces force
on actin filaments (Fig. 36.3). One or two heads are
attached to various types of tails that are adapted
for diverse purposes, including polymerization into
filaments, binding membranes, and interacting with
various cargos.
Myosin heads consist of two parts. A catalytic domain
at the N-terminus of the myosin heavy chain binds and
hydrolyzes ATP and interacts with actin filaments. Light
chain domains consist of an -helical extension of the
heavy chain from the catalytic domain associated with
one to seven light chains. Light chains are related to
calmodulin (see Fig. 3.12), which also serves as a light
chain for many myosins.
Myosin Mechanochemistry
Myosin was discovered in skeletal muscle and used to
establish general principles that apply, with interesting
variations, to energy transduction by all myosins. Muscle
myosin is responsible for the forceful contraction of
skeletal muscle (see Chapter 39). Like other types of
myosin-II, it has two heads on a long tail formed from an
-helical coiled-coil. These tails polymerize into bipolar
filaments (see Figs. 5.7 and 39.6).
The head of muscle myosin was originally isolated as
a proteolytic fragment called subfragment-1 (Fig. 36.3).
The N-terminal 710 residues of the heavy chain form the
globular catalytic domain. The nucleotide binding site
in the core of the catalytic domain is formed by a -sheet

Generic motor with stretched spring
Force
Cytoskeletal
fiber
Motor
ATP Spring
ADP + Pi
Force Support or
cargo
Resulting movement with anchored motor
Fiber moves
Support
Resulting movement with anchored fiber
Motor and cargo move
Cargo
Result with anchored fiber and anchored motor
Force
Force
Support
Spring stretched, force transmitted through
fiber to anchoring sites, no movement,
energy lost as heat
FIGURE 36.2 GENERAL FEATURES OF ATPase MOTORS.
Motors bind stably to a support or cargo and transiently to a cytoskel-
etal fiber (actin filament or microtubule). Energy liberated by adenosine
triphosphate (ATP) hydrolysis produces force to stretch an elastic
element somewhere in the physical connection between the cargo and
the cytoskeletal fiber. The resulting motion depends on whether the
force in the spring exceeds the resistance of the fiber or the cargo.
flanked by -helices with a topology similar to Ras
GTPases (see Fig. 4.6) despite little sequence similarity.
The -phosphate of ATP inserts deeply into the nucleotide-
binding site with the adenine exposed on the surface.
Actin binds more than 4nm away from the nucleotide
on the other side of the head. A region of the heavy chain
called the converter subdomain is attached to the light-
chain domain composed of an essential light chain and
a regulatory light chain wrapped around and stabilizing
a long -helix formed by the heavy chain. The inter
action of light chains with the heavy chain -helix
CHAPTER 36 n Motor Proteins 625
BOX 36.1 AAA Adenosine Triphosphatases
The common ancestor of life on the earth had a gene for
a versatile adenosine triphosphate (ATP)-binding domain.
Through gene duplication and divergence this progenitor
gave rise to the AAA family of adenosine triphosphatases
(ATPases) in all branches of the phylogenic tree. Given
the remarkable variety of functions of the contemporary
proteins, the name ATPases Associated with Diverse
Activities is apt. The family now includes regulatory
subunits of proteasomes (see Fig. 23.8); proteases from
prokaryotes, chloroplasts, and mitochondria; Hsp100
protein folding chaperones; dynein microtubule motors
(Fig. 36.14); the microtubule severing protein katanin (see
Fig. 34.8); activators of origins of replication (including
ORC1, 4, and 5 and Mcm-7 [see Fig. 42.8]); clamp loader
proteins for DNA polymerase processivity factors (see
Fig. 42.12); two proteins required for peroxisome bio
genesis (see Table 18.1); and proteins involved in vesicular
traffic such as NSF (the N-ethylmaleimide-sensitive factor
[see Fig. 21.15]).
AAA domains have a common fold with a catalytic site
that binds and hydrolyzes ATP. A Walker A motif of
conserved residues interacts with the - and -phosphates
of ATP, and Walker B motif residues participate in ATP
hydrolysis. Many AAA ATPases form ring-shaped hexamers
of identical subunits or up to six different AAA subunits
although the dynein heavy chain has six AAA domains in
one large polypeptide. Often, an arginine residue from the
adjacent subunit in the hexamer inserts into the active site
and facilitates conformational changes in response to ATP
binding and release of the -phosphate.
A Actin-binding B Actin-binding
site site
Active Active
site site
ELC ELC
RLC RLC
FIGURE 36.3 ATOMIC STRUCTURE OF THE HEAD OF
MUSCLE MYOSIN. A, Ribbon drawing of the polypeptide back-
bones. B, Space-filling model. Heavy chain residues 4204 (green);
heavy chain residues 216626 (red); heavy chain residues 647843
(purple): essential light chain (ELC [yellow]); regulatory light chain (RLC
[orange]). The myosin light chains consist of two globular domains
connected by an -helix, like calmodulin and troponin C. (For refer-
ence, see Protein Data Bank [PDB; www.rcsb.org] file 2MYS.)
626 SECTION IX n Cytoskeleton and Cellular Motility

Beginning Pointed end

A B C D of stroke

Catalytic
domain

End of
stroke

FIGURE 36.4 ACTIN FILAMENTS DECORATED WITH MYOSIN HEADS. A, Electron micrograph of frozen-hydrated actin filaments fully
occupied with myosin heads. B, Three-dimensional reconstruction from electron micrographs of an actin filament saturated with myosin heads.
C, Superimposition of atomic models of the actin filament and one myosin head on the reconstruction of the decorated filament (blue cage-like
surface). D, Space-filling atomic model of an actin filament with one attached muscle myosin head showing the light-chain domain in two posi-
tions: (1) the end of the power stroke as observed in the absence of ATP (blue), and (2) the postulated beginning of the power stroke (pink)
deduced from X-ray structures of isolated heads and spectroscopic studies. The catalytic domain (red) is fixed in one position on actin (yellow).
(Courtesy R. Milligan, Scripps Research Institute, La Jolla, CA.)

