Chapter 16
Molecular Motors
Biochemistry
by
Reginald Garrett and Charles Grisham
Garrett and Grisham, Biochemistry, Third Edition
Essential Question
• How can biological macromolecules, carrying
out conformational changes on the
microscopic, molecular level, achieve these
feats of movement that span the molecular and
macroscopic worlds?
Garrett and Grisham, Biochemistry, Third Edition
Outline
• What Is a Molecular Motor?
• What Are the Molecular Motors That
Orchestrate the Mechanochemistry of
Microtubules?
• How Do Molecular Motors Unwind DNA?
• What Is the Molecular Mechanism of Muscle
Contraction?
• How Do Bacterial Flagella Use a Proton
Gradient to Drive Rotation?
Garrett and Grisham, Biochemistry, Third Edition
Figure 16.2
(a) The structure of the tubulin  heterodimer. (b) Microtubules may be viewed as
consisting of 13 parallel, staggered protofilaments of alternating -tubulin and -tubulin
subunits. The sequences of the - and - subunits of tubulin are homologous, and the tubulin dimers are quite stable if Ca2+ is present. The dimer is dissociated only by strong
denaturing agents.
Figure 16.3
A model of the GTPdependent treadmilling
process. Both - and tubulin possess two
different binding sites for
GTP. The polymerization
of tubulin to form
microtubules is driven by
GTP hydrolysis in a
process that is only
beginning to be
understood in detail.
16.1 – What Is a Molecular Motor?
• MTs are the fundamental structural unit in
cilia and flagella (see axoneme structure,
Fig 16.5)
• Dynein proteins walk or slide along MTs to
cause bending of one MT relative to
another
• Dynein movement is ATP-driven
• See Figures 16.6 and 16.7
Garrett and Grisham, Biochemistry, Third Edition
Figure 16.5
The structure of an
axoneme. Note the
manner in which two
microtubules are joined
in the nine outer pairs.
The smaller-diameter
tubule of each pair,
which is a true cylinder,
is called the A-tubule
and is joined to the
center sheath of the
axoneme by a spoke
structure. Each outer
pair of tubules is joined
to adjacent pairs by a
nexin bridge. The Atubule of each outer pair
possesses an outer
dynein arm and an inner
dynein arm. The largerdiameter tubule is known
as the B-tubule.
Figure 16.6 (a) Diagram showing dynein interactions between adjacent microtubule pairs. (b)
Detailed views of dynein crosslinks between the A-tubule of one microtubule pair and the Btubule of a neighboring pair. (The B-tubule of the first pair and the A-tubule of the neighboring
pair are omitted for clarity.) Isolated axonemal dyneins, which possess ATPase activity, consist
of two or three “heavy chains” with molecular masses of 400 to 500 kD, referred to as  and 
(and g when present), as well as several chains with intermediate (40 to 120 kD) and low (15 to
25 kD) molecular masses. Each outer-arm heavy chain consists of a globular domain with a
flexible stem on one end and a shorter projection extending at an angle with respect to the
flexible stem. In a dynein arm, the flexible stems of several heavy chains are joined in a
common base, where the intermediate- and low-molecular-weight proteins are located.
Figure 16.7
A mechanism for ciliary motion. The sliding motion of dyneins along one microtubule while
attached to an adjacent microtubule results in a bending motion of the axoneme.
16.2 – What Are the Molecular Motors
That Orchestrate the
Mechanochemistry of Microtubules?
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•
•
•
Highways for "molecular motors"
MTs also mediate motion of organelles
and vesicles through the cell
In axons, dyneins move organelles + to -,
i.e., toward the nucleus
Kinesins move organelles - to + , i.e.,
away from the nucleus
See Figure 16.8 and compare (a) and (b)
Garrett and Grisham, Biochemistry, Third Edition
Figure 16.8
(a) Rapid axonal
transport along
microtubules permits the
exchange of material
between the synaptic
terminal and the body of
the nerve cell. (b)
Vesicles, multivesicular
bodies, and
mitochondria are carried
through the axon by this
mechanism. (Adapted
from a drawing by
Ronald Vale)
Figure 16.9
The structure of the tubulin-kinesin complex, as revealed by
image analysis of cryoelectron microscopy data. (a) The
computed, three-dimensional map of a microtubule, (b) the
kinesin globular head domain-microtubule complex, (c) a
contour plot of a horizontal section of the kinesin-microtubule
complex, and (d) a contour plot of a vertical section of the
same complex. (Taken from Kikkawa et al., 1995. Nature
376:274-277. Photo courtesy of Nobutaka Hirokawa.)
