Biochemistry, 4th Edition P53 ppsx

These cross-bridges are actually extensions of the myosin molecules, and muscle contraction is accomplished by the sliding of the cross-bridges along the thin filaments, a mechanical move

1 to 2

basic structural units of muscle contraction Each myofibril is surrounded by a

spe-cialized endoplasmic reticulum called the sarcoplasmic reticulum (SR) The SR

con-tains high concentrations of Ca2, and the release of Ca2 from the SR and its

interactions within the sarcomeres trigger muscle contraction The muscle fiber is

surrounded by the sarcolemma, a specialized plasma membrane Extensions of the

sarcolemma, called transverse tubules, or t-tubules, reach deep into the muscle fiber,

enabling the sarcolemmal membrane to be in contact with each myofibril

Skeletal muscle contractions are initiated by nerve stimuli that act directly on the

muscle Nerve impulses produce an electrochemical signal (see Chapter 32) called

an action potential that spreads over the sarcolemmal membrane and into the fiber

along the t-tubule network This signal induces the release of Ca2ions from the SR

These Ca2ions bind to proteins within the muscle fibers and induce contraction

The Molecular Structure of Skeletal Muscle Is Based on Actin

and Myosin

Examination of myofibrils in the electron microscope reveals a banded or striated

structure The bands are traditionally identified by letters (Figure 16.2) Regions of

high electron density, denoted A bands, alternate with regions of low electron

den-sity, the I bands Small, dark Z lines lie in the middle of the I bands, marking the

ends of the sarcomere Each A band has a central region of slightly lower electron

density called the H zone, which contains a central M disc (also called an M line).

Electron micrographs of cross sections of each of these regions reveal molecular

de-tails The H zone shows a regular, hexagonally arranged array of thick filaments of

myosin (15 nm diameter), whereas the I band shows a regular, hexagonal array of

thin filaments of actin, together with proteins known as troponin and tropomyosin

(7 nm diameter) In the dark regions at the ends of each A band, the thin and thick

One sarcomere

Thin filaments Thick filaments Thick and thin

filaments

M disc

FIGURE 16.2 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 disc, and the Z lines Cross sections from the

H zone show a hexagonal array of thick filaments, whereas the I band cross section shows a hexagonal array of thin filaments.

filaments interdigitate, as shown in Figure 16.2 The thin and thick filaments are

joined by cross-bridges These cross-bridges are actually extensions of the myosin

molecules, and muscle contraction is accomplished by the sliding of the cross-bridges along the thin filaments, a mechanical movement driven by the free energy

of ATP hydrolysis

The Composition and Structure of Thin Filaments Actin, the principal component

of thin filaments, is found in substantial amounts in most eukaryotic cells At low ionic

strength, actin exists as a 42-kD globular protein, denoted G-actin (Figure 16.3)

Un-der physiological conditions (higher ionic strength), G-actin polymerizes to form a

fi-brous form of actin, called F-actin As shown in Figure 16.4, F-actin is a right-handed

helical structure, with a helix pitch of about 72 nm per turn The F-actin helix is the

core of the thin filament, to which tropomyosin and the troponin complex also add.

Tropomyosin winds around actin filaments and prevents myosin binding in resting muscle When a nerve impulse arrives at the sarcolemmal membrane, Ca2ions re-leased from the sarcoplasmic reticulum bind to the troponin complex, inducing a conformation change that allows myosin to bind to actin, initiating contraction In nonmuscle cells, actin filaments are the highways across which a variety of cellular cargo is transported

The Composition and Structure of Thick Filaments Myosin, the principal compo-nent of muscle thick filaments, is a large protein consisting of six polypeptides, with

an aggregate molecular weight of approximately 540 kD As shown in Figure 16.5, the

six peptides include two 230-kD heavy chains, as well as two pairs of different 20-kD light chains, denoted LC1 and LC2 The heavy chains consist of globular amino-terminal myosin heads, joined to long -helical carboxy-terminal segments, the tails.