resembles calmodulin binding to its target proteins (see
Fig. 3.12).
Myosin heads bind tightly and rigidly to actin fila
ments in the absence of ATP. This is called a rigor
complex, because it forms in muscle during rigor mortis
when ATP is depleted after death. Myosin heads bound
along an actin filament form a polarized structure, resem
bling a series of arrowheads when viewed from the side
(Fig. 36.4). The heads bind at an angle and wrap around
the filament. Their orientation defines the barbed and
pointed ends of the actin filament (see Fig. 33.8). All
known myosins, except myosin-VI, move toward the
barbed end of the filament.
The atomic structures of the myosin head and
actin filament fit nicely into the three-dimensional struc
ture of the decorated filament determined by electron
microscopy, providing the structural starting point
for understanding the mechanics of force production
(Fig. 36.4). Each myosin head contacts two adjacent
actin subunits.
Actomyosin Adenosine Triphosphatase Cycle
Myosin uses energy from ATP hydrolysis to move actin
filaments, so an appreciation of the mechanism requires
an understanding of the biochemical steps along the
reaction pathway. Fig. 36.5A looks intimidating, but
working through it one step at a time reveals its logic
and simplicity. Note that the mechanism consists of two
parallel lines of chemical intermediates. First, consider
the bottom line showing the reactions that explain why
myosin alone turns over ATP remarkably slowly, at a rate
of only approximately 0.02s1:
Step 1. At physiological concentrations of ATP, myosin
binds ATP in less than 1 millisecond. Energy from ATP
binding allows a conformational change in the myosin
that can be detected by a change in the intrinsic fluo
rescence of the protein itself.
Step 2. The enzyme catalyzes the hydrolysis of ATP. This
reaction is moderately fast (>100s1) and readily revers
ible. The equilibrium constant for hydrolysis on the
enzyme is near 1, so each ATP is hydrolyzed to adenos
ine diphosphate (ADP) and inorganic phosphate and
the triphosphate is resynthesized several times before
the products dissociate from the enzyme. ATP splitting
provides energy for a second conformational change,
reflected in a further increase in the fluorescence of
the myosin. These conformational changes reorient
the converter subdomain and the light chain domain
poised to undergo the molecular rearrangements that
subsequently produce movement.
Step 3. Inorganic phosphate (P) slowly dissociates from
the active site (at a rate of approximately 0.02s1) by
escaping through a narrow back door on the far side
of the enzyme. This is the rate-limiting step in the
pathway. The loss of phosphate is coupled to confor
mational changes that return myosin toward its basal
state. The phosphate dissociation step has the largest
negative free energy change, so it is presumed that
energy derived from ATP binding and hydrolysis and
stored in conformational changes in the myosin head
is used to do work or dissipated as heat at this point
in the reaction pathway.
Step 4. Once phosphate dissociates, ADP leaves rapidly
from the front door.
To summarize, in the absence of actin filaments, ATP
binds rapidly to myosin and is rapidly but reversibly split,
and the products slowly dissociate from the active site.
The overall cycle of the enzyme is limited by the slow
conformational change coupled to phosphate dissocia
tion. Energy derived from ATP binding and hydrolysis is
 CHAPTER 36 n Motor Proteins 627

A Strong Weak Strong

AM AM*T AM**DP AMD AM Myosin bound to actin

1 2 3 4
1 2 3 4
M M*T M**DP MD M Free myosin

1000 s-1 100 s-1 10 s-1 1 s-1 0.1 s-1

B P

ADP
ATP

ADP ADP
B + Pi
+ Pi

Pi

Rapid equilibrium Phosphate ADP dissociates ATP binding

free and bound dissociates Head dissociates
Light chain
domain rotates

ATP hydrolysis
FIGURE 36.5 MYOSIN ATPase MECHANISMS. A, A diagram of the actomyosin ATPase cycle of striated muscle myosin-II showing the
actin filament (A), myosin head (M), ATP (T), adenosine diphosphate (ADP) (D), and inorganic phosphate (P). Transient-state kinetics revealed the
major chemical intermediates and the rate constants for their transitions. Arrow sizes are proportional to the rates of the reactions, with second-
order reactions adjusted for physiological concentrations of reactants. One or two asterisks indicate conformational changes in the myosin head
induced by ATP binding and hydrolysis. Myosin without nucleotide (M) and myosin with ADP (MD) bind much more tightly to actin filaments than
do AMT and AMDP. The weakly bound AMT and AMDP intermediates are in a rapid equilibrium with free MT and MDP. The beige shading shows
the main pathway through the reaction. B, The postulated force-producing structural changes in the orientation of the light-chain domain (purple
and blue) coupled to the myosin ATPase cycle. (B, Data from R. Vale, University of California, San Francisco, and R. Milligan, Scripps Research
Institute, La Jolla, CA.)

used for a conformational change in the myosin head
that is dissipated when phosphate dissociates.
The upper line in Fig. 36.5A shows myosin associated
with an actin filament. The chemical intermediates
are the same, but some of the key rate constants differ
for the actin-bound and free myosin. Steps 1 and 2 are
similar to those of free myosin, but step 3the dissocia
tion of phosphateis much faster when a head is bound
to an actin filament. As a result, myosin bound to actin
traverses the ATPase cycle approximately 200 times
faster than myosin free in solution, and ATP hydrolysis
becomes the rate-limiting step. This effect of actin is
referred to as actin activation of the myosin ATPase. A
practical advantage of this mechanism is that the ATPase
cycle is essentially turned off unless the head interacts
with an actin filament.
Finally, consider the vertical arrows representing tran
sitions between bound and free states of each myosin
chemical intermediate. All myosin intermediates bind
rapidly to actin filaments, but the dissociation rate
constants vary over a wide range depending on the
nucleotide that is bound to the active site of the myosin.
Myosin with no nucleotide or with bound ADP alone
dissociates very slowly and therefore binds tightly to
actin filaments. Myosin with bound ATP or ADP+Pi
dissociates rapidly from actin, so these states bind
actin weakly.
One cycle of ATP hydrolysis takes about 50 millisec
onds, but a single pathway cannot be drawn through the
reaction mechanism of ATP, myosin, and actin owing to
the rapid equilibrium of myosin intermediates (MT and
MDP) hopping on and off actin filaments on a millisec
ond time scale. Starting with AM, ATP binds very rapidly
and sets up a rapid, four-way equilibrium including AMT,
MT, AMDP, and MDPthe major intermediates during
steady-state ATP turnover in muscle (see Chapter 39).
Because the products of ATP hydrolysis dissociate much
more rapidly from AMDP than from MDP, the favored
pathway out of this four-way equilibrium is through
AMDP to AMD and back to AM. The overall ATPase rate
depends on the actin concentration, which determines
the fraction of myosin heads bound to actin in the
628 SECTION IX n Cytoskeleton and Cellular Motility
AMDP state. At the high actin concentrations in cells, a
significant fraction of myosin heads is associated with
actin (approximately 10% in contracting muscle), but
each molecule continues to exchange on and off
actin filaments.
Transduction of Chemical Energy Into
Molecular Motion
Myosin heads produce force during the transition from
the AMDP state to the AMD and AM states. Production
of force at this step makes sense for two reasons: First,
the large free-energy difference between AMDP and
AMD provides sufficient energy to produce force;
second, the force-producing AMD and AM intermediates
bind tightly to actin, so any force between the motor and
the actin track is not dissipated. However, for many
myosins, including skeletal muscle myosin, these force-
producing states occupy a small fraction of the whole
ATPase cycle. The fraction of the time in force producing
states is called the duty cycle. ADP dissociates rapidly
from AMD, and ATP binds rapidly to AM, dissociating
myosin from the actin filament and initiating another
ATPase cycle.
Fifty years of research using a combination of mechan
ical measurements, static atomic structures of myosin
heads with various bound nucleotides, and spectro
scopic observations of contracting muscle revealed the
structural basis for the conversion of free energy into
force: a dramatic conformational change in the orienta
tion of the light-chain domain associated with phosphate
dissociation (Fig. 36.5B).
Elegant mechanical experiments measured the size
of the mechanical step produced by a myosin during
one cycle of ATP hydrolysis. These experiments on
live muscles first suggested that each cycle of ATP
hydrolysis moves an actin filament approximately 5
to 10nm relative to myosin. Now one may observe
myosin moving single actin filaments by fluorescence
microscopy. An array of myosin heads attached to a
microscope slide can use ATP hydrolysis to push actin
filaments over the surface (Fig. 36.6AC). Assays with
single myosin molecules show that each cycle of ATP
hydrolysis can move an actin filament up to 5 to 15nm
and develop a force of about 3 to 7 piconewtons (pN)
(Fig. 36.6D). At low ATP concentrations, the interval
between the force-producing step and the binding
of the next ATP is relatively long, so single steps can
be observed.
Further insights emerged from biophysical studies of
muscle and purified proteins using x-ray diffraction (see
Fig. 39.11), electron microscopy, electron spin reso
nance spectroscopy, and fluorescence spectroscopy.
These experiments showed that the light-chain domain
pivots around a fulcrum, the converter subdomain within
the catalytic domain, which is stationary relative to the
actin filament. For example, spectroscopic probes on
light chains revealed a change in orientation when
muscle is activated to contract, whereas probes on the
catalytic domain do not rotate. Crystal structures of
myosin heads with various bound nucleotides and nucle
otide analogs show that the light-chain domain can pivot
up to 90 degrees (Fig. 36.4D). The light-chain domain is
bent more acutely in the AMT and AMDP intermediates
and pivots to a more extended orientation when phos
phate dissociates (Fig. 36.3). ADP dissociation extends
this rotation of some classes of myosin. Consistent with
rotation of the light-chain domain producing movement,
the rate of actin filament gliding in an in vitro assay is
proportional to the length of the light-chain domain. The
observed range of orientations of the light-chain domain
relative to the catalytic domain can account for the
observed step size of 10nm for muscle myosin. Some
aspects of these conformational changes and their rela
tion to phosphate release are similar to Ras family
GTPases (see Fig. 4.6).
Rotation of the light-chain domain is believed to
produce movement indirectly in the sense that force-
producing intermediates stretch elastic elements in the
system. This mechanism is represented by a spring
in Fig. 36.2. The elastic elements in the myosin-actin
complex are most likely to be mainly in the myosin head,
with small contributions from the actin and myosin fila
ments. Movement of the light-chain domain tensions the
spring transiently in the AMD and AM states. Dissociation
of ADP and rebinding of ATP to the AM intermediate
reverts the system to the rapid equilibrium of mostly
dissociated weakly bound intermediates. Any force left
in the spring is lost as soon as the head dissociates from
the actin filament.
The actual motion produced depends on the mechani
cal resistance in the system (Fig. 36.2). If both myosin
and actin are fixed, elastic elements are stretched for the
life of the force-producing states (AMD and AM), and the
energy is lost as heat when the head dissociates. This
happens when one tries to lift an immovable object. If
the resistance is less than the force in the stretched
elastic elements, the actin filament moves relative to
myosin, as in muscle contraction. The distance moved
in each step depends on the resistance, as the spring
stops shortening when the forces are balanced.
Myosin Superfamily
Eukaryotes have 35 classes of myosin and many other
examples of unique myosins in single species (Fig. 36.7).
All arose from a gene similar to myosin-I in the last
eukaryote common ancestor more than a billion years
ago. The primordial gene then gave rise to the gene for
myosin-V, so this class is also widespread. Gene duplica
tion, divergence, and acquisition of extra domains pro
duced many other myosin genes encoding proteins
specialized for particular biological functions made pos
sible by variations of the mechanochemical ATPase cycle
 CHAPTER 36 n Motor Proteins 629