Polymerization Inhibitors
Therapeutic agents for gout and cancer
• Colchicine, from autumn crocus, inhibits
MT polymerization, mitosis and also white
cell movement - it is a remedy for gout and
an inducer of larger, healthier plants
• Vinblastine, vincristine also inhibit MT
polymerization - anticancer agents
• Taxol, from yew tree bark, stimulates
polymerization, stabilizes microtubules and
inhibits tumor growth, (esp. breast and
ovarian)
Garrett and Grisham, Biochemistry, Third Edition
The structures of
vinblastine, vincristine,
colchicine, and taxol.
16.3 – How Do Molecular Motors
Unwind DNA?
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•
•
•
Four types: skeletal, cardiac, smooth and
myoepithelial cells
A fiber bundle contains hundreds of myofibrils
that run the length of the fiber
Each myofibril is a linear array of sarcomeres
Each sarcomere is capped on ends by a
transverse tubule (t-tubule) that is an
extension of sarcolemmal membrane
Surfaces of sarcomeres are covered by SR
Garrett and Grisham, Biochemistry, Third Edition
Figure 16.10
(a) A hand-over-hand model for movement along (and unwinding of) DNA by E. coli Rep
helicase. The P2S state consists of a Rep dimer bound to ssDNA. The P2S state consists of a
Rep dimer bound to ssDNA. The P2SD state involves on Rep monomer bound to ssDNA and
the other bound to dsDNA. The P2S2 state has ssDNA bound to each Rep monomer. ATP
binding and hydrolysis control the interconversion of these states and walking along the DNA
substrate. (b) Crystal structure of the E. coli Rep helicase dimer. (With permission from
Korolev,S., Hsieh,J., Gauss,G., Lohman,T.L., and Waksman,G., 1997. Major domain swiveling revealed by
the crystal structues of complexes of E.coli Rep helicase bound to single-stranded DNA and ADP. Cell
90:635-647.)
Figure 16.11
The structure of a
skeletal muscle cell,
showing the manner in
which t-tubules enable
the sarcolemmal
membrane to contact the
ends of each myofibril in
the muscle fiber. The
foot structure is shown in
the box.
What are t-tubules and SR
for?
The morphology is all geared to Ca
release and uptake!
• Nerve impulses reaching the muscle
produce an "action potential" that spreads
over the sarcolemmal membrane and into
the fiber along the t-tubule network
Garrett and Grisham, Biochemistry, Third Edition
What are t-tubules and SR
for?
The morphology is all geared to Ca
release and uptake!
• The signal is passed across the triad
junction and induces release of Ca2+ ions
from the SR
• Ca2+ ions bind to sites on the fibers and
induce contraction; relaxation involves
pumping the Ca2+ back into the SR
Garrett and Grisham, Biochemistry, Third Edition
16.4 – What Is the Molecular
Mechanism of Muscle Contraction?
•
•
•
•
•
•
Be able to explain the EM in Figure 16.12 in
terms of thin and thick filaments
Thin filaments are composed of actin polymers
F-actin helix is composed of G-actin monomers
F-actin helix has a pitch of 72 nm
But repeat distance is 36 nm
Actin filaments are decorated with tropomyosin
heterodimers and troponin complexes
Troponin complex consists of: troponin T (TnT),
troponin I (TnI), and troponin C (TnC)
Garrett and Grisham, Biochemistry, Third Edition
Figure 16.12
Electron micrograph of a
skeletal muscle myofibril
(in longitudinal section).
The length of one
sarcomere is indicated,
as are the A and I bands,
the H zone, the M disk,
and the Z lines. Crosssections from the H zone
show a hexagonal array
of thick filaments,
whereas the I band
cross-section shows a
hexagonal array of thin
filaments. (Photo courtesy
of Hugh Huxley, Brandeis
University)
Figure 16.13
The three-dimensional
structure of an actin
monomer from skeletal
muscle. This view shows
the two domains (left
and right) of actin.
Figure 16.14
The helical
arrangement of actin
monomers in F-actin.
The F-actin helix has
a pitch of 72 nm and
a repeat distance of
36 nm. (Electron
micrograph courtesy
of Hugh Huxley,
Brandeis University)
Figure 16.15
(a) An electron
micrograph of a thin
filament, (b) a
corresponding image
reconstruction, and (c) a
schematic drawing
based on the images in
(a) and (b). The
tropomyosin coiled coil
winds around the actin
helix, each tropomyosin
dimer interacting with
seven consecutive actin
monomers. Troponin T
binds to tropomyosin at
the head-to-tail junction.