These tails are intertwined to form a left-handed coiled coil approximately 2 nm in diameter and 130 to 150 nm long Each of the heads in this dimeric structure is

asso-Globular heads

Light chains

Coiled-coil rod

Converter domain Relay helix

2 nm COO –

COO –

NH3

NH3

(a)

(b)

150 nm

FIGURE 16.5 (a) A schematic drawing of a myosin hexamer, showing the two heavy chains

and four light chains 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 myosin head The head and

neck domains of the heavy chain are red; the essential light chain is yellow and the regulatory light chain is blue (pdb id  1B7T).

FIGURE 16.3 The three-dimensional structure of an

actin monomer from skeletal muscle This view shows

the two domains (left and right) of actin

(pdb id  1J6Z).

FIGURE 16.4 A molecular model of an actin polymer,

based on the actin monomer structure shown in

Figure 16.3 (pdb id  1A5X).

ciated with an LC1 and an LC2 The myosin heads exhibit ATPase activity, and

hy-drolysis of ATP by the myosin heads drives muscle contraction LC1 is also known as

the essential light chain, and LC2 is designated the regulatory light chain Both light

chains are homologous to calmodulin and troponin C Dissociation of LC1 from the

myosin heads by alkali cations results in loss of the myosin ATPase activity

The myosin head consists of a globular domain, where ATP is bound and

hy-drolyzed, and a long -helical neck, to which the light chains are bound The most

prominent feature of the globular head is the actin-binding cleft between the

so-called upper and lower domains The N-terminal domain and the upper domain

to-gether form a seven-stranded -sheet The ATP-binding site is partially defined by

three loops: switch 1, switch 2, and the P-loop Conformation changes driven by

ATP hydrolysis cause a rotation of the converter domain and the long-neck helix—

the fundamental event in contraction

Repeating Structural Elements Are the Secret of Myosin’s Coiled Coils Several

key features of the myosin sequence are responsible for the -helical coiled coils

formed by myosin tails Several orders of repeating structure are found in all myosin

tails, including 7-residue, 28-residue, and 196-residue repeating units Large

stretches of the tail domain are composed of 7-residue repeating segments The first

and fourth residues of these 7-residue units are generally small, hydrophobic amino

acids, whereas the second, third, and sixth are likely to be charged residues The

consequence of this arrangement is shown in Figure 16.6 Seven residues form two

turns of an -helix, and in the coiled coil structure of the myosin tails, the first and

fourth residues face the interior contact region of the coiled coil Residues b, c, and

f (2, 3, and 6) of the 7-residue repeat face the periphery, where charged residues

can interact with the water solvent At the 28 (4  7) residue and 196 (28  7)

residue levels, specialized amino acid sequence patterns promote packing of large

numbers of myosin tails in offset or staggered arrays (Figure 16.7)

FIGURE 16.6 An axial view of the two-stranded,-helical

coiled coil of a myosin tail Hydrophobic residues a and

d of the 7-residue repeat sequence align to form a hydrophobic core Residues b, c, and f face the outer sur-face of the coiled coil and are typically ionic.

A DEEPER LOOK

The P-Loop: A Common Motif in Enzymes That Hydrolyze Nucleoside Triphosphates

Skeletal muscle myosin is just one member of a large class of

en-zymes that convert the free energy of NTP hydrolysis into

chemi-cal signaling, mechanichemi-cal work, or both These enzymes all employ

a polypeptide loop between a -strand and an -helix with the

sequence GxxxxGK(S/T) or GxxGK(S/T) This sequence, the

so-called P-loop, coordinates the triphosphate chain of the NTP to be

cleaved Side-chain (lysine amino and serine/threonine–OH)

groups, as well as backbone (amide–NH) groups of the P-loop,

position the - and -phosphate groups of the substrate so as to

facilitate hydrolysis Genomic analysis reveals that 10% to 18% of

predicted gene products are P-loop NTPases P-loops are found in

a variety of motor proteins, including myosins and kinesins, as well

as in the ABC ATPases (see Chapter 9), the AAA ATPases (see

Section 16.4), the F1 ATPase (a rotary motor; see Chapter 20), the

GTP-binding proteins known as G-proteins (for example, EF-Tu

discussed in Chapter 30 and Ras discussed in Chapter 32), and

adenylate kinase (see Chapter 27)

Location of M disc region

FIGURE 16.7 The packing of myosin molecules in a thick filament Adjoining molecules are offset by approxi-mately 14 nm, a distance corresponding to 98 residues

of the coiled coil.