A B D

Actin
filament

Step

Return
Myosin
C

40
Actin Events

Distance (nm)
20

ADP Myosin
+ Pi 0
ATP

20
0 0.5 1.0 1.5
GLASS Time (s)

FIGURE 36.6 IN VITRO MOTILITY ASSAYS WITH PURIFIED MUSCLE MYOSIN AND ACTIN FILAMENTS. AC, Actin filament gliding
assays. A, Filaments are labeled with rhodamine-phalloidin to render them visible by light microscopy. ATP hydrolysis by myosin moves actin fila-
ments over the surface with the pointed end leading as the myosins walk toward the barbed end of the filaments. BC, Drawings of actin filaments
moving over myosin heads immobilized on a glass coverslip. D, Measurement of the muscle myosin step size. An actin filament is attached
between two plastic beads, which are suspended by laser optical traps. The optical traps move the filament near a myosin molecule on the
surface of another bead attached to the microscope slide, allowing a myosin head to attach to the actin filament. When supplied with ATP, a
single myosin head can move the actin filament a short distance corresponding to the step size. The graph shows the time course of displace-
ments of the actin filament and attached beads. Brownian motion limits the precision of the measurement of the size of these steps to a range
of 5 to 15nm. The duration of the step depends on the ATP concentration, because ATP dissociates the force-producing AM state, allowing
the force of the optical traps to return the beads and the actin filament to their original position. Pi, inorganic phosphate.
(A, Courtesy A. Bresnick, Albert Einstein College of Medicine, New York. D, For reference, see Finer JT, Simmons RM, Spudich JA. Single myosin
molecule mechanics: piconewton forces and nanometer steps. Nature. 1994;368:113119.)

and acquisition of diverse tails to interact with cargo.
Within a myosin class, the tails are similar to each other,
but between classes, tails are diverse in terms of their
ability to polymerize and interact with other cellular
components including membranes and ribonucleopro
tein particles.
No organism has genes for all 35 classes of myosin
and a few species, including Giardia lamblia, have no
myosin genes. Myosin-I is most widespread, but plants
and related organisms lost this gene. A primitive myosin-V
gene gave rise to plant myosin-VIII and myosin-XI, which
move at very high speeds (see Fig. 37.9). Organisms on
the branch including amoebas, yeast and animals have
genes for myosins types I, II, and V, but myosin genes
diversified in animals, so humans have 40 myosin genes
from 13 classes. Gene duplications gave rise to multiple
isoforms within most classes of myosin. For instance, the
vertebrate smooth muscle myosin gene arose from dupli
cation of a gene for a cytoplasmic myosin-II.
Establishing the biological functions of the various
myosins has been challenging. Biochemical characteriza
tion of cargo and localization in cells provide some clues,
but genetic or biochemical knockouts often have mild
effects, probably owing to overlapping functions of the
myosins and the capacity of some cells to adapt to their
loss, at least under laboratory conditions.
Myosin-I was the first unconventional myosin
discoveredunconventional in the sense that it differed
from the type II myosin originally isolated from skeletal
muscle. These myosins have one head and short tails
with various types of domains, including a basic domain
with affinity for acidic phospholipids. The presence of
630 SECTION IX n Cytoskeleton and Cellular Motility

Class Example Heavy chain domains Architecture Distribution Absent

Membrane
Head binding GPQ
I. Dictyostelium MyoB SH3 Most eukaryotes, Plants, ampicomplexa
IQ motif
stramenopiles, others
I. Bovine BB myosin-I ++

Coiled-coil
II. Chicken muscle myosin-II Amoebas, fungi, animals Plants, others
Kinase
III. Drosophila ninaC long ++ Arthropods, chordates Fungi, plants, others