(a and b, courtesy of
Linda Rost and David
DeRosier, Brandeis
University; c, courtesy of
George Phillips, Rice
University)
The Composition and Structure
of Thick Filaments
•
•
•
•
•
Myosin - 2 heavy chains, 4 light chains
Heavy chains - 230 kD each
Light chains - 2 pairs of different 20 kD chains
The "heads" of heavy chains have ATPase
activity and hydrolysis here drives contraction
Light chains are homologous to calmodulin
and also to TnC
See structure of heads in Figure 16.16
Garrett and Grisham, Biochemistry, Third Edition
Figure 16.16
(a) An electron micrograph of a
myosin molecule and a
corresponding schematic
drawing. The tail is a coiled coil
of intertwined -helices
extending from the two globular
heads. One of each of the
myosin light chain proteins, LC1
and LC2, is bound to each of the
globular heads. (b) A ribbon
diagram shows the structure of
the S1 myosin head (green, red,
and purple segments) and its
associated essential (yellow) and
regulatory (magenta) light chains.
(a, Electron micrograph courtesy of
Henry Slayter, Harvard Medical
School; b, courtesy of Ivan Rayment
and Hazel M. Holden, University of
Wisconsin, Madison)
Repeating Structural Elements Are
the Secret of Myosin’s Coiled Coils
The secret to ultrastructure
• 7-residue, 28-residue and 196-residue
repeats are responsible for the
organization of thick filaments
• Residues 1 and 4 (a and d) of the sevenresidue repeat are hydrophobic; residues
2,3 and 6 (b, c and f) are ionic
• This repeating pattern favors formation of
coiled coil of tails. (With 3.6 - NOT 3.5 residues per turn, -helices will coil!)
Garrett and Grisham, Biochemistry, Third Edition
Figure 16.17
An axial view of the two-stranded, -helical coiled coil of a myosin tail. Hydrophobic
residues a and d of the seven-residue repeat sequence align to form a hydrophobic core.
Residues b, c, and f face the outer surface of the coiled coil and are typically ionic.
More Repeats!
• 28-residue repeat (4 x 7) consists of
distinct patterns of alternating side-chain
charge (+ vs -), and these regions pack
with regions of opposite charge on
adjacent myosins to stabilize the filament
• 196-residue repeat (7 x 28) pattern also
contributes to packing and stability of
filaments
Garrett and Grisham, Biochemistry, Third Edition
Figure 16.18
The packing of myosin molecules in a thick filament. Adjoining molecules are offset by
approximately 14 nm, a distance corresponding to 98 residues of the coiled coil.
Associated proteins of Muscle
-Actinin, a protein that contains several
repeat units, forms dimers and contains
actin-binding regions, and is analogous in
some ways to dystrophin
• Dystrophin is the protein product of the
first gene to be associated with muscular
dystrophy - actually Duchennes MD
• See the box on pages 524-525

Garrett and Grisham, Biochemistry, Third Edition
Dystrophin
New Developments!
Dystrophin is part of a large complex of
glycoproteins that bridges the inner
cytoskeleton (actin filaments) and the
extracellular matrix (via a protein called
laminin)
• Two subcomplexes: dystroglycan and
sarcoglycan
• Defects in these proteins have now been linked
to other forms of muscular dystrophy
Garrett and Grisham, Biochemistry, Third Edition
A model for the actin - dystrophin glycoprotein complex in skeletal
muscle. Dystrophin is postulated to
form tetramers of antiparallel
monomers that bind actin at their Ntermini and a family of dystrophinassociated glycoproteins at their Ctermini. This dystrophin-anchored
complex may function to stabilize
the sarcolemmal membrane during
contraction - relaxation cycles, link
the contractile force generated in
the cell (fiber) with the extracellular
environment, or maintain local
organization of key proteins in the
membrane. The dystrophinassociated membrane proteins
(dystroglycans and sarcoglycans)
range from 25 to 154 kD . (Adapted
from Ahn, A. H., and Kunkel, L. M.,
1993. Nature Genetics 3:283-291,
and Worton, R., 1995. Science
270:755-756.)
The Dystrophin Complex
Links to disease
 -Dystroglycan - extracellular, binds to
merosin (a component of laminin) mutation in merosin linked to severe
congenital muscular dystrophy
 -Dystroglycan - transmembrane protein
that binds dystrophin inside
• Sarcoglycan complex - , , g - all
transmembrane - defects linked to limbgirdle MD and autosomal recessive MD
Garrett and Grisham, Biochemistry, Third Edition
The Sliding Filament Model
•
•
•
•
•
Many contributors!