V. Chicken myosin V/dilute Amoebas, fungi, animals Plants, stramenopiles

VI. Porcine myosin VI Animals Fungi, plants, others

TH4 Talin
VII. Human myosin VIIA Animals Fungi, plants, others

VIII. Arabidopsis ATM1 Algae, plants Fungi, plants, others

IX. Human myosin IXb Animals Fungi, plants, others

pH domains
X. Bovine myosin X Deuterostomes, Cnidaria Other animals, others

XI. Arabidopsis MYA1 Algae, plants Fungi, animals, others

XII. C. elegans Myo12 Nemotodes only All others

XIV. Toxoplasma gondii myosin A + = 100 amino acids Ampicomplexa only All others

FIGURE 36.7 THE MYOSIN FAMILY. Drawing of myosin heavy chain domains and molecular models of myosin isoforms showing catalytic
domains (rose); IQ motifs, light-chainbinding sites (rose bars); basic domains with affinity for membrane lipids (violet); SH3 (Src homology 3)
domains (dark green); coiled-coil (orange); kinase domain (light blue); and pleckstrin homology domain (blue). (For reference, see Odronitz F,
Kollmar M. Drawing the tree of eukaryotic life based on the analysis of 2,269 manually annotated myosins from 328 species. Genome Biol.
2007;8:R196. See also Myosin Home Page, available at http://www.mrc-lmb.cam.ac.uk/myosin/myosin.html.)

an Src homology 3 (SH3) domain (see Fig. 25.10) allows
some type I myosins to bind proline-rich sequences in
other proteins. Those with an actin filamentbinding
domain separate from the motor domain can crosslink
actin filaments. With duty cycles of less than 10%, mul
tiple myosin heads must work together in concert to
move membranes. Mutations show that myosin-I partici
pates in endocytosis, as expected from its concentration
at sites of phagocytosis and macropinocytosis. In micro
villi of intestinal epithelial cells, myosin-I links actin fila
ments laterally to the plasma membrane (see Fig. 33.2B).
Heavy-chain phosphorylation activates myosin-I from
lower eukaryotes, whereas calcium binding to calmodu
lin light chains regulates myosin-I from the intestinal
brush border.
The myosin-II class includes various muscle and
cytoplasmic myosins that also have two heads, two IQ
motifs, and long coiled-coil tails. Assembly of tails into
bipolar filaments (see Fig. 5.7) allows myosin-II to pull
together oppositely polarized actin filaments during
muscle contraction (see Chapter 39) and cytokinesis (see
Fig. 44.24). As in smooth muscle (see Fig. 39.23), phos
phorylation of the regulatory light chain activates
myosin-II in animal nonmuscle cells. In addition, phos
phorylation of the heavy chain regulates the enzyme
activity and/or polymerization of some myosin-IIs.
Myosin-V moves pigment granules, ribonucleopro
tein particles and other cellular components (see Fig.
37.11). A long light-chain domain with seven IQ motifs
allows myosin-V to take long steps along the actin fila
ment (Fig. 36.8). These steps are processive, because
slow ADP dissociation from the AMD intermediate allows
time for the other head to take a long step and bind an
actin subunit 36nm beyond the first head toward the
barbed end of the filament. Mechanical strain after the
step may modestly increase the rate of ADP dissociation
from the trailing head. This cooperation between the
heads initiates ATP binding and the next ATPase cycle,
as the motor walks deliberately along the filament. These
features make myosin-V a valuable model for the lever
arm for movements of the whole myosin family.
Myosin-VI arose in metazoan cells and is the only
myosin known to move toward the pointed end of actin
filaments. Unique features of the converter domain result
in the lever arm swinging opposite to the conventional
direction. The lever arm is a long, single -helix begin
ning with a single IQ-motif associated with calmodulin.
Lacking coiled-coil, myosin-VI is a monomer unless
adapter proteins bring together the C-terminal globular
cargo-binding domains of two molecules. These dimers
can take huge steps of approximately 30nm, but
myosin-VI can also act as a tether, because the force-
producing AMD and AM states occupy a large fraction of
the ATPase cycle, owing to slow ADP dissociation from
AMD state and slow ATP binding to AM. These features
allow myosin-VI to move endocytic vesicles from the
plasma membrane into the cytoplasm and to contribute
to the formation of autophagosomes. Myosin-VII and
 CHAPTER 36 n Motor Proteins 631

A Strong Weak Strong

AM AM*T AM**DP AMD AM

M M*T M**DP MD M

1000 s-1 100 s-1 10 s-1 1 s-1 0.1 s-1

B Pointed
ADP
ADP ATP

ADP ADP
ADP ADP ADP

ADP-Pi ADP
Barbed

Force produced by ATP binds trailing Trailing head steps New leading head
leading head head which forward 72 nm and binds actin
dissociates ADP dissociates hydrolyzes ATP
from trailing head

Pi dissociates
FIGURE 36.8 MYOSIN-V MECHANISM. A, ATPase cycle with ADP release as the rate-limiting step rather than phosphate dissociation as
for muscle myosin (see Fig. 36.5A). B, Relationship of mechanical steps to the ATPase cycle. Shown are actin filament (A), myosin head (M), ATP
(T), ADP (D), and inorganic phosphate (Pi). (For reference, see De La Cruz EM, Ostap EM. Relating biochemistry and function in the myosin
superfamily. Curr Opin Cell Biol. 2004;16:6167. For a movie of myosin-V stepping on actin filaments, see Kodera N, Yamamoto D, Ishikawa R,
etal. Video imaging of walking myosin V by high-speed atomic force microscopy. Nature. 2010;468:7276.)

myosin-X are also monomers with single chain -helices
as lever arms.
Myosin mutations cause human diseases. Loss-of-func
tion mutations in the genes for myosins-IIA, -IIIA, -VI,
-VIIA, and -XV cause deafness and vestibular dysfunction.
Mutations in the genes for cardiac muscle myosin heavy
and light chains are responsible for many cases of car
diomyopathies (see Table 39.1).
Microtubule Motors
The kinesin and dynein families of molecular motors use
energy from ATP hydrolysis to move vesicles, membrane-
bound organelles, chromosomes, and other cargo along
microtubules (see Fig. 37.1). Dynein also powers bend
ing motions of eukaryotic flagella and cilia (see Fig.
38.14). Dyneins move themselves and any cargo toward
the minus end of microtubules. Most kinesins move
in the opposite direction, toward the plus end, but
some kinesin family members are minus-end-directed
motors and others promote microtubule disassembly.
Like myosins, microtubule motors have heads with
ATPase activity and tails that interact with cargo.
Kinesins
Kinesin-1 is a processive motor that moves cargo, such
as an organelle, continuously toward the plus end of a
microtubule. The two heads are attached to an -helical
coiled-coil tail, much like myosin-II, except both the
heads and coiled-coil are smaller (Fig. 36.9). Each head,
consisting of approximately 340 residues, is a motor unit
that binds microtubules and catalyzes ATP hydrolysis.
Light chains associated with the C-terminal bifurcation
of the tail bind cargo molecules (Fig. 36.9E).
Because the kinesin head is less than half the size of
a myosin head and because the proteins lack appreciable
sequence homology, the atomic structure of kinesin-1
(Fig. 36.9C) revealed a major surprise: The small kinesin
head is folded like the core of the catalytic domain of
myosin! In fact, this core, which consists of a central,
mixed -sheet flanked by helices, is similar to the
632 SECTION IX n Cytoskeleton and Cellular Motility

A B D. Structural overlap E. Kinesin light chain with

N cargo peptide
1 3
4

Head Myosin 6
7

Kinesin 3 2

Coiled-coil
8
Neck 1
1
Tail 2
Cargo
C. Kinesin structure peptide
Tail

ATP

955
C
Light
chains

FIGURE 36.9 STRUCTURE OF KINESINS. A, Domain architecture of the polypeptide sequence of the heavy chain of kinesin-1. B, Sketch
of kinesin-1 showing two heads and the coiled-coil tail with light chains bound at the distal end. C, Ribbon diagram of the polypeptide backbone
of the kinesin head showing ATP as a space-filling model (green), the neck-linker residues (red), and the proximal part of the coiled-coil tail.
D, Superimposition of the core of the kinesin-1 head on the catalytic domain of myosin showing the structural homology of the proteins. The
detailed ribbon diagram shows only the homologous elements of secondary structure. The overview (right) shows kinesin-1 (blue) superimposed
on the structure of the whole head of skeletal muscle myosin (pink). E, Ribbon model of a kinesin-1 light chain (purple) with a bound cargo
peptide (green) with a DWED motif. (C, For reference, see PDB file 3KIN and Sack S, Muller J, Marx A, etal. X-ray structure of motor and neck
domains from rat brain kinesin. Biochemistry. 1997;36:1615516165. D, Superimposed ribbon diagrams courtesy of R. Vale, University of
California, San Francisco. E, For reference, see PDB file 3ZFW and Pernigo S, Lamprecht A, Steiner RA, etal. Structural basis for kinesin-1:
cargo recognition. Science. 2013;340:356359.)

considerably smaller Ras family GTPases (see Fig. 4.6). Disordered

This provided strong evidence that all three families of neck linker for
Docked neck linker kinesin without
nucleoside triphosphatases evolved from a common for kinesin-ATP nucleotide
ancestor (Fig. 36.1). ATP binds to a site on kinesin that
is homologous to the guanosine triphosphate (GTP)-
binding site of Ras, but the enzyme mechanisms differ
in important ways. The microtubule-binding site is some
distance from the ATP-binding site (Fig. 36.10).

Kinesin Mechanochemistry
Single kinesin-1 heads, produced experimentally from
truncated cDNAs, traverse a microtubule-stimulated
ATPase cycle much like myosin (Fig. 36.11A). Both bind
and hydrolyze ATP rapidly followed by slower release of
() -tubulin -tubulin (+)
phosphate and ADP. However, in contrast to muscle
myosin, kinesin heads may remain bound to the micro FIGURE 36.10 INTERACTION OF KINESIN-1 WITH MICRO-
tubule through multiple cycles of ATP hydrolysis rather TUBULES. A, Three-dimensional reconstructions from electron micro-
than dissociating when bound to ATP or ADP-Pi. graphs of kinesin-1 heads (blue) bound to a microtubule (yellow and
red). The kinesin head on the left has bound ATP and the neck linker
In vitro motility assays (Fig. 36.12) revealed that a (green) docked. The kinesin head on the right has no bound nucleotide
two-headed kinesin-1 can move along a single (or two and the neck linker (red) is disordered. Ribbon diagrams show how
parallel) microtubule protofilaments for long distances the neck linker of the leading head (right) must be unfolded to connect
at 0.8 m/s. The motor makes discrete steps of 8nm, to the trailing head at the beginning of the coiled-coil tail. (From Charles
the spacing of successive tubulin dimers in a microtu Sindelar, Yale University, based on Shang Z, Zhou K, Xu C, etal. High-
resolution structures of kinesin on microtubules provide a basis for
bule. Each step takes 10 milliseconds when kinesin is nucleotide-gated force-generation. Elife. 2014;3:e04686.)
moving at full speed. This step is remarkably large for
the small (<10nm) kinesin heads linked together at the
neck region. Kinesin is very powerful, stalling at a force
of 6 pN. This allows single molecules to move large
organelles through the cytoplasm.
 CHAPTER 36 n Motor Proteins 633

A Strong Weak Strong

> 100 s-1 80 s-1 300 s-1

MtK MtKT MtKDP MtKD MtK

KDP KD

1000 s-1 100 s-1 10 s-1 1 s-1 0.1 s-1

(+)
B
ADP ADP

ATP
0
ATP ADP
+ Pi
Pi ADP

ADP
ADP
()

Trailing head weakly ATP binds New trailing head Pi dissociates

associates with MT leading head hydrolyzes ATP from trailing head
weakening head's
Trailing head New leading head binding to MT
rotates binds MT and
dissociates ADP

FIGURE 36.11 KINESIN-1 ATPase MECHANISM. A, A diagram of the kinesin-microtubule ATPase cycle for a single kinesin-1 head showing
the kinesin (K), microtubule (Mt), ATP (T), ADP (D), and inorganic phosphate (P). Arrow sizes are proportional to the rates of the reactions, with
second-order reactions adjusted for physiological concentrations of reactants. The beige shading shows two pathways through the reaction, one
along the top line without dissociation, and the other with dissociation from the microtubule. B, Hand-over-hand, processive stepping of kinesin
along a microtubule. The empty, microtubule-bound head binds and hydrolyzes ATP resulting in a conformational change favoring docking of its
neck-linker (green), thereby moving forward the detached head with its undocked (pink) neck-linker. Binding of the new leading head to the
microtubule causes a conformation change that dissociates ADP. Dissociation of the -phosphate from the trailing head results in its dissociation
from the microtubule. This returns the heads to their original condition, but with the motor advanced 8nm with the heads in the opposite chemi-
cal states. Pi, inorganic phosphate. (B, Data from R. Vale, University of California, San Francisco, and R. Milligan, Scripps Research Institute, La
Jolla, CA. For reference, see Cao L, Wang W, Jiang Q, etal. The structure of apo-kinesin bound to tubulin links the nucleotide cycle to move-
ment. Nat Commun. 2014;5:5364.)

Processive movement depends on the ability of
kinesin to remain associated with the microtubule
through more than a hundred cycles of ATP hydrolysis.
This is made possible by cooperation between the two
heads that ensures at least one head is bound to the
microtubule throughout the ATPase cycle (Fig. 36.11B).
Reciprocal affinities for nucleotide and microtubules
allow the two heads to alternate between microtubule
binding and dissociation. For example, if kinesin-1 with
ADP bound to both heads is mixed with microtubules,
one head binds a microtubule and rapidly dissociates its
ADP, leaving the other head dissociated with bound
ADP. Binding and hydrolysis of ATP on the open site of
the head associated with the microtubule, drives a con
formational change that repositions the trailing head
forward so it can pass the bound head and bind the next
tubulin dimer toward the plus end of the microtubule.
Association of the new leading head with the microtu
bule promotes dissociation of its bound ADP. Dissocia
tion of the -phosphate from the new trailing head
weakens its affinity for the microtubule. Its detachment
from the microtubule with bound ADP brings the cycle
back to its starting point with kinesin having advanced
8nm (Fig. 36.12). Experiments with single kinesin-1
molecules labeled with fluorescent dyes showed that
they alternate steps on the right and left sides of the
microtubule like a gymnast walking on a balance beam.
The mechanism of stepping is postulated to be the
docking and undocking of a segment of the kinesin-1
heavy chain linking the motor domain to the coiled-coil
neck and tail (Fig. 36.10). With ATP bound the motor
domain has a conformation that favors association of the
neck-linker peptide, as in the X-ray structure of dimeric
kinesin (Fig. 36.9C). When either no nucleotide or ADP
is bound, the conformation of the motor domain releases
the neck-linker peptide.
Kinesin Superfamily
The last eukaryotic common ancestor had only a single
myosin but already had at least 11 families of kinesins
634 SECTION IX n Cytoskeleton and Cellular Motility