Hugh Huxley and Jean Hanson
Andrew Huxley and Ralph Niedergerke
Albert Szent-Gyorgyi showed that actin and
myosin associate (actomyosin complex)
Sarcomeres decrease length during
contraction (see Figure 16.19)
Szent-Gyorgyi also showed that ATP causes
the actomyosin complex to dissociate
Garrett and Grisham, Biochemistry, Third Edition
Figure 16.19
The sliding filament model of skeletal muscle contraction. The decrease in sarcomere
length is due to decreases in the width of the I band and H zone, with no change in the
width of the A band. These observations mean that the lengths of both the thick and thin
filaments do not change during contraction. Rather, the thick and thin filaments slide
along one another.
The Contraction Cycle
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•
•
•
Study Figure 16.20!
Cross-bridge formation is followed by power
stroke with ADP and Pi release
ATP binding causes dissociation of myosin
heads and reorientation of myosin head
Details of the conformational change in the
myosin heads are coming to light!
Evidence now exists for a movement of at
least 35 Å in the conformation change
between the ADP-bound state and ADP-free
state
Garrett and Grisham, Biochemistry, Third Edition
Figure 16.20
The mechanism of skeletal muscle
contraction. The free energy of ATP
hydrolysis drives a conformational
change in the myosin head, resulting
in net movement of the myosin heads
along the actin filament. (Inset) A
ribbon and space-filling
representation of the actin-myosin
interaction. (S1 myosin image
courtesy of Ivan Rayment and Hazel
M. Holden, University of Wisconsin,
Madison.)
Similarities in Motor Proteins
• Initial events of myosin and kinesin action
are similar
• But the conformational changes that
induce movement are different in myosins,
kinesins, and dyneins
Garrett and Grisham, Biochemistry, Third Edition
Figure 16.21
Ribbon structures of the myosin and kinesin
motor domains and the conformational
changes triggered by the g-P sensor and the
relay helix.The upper panels represent the
motor domains of myosin and kinesin,
respectively, in the ATP-or ADP-Pi-like state.
Similar structural elements in the catlytic
cores of the two domains are shown in blue,
the relay helices are dark green, and the
mechanical elements (neck linker for
kinesin, lever arm domains for myosin) are
yellow. The nucleotide is shown as a white
space-filling model. The similarity of the
conformation changes caused by the relay
helix in going from the ATP/ADP-Pi-bound
state to the ADP-bound or nucleotide-free
state is shown in the lower panels. In both
cases, the mechanical elements of the
protein shift their positions in response to
relay helix motion. Note that the direction of
mechanical element motion is nearly
perpendicular to the relay helix motion.
(Adapted from Vale,R.D. and Milligan,R.A.,
2000.The way things move: Looking under the
hood of molecular motor proteins. Science 288:8895.)
Figure 16.22
Models for the intramolecular
communication and conformational
changes that lead to movement within the
motor domains of myosin, kinesin, and
dynein. In both myosin (a) and kinesin (b),
ATP hydrolysis causes a conformational
change near the ATP-binding site that is
communicated to the track-binding site
(green arrow). The information is then
relayed (red arrow) via homologous
structural elements to a mechanical
amplifier. (In myosin, the amplifier is the
long helix stabilized by light chains; in
kinesin, it is a flexible peptide segment,
the “neck-linker,” that connects the motor
domain with the neck helix.) (c) The
mechanism of intramolecular
communication in dynein is not well
understood, but a conformational change
at the ATP-binding site must be
communicated to the stalk that contains
the microtubule (MT)-binding site, inducing
an angular swinging of the stalk.
Muscle Contraction Is Regulated
by Ca2+
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•
•
•
Ca2+ Channels and Pumps
Release of Ca2+ from the SR triggers
contraction
Reuptake of Ca2+ into SR relaxes muscle
So how is calcium released in response to
nerve impulses?
Answer has come from studies of
antagonist molecules that block Ca2+
channel activity
Garrett and Grisham, Biochemistry, Third Edition
Figure 16.23
Ca2+ is the trigger signal for muscle
contraction. Release of Ca2+ through
voltage- or Ca2+-sensitive channels
activates contraction. Ca2+ pumps induce
relaxation by reducing the concentration
of Ca2+ available to the muscle fibers.