A Microtubule movement B C
96

80
Bead

Distance (nm)
() movement 64

Kinesin stepping (+)

48
toward (+) end
32
()
16
Kinesin stepping
toward (+) end 0
(+) 1 2 3 4 5
Time (s)

FIGURE 36.12 IN VITRO MOTILITY ASSAYS FOR MICROTUBULE MOTORS. A, Gliding assay. Kinesin or dynein that is attached to a
microscope slide uses ATP hydrolysis to move microtubules over the surface. A single kinesin-1 molecule can move a microtubule in this assay.
Microtubules can be imaged by video-enhanced differential interference contrast microscopy (see Fig. 34.6) or fluorescence microscopy. B, Bead
assay. Kinesin or dynein that is attached to a plastic bead uses ATP hydrolysis to move the bead along a microtubule attached to the microscopic
slide. C, Experimental measurement of the kinesin-1 step size using the bead assay. The bead is held in a laser optical trap so that 8-nm steps
can be recorded, as a single, two-headed kinesin-1 moves a bead processively along a microtubule, as in B. The position of the bead is recorded
with nanometer precision by interferometry. (For reference, see Svoboda K, Schmidt CF, Schnapp BJ, etal. Direct observation of kinesin stepping
by optical trapping interferometry. Nature. 1993;365:721727.)

Kinesin structures
N-terminal motor Example Heavy chain domains Architecture

Kinesin-1 Hs Kif5B
Head Coiled-coil Tail

Kinesin-2 Sp Krp85/95 85
95

Kinesin-3 Mm Kif1b

Kinesin-4 Xl KIp1

Kinesin-5 Dm Klp61f

Kinesin-7 Hs CENP-E

Internal motor
Kinesin-13 Xl MCAK

C-terminal motor
Kinesin-14 Dm Ncd

FIGURE 36.13 KINESIN FAMILY. A, Phylogenetic relationships of some of the kinesins based on the sequences of the motor domains.
B, Drawing of kinesin heavy chain domains and molecular models of kinesin isoforms showing the catalytic domain (red), coiled-coil tail (orange),
and tail piece (blue). (Data from R. Case and R. Vale, University of California, San Francisco. For reference, see Lawrence CJ, Dawe RK, Christie
KR, etal. Standardized kinesin nomenclature. J Cell Biol. 2004;167:1922; and Dagenbach EM, Endow SA. A new kinesin tree. J Cell Sci.
2004;117:37. See also the Kinesin Home Page at https://labs.cellbio.duke.edu/kinesin.)

with motor domains associated with a variety of coiled-
coil stalks and tails (Fig. 36.13 and Table 36.2). Thus
the microtubule system was much more developed than
the actin system at this point in evolution. Contempo
rary eukaryotes have genes for 17 families of kinesins
(often with multiple isoforms) plus a few more para
logs identified in isolated species. On the other hand,
many eukaryotes have lost one or more kinesin genes;
for example, amoebas lack kinesin-2 and alveolates
lack kinesin-7.

TABLE 36.2 Kinesin Superfamily: Classification and Examples of Kinesin-Family Motor Proteins
Class Examples Subunits (kD)
N-Terminal Motor
Kinesin-1 Human KHC 2 110, 2 70
Kinesin-2 Urchin KRP85/95 1 79, 1 84, 1 115
Kinesin-3 Mouse KIF1B 1 130
Kinesin-4 Xenopus Kp11 2 139
Kinesin-5 Fly KLP61F 4 121
Kinesin-7 Human CENP-E 2 340
Internal Motor
Kinesin-13 MCAK 2 83
C-Terminal Motor
Kinesin-14 Fly Ncd 2 78
Modified from Vale RD, Fletterick RJ. The design plan of kinesin motors. Annu Rev Cell Dev Biol. 1997;13:745777. More data on kinesins are available
at the Kinesin Home Page, https://labs.cellbio.duke.edu/kinesin.
All members of the kinesin family have similar motor
domains attached to a variety of tails that interact
with cargo (Fig. 36.13). Motor domains are generally
found at the N-terminus, but may be located in the
middle (kinesin-13) or at the C-terminus (kinesin-14).
Regardless of their location, motor domains have similar
structures.
Most kinesins are dimeric, with two polypeptides
joined in a coiled-coil. Most are homodimers, but
kinesin-2 not only forms homodimers but also heterotri
mers consisting of two different polypeptides with
motor domains plus another large subunit. Tetrameric
kinesin-V molecules bind to two microtubules with
opposite polarities and move them apart.
Most kinesins move toward the plus end of the micro
tubule, but C-terminal kinesin-14 motors move toward
the minus end. The reverse direction of kinesin-14 move
ment is not explained by either the architecture of the
motor domain, the ATPase mechanism, which is similar
to kinesin-1, or the attachment of the N-terminus of the
motor domain to the stalk. Instead, the proximal part of
the Ncd coiled-coil stalk rotates approximately 70
degrees toward the minus end of the microtubule when
ATP binds to the active site.
Kinesins transport a variety of cargo, including chro
mosomes and organelles, along microtubules. Kinesin-1
moves membrane vesicles toward the plus end of micro
tubules away from the centrosome and along axons of
neurons (see Fig. 37.1). The light chain links the end
of the kinesin-1 tail to cargo proteins (Fig. 36.9E).
Kinesin-2 is the motor for anterograde intraflagellar
transport, movement of particles toward the tip of
the axoneme in cilia and flagella (see Fig. 38.18). It
also transports a variety of cargos over long distances in
the cytoplasm. Kinesin-4 motors (also called chromo-
kinesins) bind both DNA and microtubules. In neurons,
they move cargo along axons. In dividing cells they
participate in the formation of condensed mitotic
CHAPTER 36 n Motor Proteins 635
Velocity (m s1) Functions
+0.9 Organelle movement
+0.4 Organelle movement
+0.7 Mitochondria movement
+0.2 Chromosome movement
+0.04 Pole separation, mitosis
+0.1 Kinetochore-microtubule binding
Microtubule disassembly
0.2 Mitotic/meiotic spindle
chromosomes (see Fig. 44.7). Kinesin-7 (originally called
CENP-E) concentrates at kinetochores where it helps
move the chromosome toward the middle of the mitotic
spindle prometaphase (see Fig. 44.5). Bipolar kinesin-5
motors form an antiparallel tetramer of two dimeric kine
sins that bridge a pair of oppositely polarized microtu
bules and push apart the poles of the mitotic spindle (see
Fig. 44.7).
Both kinesin-8 and kinesin-13 use cycles of ATP hydro
lysis to remove tubulin dimers from the ends of micro
tubules. Kinesin-8 motors to the plus end where it works,
whereas kinesin-14 lacks motor activity but can diffuse
on the microtubule surface to reach either end. This
depolymerizing activity is important for mitosis.
Most kinesins appear to be constitutively active, but
intramolecular interactions autoinhibit kinesin-1. The tail
folds back and binds between the heads, shutting off the
motor. Adapter proteins compete the tail from the head,
freeing the motor to be active.
Dyneins
Dynein microtubule-based motors are AAA ATPases (Box
36.1), so their evolutionary origin differs from myosins
and kinesins (Fig. 36.1). Most AAA ATPases consist of six
separate ATPase domains, but in dynein these domains
(AAA1AAA6) are concatenated in a giant heavy chain of
nearly 500kD rather than separate polypeptides (Fig.
36.14A). Cytoplasmic dynein is a dimer of two heavy
chains plus accessory polypeptides that bring the total
molecular weight to approximately 1.4 MDa. The
N-terminal quarter of the heavy chain forms a tail that
interacts with intermediate chains, light intermediate
chains, dimers of light chains, and cargo molecules (Fig.
36.14B). A linker domain connects the tail to AAA1. A
segment of the dynein heavy chain within AAA4 forms
an antiparallel coiled-coil stalk with a small microtubule-
binding domain at the tip.
636 SECTION IX n Cytoskeleton and Cellular Motility

A N 0 B. Whole dynein molecule C D Pre power stroke Post power stroke

Linker ATP analog ADP in AAA1
DHC AAA1 in AAA1
Tail dimerization
domain
DIC WD40 Tail
AAA2
DLIC AAA6
1388
AAA5
Linker AAA3

1 LC8
AAA-ATPase domains

Buttress AAA4
2 Tctex of AAA5
Motors
3

4
Coiled-coil Coiled-coil
stalk stalk of AAA4
Microtubule
binding site
5

6 Microtubule binding
domain of AAA4
C 4730

FIGURE 36.14 DYNEIN STRUCTURE. A, Domain organization of a dynein heavy chain showing the six AAA ATPase modules and two
sequences that form an antiparallel coiled-coil stalk with an ATP-sensitive microtubule-binding site at the tip. The first AAA domain is the catalytic
site. AAA domains 2, 3, and 4 bind ATP, but hydrolysis is not coupled to movement. AAA domains 5 and 6 do not bind ATP. B, Model for cyto-
plasmic dynein-1 based on crystal structures of motor domains and reconstructions of electron micrographs of the dynactin complex. C, Ribbon
diagram of crystal structures of cytoplasmic dynein-1 motor domains (Apo) without nucleotide bound to AAA1. The linker domain is purple. AAA
domains are color coded as in A, with the stalk and microtubule binding domain extending from AAA3. D, Space-filling models of dynein with
(ADP-Vo) with ADP and the phosphate analog vanadate bound to AAA1 the pre power stroke state and ADP bound to AAA1 the post power
stroke state. The AAA hexamer is more compact with bound ADP-Vo. These two structures differ in the conformations of the AAA hexamer,
linker domain and stalk. (For reference, see EMData Bank files 2861 and 2862 and Schmidt H, Zalyte R, Urnavicius L, etal. Structure of human
cytoplasmic dynein-2 primed for its power stroke. Nature. 2015;518:435438; and Urnavicius L, Zhang K, Diamant AG, etal. The structure of
the dynactin complex and its interaction with dynein. Science. 2015;347:14411446.)

Dynein Mechanochemistry weakly to microtubules, but phosphate dissociation

Sufficient information is available from enzyme kinetics, during one of the transient interactions with a microtu
crystal structures, and motility assays to construct a bule reverses both conformational changes produced by
mechanochemical cycle for dynein interacting with a ATP. The linker domain straightens out, producing a
microtubule (Fig. 36.15A). The AAA1 domain binds and power stroke that moves the tail and any associated
hydrolyzes ATP during force-producing interactions with cargo (including the other subunit of a dynein dimer)
microtubules. Full motor function requires ADP or ATP toward the minus end of the microtubule. In addition,
binding to AAA domains 2 to 4, but ATP hydrolysis by the affinity for microtubules increases, allowing trans
these domains is not coupled directly to motility. AAA mission of force from the microtubule to the cargo.
domains 5 and 6 do not bind nucleotides. The dynein Binding to a microtubule also stimulates the rate of ADP
ATPase cycle of AAA1 resembles the actomyosin ATPase dissociation from dynein approximately 10-fold, from
mechanism in broad outline. approximately 3s1 to approximately 33s1, restarting
When AAA1 is free of nucleotide, dynein binds tightly the ATPase cycle.
to a microtubule at a site between the - and -tubulin Yeast dynein dimers can walk processively toward the
subunits with the stalk pointing toward the minus end minus end of a microtubule in in vitro motility assays.
of the polymer. ATP binding to AAA1 causes compaction The mechanism likely involves steps by the two motor
of the whole AAA hexamer and produces two important domains on adjacent protofilaments of the microtubule.
conformational changes (Fig. 36.14). First, the buttress The size of the mechanical step associated with each
on AAA5 communicates the conformational change in ATP hydrolysis in most often 8nm, but cytoplasmic
the hexamer to the stalk by displacing the stalk helices dynein can take larger steps up to 24nm when the load
relative to each other. This, in turn, changes the confor is low. Yeast dynein produces a force of 5 to 7 pN,
mation of the microtubule-binding domain more than similar to kinesin.
20nm distant from the ATP binding site and reduces its
affinity for the microtubule. Thus, dynein-ATP dissoci Dynein Superfamily
ates from the microtubule. Second, the linker domain Dynein genes are ancient, arising well before the last
bends in the middle, moving to the pre power stroke common eukaryotic ancestor (see Fig. 2.4B), but they
state. After ATP hydrolysis, dynein-ADP-Pi also binds were lost multiple times during evolution, so neither red