Dihydropyridine Receptor
In t-tubules of heart and skeletal muscle
• Nifedipine and other DHP-like molecules
bind to the "DHP receptor" in t-tubules
• In heart, DHP receptor is a voltage-gated
Ca2+ channel
• In skeletal muscle, DHP receptor is
apparently a voltage-sensing protein and
probably undergoes voltage-dependent
conformational changes
Garrett and Grisham, Biochemistry, Third Edition
Ryanodine Receptor
The "foot structure" in terminal cisternae of
SR
• Foot structure is a Ca2+ channel of unusual
design
• Conformation change or Ca2+ -channel
activity of DHP receptor apparently gates
the ryanodine receptor, opening and
closing Ca2+ channels
• Many details are yet to be elucidated!
Garrett and Grisham, Biochemistry, Third Edition
Muscle Contraction Is Regulated
by Ca 2+
•
•
•
•
Tropomyosin and troponins mediate the
effects of Ca2+
See Figure 16.24
In absence of Ca2+, TnI binds to actin to
keep myosin off
TnI and TnT interact with tropomyosin to
keep tropomyosin away from the groove
between adjacent actins
But Ca2+ binding changes all this!
Garrett and Grisham, Biochemistry, Third Edition
Ca 2+ Turns on Contraction
• Binding of Ca2+ to TnC increases binding
of TnC to TnI, simultaneously decreasing
the interaction of TnI with actin
• This allows tropomyosin to slide down into
the actin groove, exposing myosin-binding
sites on actin and initiating contraction
• Since troponin complex interacts only with
every 7th actin, the conformational
changes must be cooperative
Garrett and Grisham, Biochemistry, Third Edition
Figure 16.24
A drawing of the thick and thin filaments of
skeletal muscle in cross-section showing the
changes that are postulated to occur when
Ca2+ binds to troponin C.
Binding of Ca 2+ to Troponin C
• Four sites for Ca2+ on TnC - I, II, III and IV
• Sites I & II are N-terminal; III and IV on C
term
• Sites III and IV usually have Ca2+ bound
• Sites I and II are empty in resting state
• Rise of Ca2+ levels fills sites I and II
• Conformation change facilitates binding of
TnC to TnI
Garrett and Grisham, Biochemistry, Third Edition
Figure 16.25
(a) A ribbon diagram and (b) a
molecular graphic showing two
slightly different views of the
structure of troponin C. Note the
long -helical domain
connecting the N-terminal and
C-terminal lobes of the
molecule.
Smooth Muscle Contraction
No troponin complex in smooth muscle
• In smooth muscle, Ca2+ activates myosin light
chain kinase (MLCK) which phosphorylates
LC2, the regulatory light chain of myosin
• Ca2+ effect is via calmodulin - a cousin of TnC
• Hormones regulate contraction - epinephrine, a
smooth muscle relaxer, activates adenylyl
cyclase, making cAMP, which activates protein
kinase, which phosphorylates MLCK,
inactivating MLCK and relaxing muscle
Garrett and Grisham, Biochemistry, Third Edition
Smooth Muscle Effectors
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•
•
•
Useful drugs
Epinephrine (as Primatene) is an over-thecounter asthma drug, but it acts on heart as
well as on lungs - a possible problem!
Albuterol is a more selective smooth muscle
relaxer and acts more on lungs than heart
Albuterol is used to prevent premature labor
Oxytocin (pitocin) stimulates contraction of
uterine smooth muscle, inducing labor
Garrett and Grisham, Biochemistry, Third Edition
The structure of oxytocin.
16.5 – How Do Bacterial Flagella
Use a Proton Gradient to Drive
Rotation?
• Motor proteins drive flagellar rotation!
• In E. coli, a proton gradient, not ATP,
drives the flagellar motor
• 800-1200 protons must flow through this
complex during a single rotation of the
flagellar filament!
Garrett and Grisham, Biochemistry, Third Edition
Figure 16.26
A model of the flagellar motor assembly of
Escherichia coli. The M ring carries an array
of about 100 motB proteins at its periphery.
These juxtapose with motA proteins in the
protein complex that surrounds the ring
assembly. Motion of protons through the
motA/motB complexes drives the rotation of
the rings and the associated rod and helical
filament.
Figure 16.27
Howard Berg’s model for coupling between
transmembrane proton flow and rotation of the
flagellar motor. A proton moves through an
outside channel to bind to an exchange site
on the M ring. When the channel protein
slides one step around the ring, the proton is
released and flows through an inside channel
and into the cell, while another proton flows
into the outside channel to bind to an adjacent
exchange site. When the motA channel
protein returns to its original position under an
elastic restoring force, the associated motB
protein moves with it, causing a
counterclockwise rotation of the ring, rod, and
helical filament. (Adapted from Meister, M.,
Caplan, S. R., and Berg, H. C., 1989.
Dynamics of a tightly coupled mechanism for
flagellar rotation. Biophysical Journal 55:905914)