Apo ATP ADPP
S W W S
Strong Weak
MtDy MtDyT ? MtDyDP
Dy DyT DyDP
ATP ADPP
FIGURE 36.15 DYNEIN-MICROTUBULE ATPase MECHANISM. Chemical pathway and structures. Arrow sizes are proportional to the
rates of the reactions, with second-order reactions adjusted for physiological concentrations of reactants. The beige shading shows the main
pathway through the reaction. D, ADP; Dy, dynein; Mt, microtubule; P, inorganic phosphate; T, ATP. The drawings are interpretations of the
intermediates in the cycle based on crystal structures. (Modified from Cianfrocco MA, DeSantis ME, Leschziner AE, etal. Mechanism and regula-
tion of cytoplasmic dynein. Annu Rev Cell Dev Biol. 2015;31:83108.)
algae nor flowering plants now have dynein genes.
Animals have two genes for cytoplasmic dynein heavy
chains and multiple isoforms of intermediate, light inter
mediate, and light chains. Alternative splicing, especially
of intermediate chains, further increases the complexity.
Cytoplasmic dyneins are dimeric proteins (Fig.
36.14B). Cytoplasmic dynein-2 moves cargo for intrafla
gellar transport (see Fig. 38.18). Cytoplasmic dynein-1
has diverse functions around the entire cell cycle. During
interphase, dynein-1 transports organelles, RNAs, and
some viruses toward the minus ends of microtubules
(see Fig. 37.2) generally moving cargo toward the cell
center where the centrosome and Golgi apparatus are
located. In neurons, dynein-1 moves cargo inside axons
toward the cell body (see Fig. 37.3). During mitosis,
dynein-1 in the cell cortex and bound to kinetochores of
chromosomes applies forces to microtubules and helps
to position the mitotic spindle (see Fig. 44.7).
Given these diverse activities, it is not surprising that
dynein mutations cause serious phenotypes and contrib
ute to disease. A null mutation in the gene for a mouse
cytoplasmic dynein-1 heavy chain leaves the Golgi appa
ratus dispersed throughout the cytoplasm and is lethal
during embryogenesis. A temperature-sensitive mutation
in Caenorhabditis elegans dynein-1 causes defects
in mitosis at the restrictive temperature. Mutations of
dynein-1 and associated proteins contribute to human
neurodegenerative diseases including some cases of Par
kinson disease and spinal muscular atrophy.
Accessory proteins regulate the enzyme activity
and movements of cytoplasmic dyneins. Transport by
mammalian dynein depends on the dynactin complex
CHAPTER 36 n Motor Proteins 637
ADP Apo
S
Strong
P
30 s-1
MtDyD MtDy
P
3 s-1
DyD Dy
1000 s-1 100 s-1
10 s-1 1 s-1 0.1 s-1
(see Fig. 37.2), a huge complex of 23 subunits (11 dif
ferent proteins) with a total molecular weight of approx
imately 1.2 MDa. Dynactin and an adapter protein not
only link cytoplasmic dynein-1 to cargo, but also stimu
late its motor activity, perhaps by positioning the two
motors in a productive way.
A complex of proteins (Lis-1 and Nudel/NudE)
increases dyneins affinity for microtubules, slows the
motor, and may increase force production. Nudel/NudE
binds intermediate chains and tethers Lis-1 to dynein.
Lis-1, a -propeller protein, interacts directly with the
AAA hexamer and sterically blocks a movement of the
linker domain that is required for microtubule release.
Genetic experiments suggest that Lis1 is a dynein acti
vator, but how tight binding to microtubules activates
dynein remains unclear. Loss-of-function mutations
of the Lis1 genes interfere with development of the
cerebral cortex, which lacks gyres and is smooth in
affected humans.
Humans have 14 genes for axonemal dyneins, which
consist of one to three heavy chains. In axonemes of cilia
and flagella, at least seven different dynein isoforms bind
to unique sites on the outer doublets (see Fig. 38.14).
Calcium and a cyclic adenosine monophosphate (cAMP)
dependent protein kinase (see Fig. 25.3D) regulate
dynein in cilia and flagella.
ACKNOWLEDGMENTS
We thank Andrew Carter, Erika Holzbaur, Samara Reck-
Peterson, and Lee Sweeney for their suggestions on revi
sions to this chapter.
638 SECTION IX n Cytoskeleton and Cellular Motility
SELECTED READINGS
Bhabha G, Johnson GT, Schroeder CM, etal. How dynein moves along
microtubules. Trends Biochem Sci. 2016;41:94-105.
Bloemink MJ, Geeves MA. Shaking the myosin family tree: biochemical
kinetics defines four types of myosin motor. Semin Cell Dev Biol.
2011;22:961-967.
Buss F, Spudich G, Kendrick-Jones J. Myosin VI: Cellular functions and
motor properties. Annu Rev Cell Dev Biol. 2004;20:649-676.
Carter AP, Diamant AG, Urnavicius L. How dynein and dynactin trans
port cargos: a structural perspective. Curr Opin Struct Biol. 2016;
37:62-70.
Cianfrocco MA, DeSantis ME, Leschziner AE, etal. Mechanism and
regulation of cytoplasmic dynein. Annu Rev Cell Dev Biol. 2015;
31:83-108.
Cross RA, McAinsh A. Prime movers: the mechanochemistry of mitotic
kinesins. Nat Rev Mol Cell Biol. 2014;15:257-271.
De La Cruz EM, Ostap EM. Relating biochemistry and function in the
myosin superfamily. Curr Opin Cell Biol. 2004;16:61-67.
Erzberger JP, Berger JM. Evolutionary relationships and structural
mechanisms of AAA+ proteins. Annu Rev Biophys Biomol Struct.
2006;35:93-114.
Fu MM, Holzbaur EL. Integrated regulation of motor-driven organelle
transport by scaffolding proteins. Trends Cell Biol. 2014;24:
564-574.
Geeves MA, Holmes KC. Structural mechanism of muscle contraction.
Annu Rev Biochem. 1999;68:687-728.
Greenberg MJ, Ostap EM. Regulation and control of myosin-I by the
motor and light chain-binding domains. Trends Cell Biol. 2013;
23:81-89.
Hammer JA 3rd, Sellers JR. Walking to work: roles for class V myosins
as cargo transporters. Nat Rev Mol Cell Biol. 2011;13:13-26.
Hartman MA, Finan D, Sivaramakrishnan S, etal. Principles of uncon
ventional myosin function and targeting. Annu Rev Cell Dev Biol.
2011;27:133-155.
Hartman MA, Spudich JA. The myosin superfamily at a glance. J Cell
Sci. 2012;125:1627-1632.
King SM. Integrated control of axonemal dynein AAA(+) motors.
J Struct Biol. 2012;179:222-228.
Kull FJ, Endow SA. Force generation by kinesin and myosin cytoskeletal
motor proteins. J Cell Sci. 2013;126:9-19.
Milic B, Andreasson JO, Hancock WO, etal. Kinesin processivity is
gated by phosphate release. Proc Natl Acad Sci USA. 2014;111:
14136-14140.
Odronitz F, Kollmar M. Drawing the tree of eukaryotic life based on
the analysis of 2,269 manually annotated myosins from 328 species.
Genome Biol. 2007;8:R196.
Preller M, Manstein DJ. Myosin structure, allostery, and mechano-
chemistry. Structure. 2013;21:1911-1922.
Roberts AJ, Kon T, Knight PJ, etal. Functions and mechanics of dynein
motor proteins. Nat Rev Mol Cell Biol. 2013;14:713-726.
Schmidt H, Zalyte R, Urnavicius L, etal. Structure of human cytoplas
mic dynein-2 primed for its power stroke. Nature. 2015;518:435-
438. For video of conformational change see <http://www.nature
.com/nature/journal/v518/n7539/fig_tab/nature14023_SV6.html>.
Scholey JM. Kinesin-2: a family of heterotrimeric and homodimeric
motors with diverse intracellular transport functions. Annu Rev Cell
Dev Biol. 2013;29:443-469.
Sun Y, Goldman YE. Lever-arm mechanics of processive myosins.
Biophys J. 2011;101:1-11.
Tumbarello DA, Kendrick-Jones J, Buss F. Myosin VI and its cargo
adaptors-linking endocytosis and autophagy. J Cell Sci. 2013;126:
2561-2570.
Walczak CE, Gayek S, Ohi R. Microtubule-depolymerizing kinesins.
Annu Rev Cell Dev Biol. 2013;29:417-441.
Wickstead B, Gull K, Richards TA. Patterns of kinesin evolution reveal
a complex ancestral eukaryote with a multifunctional cytoskeleton.
BMC Evol Biol. 2010;10:110.