䊳 P-loops of histidine permease (pdb id  1B0U, orange) and HprK protein kinase (pdb id  1KKL, green).

ATP is shown in light blue.

Go to CengageNOW at www.cengage.com/login and click BiochemistryInteractive to explore myosin.

The Mechanism of Muscle Contraction Is Based on Sliding Filaments

When muscle fibers contract, the thick myosin filaments slide or walk along the thin

actin filaments The basic elements of the sliding filament model were first

de-scribed in 1954 by two different research groups: Hugh Huxley and his colleague Jean Hanson, and the physiologist Andrew Huxley and his colleague Ralph Niedergerke Several key discoveries paved the way for this model Electron micro-scopic studies of muscle revealed that sarcomeres decreased in length during con-traction and that this decrease was due to decreases in the width of both the I band and the H zone (Figure 16.8) At the same time, the width of the A band (which is the length of the thick filaments) and the distance from the Z discs to the nearby H zone (that is, the length of the thin filaments) did not change These observations made it clear that the lengths of both the thin and thick filaments were constant during contraction This conclusion was consistent with a sliding filament model

The Sliding Filament Model The shortening of a sarcomere (Figure 16.8) involves sliding motions in opposing directions at the two ends of a myosin thick filament Net

HUMAN BIOCHEMISTRY

The Molecular Defect in Duchenne Muscular Dystrophy Involves

an Actin-Anchoring Protein

Duchenne muscular dystrophy is a degenerative and fatal disorder

of muscle affecting approximately 1 in 3500 boys Victims of

Duchenne dystrophy show early abnormalities in walking and

run-ning By the age of 5, the victim cannot run and has difficulty

stand-ing, and by early adolescence, walking is difficult or impossible

The loss of muscle function progresses upward in the body,

affect-ing next the arms and the diaphragm Respiratory problems or

in-fections usually result in death by the age of 30 Louis Kunkel and

his co-workers identified the Duchenne muscular dystrophy gene

in 1986 This gene produces a protein called dystrophin, which is

highly homologous to -actinin and spectrin A defect in

dys-trophin is responsible for the muscle degeneration of Duchenne

dystrophy

Dystrophin is located on the cytoplasmic face of the muscle

plasma membrane, linked to the plasma membrane via an integral

membrane glycoprotein Dystrophin has a high molecular mass

(427 kD) but constitutes less than 0.01% of the total muscle protein

It folds into four principal domains (see accompanying figure, part a), including an N-terminal domain similar to the actin-binding do-mains of actinin (in muscle) and spectrin (in red blood cells), a long repeat domain, a -dystroglycan-binding domain, and a C-terminal domain that is unique to dystrophin The repeat domain consists of

24 triple-helical repeat units of approximately 109 residues each

“Spacer sequences” high in proline content, which do not align with the repeat consensus sequence, occur at the beginning and end of the repeat domain Spacer segments are found between repeat ele-ments 3 and 4 and 19 and 20 The high proline content of the spac-ers suggests that they may represent hinge domains The spacer/ hinge segments are sensitive to proteolytic enzymes, indicating that they may represent more exposed regions of the polypeptide The N-terminal actin-binding domain appears capable of binding to 24 actin monomers in a polymerized actin filament

(a)

-Actinin

-Spectrin

N

Actin-binding

domain

-Dystroglycan–

binding domain

-Dystroglycan–

binding domain

-Dystroglycan–

binding domain

C-terminal domain Long spectrin repeat domain

Actin-binding

domain

A comparison of the amino acid sequence of dystrophin, -actinin, and spectrin The potential

hinge segments in the dystrophin structure are indicated.

1 N

N

2 3 4 5 6 7 8 9 10 11 12

1 2 3 4

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 C

Dystrophin

Dystrophin itself appears to be part of an elaborate protein–

glycoprotein complex that bridges the inner cytoskeleton (actin

fil-aments) and the extracellular matrix (via a matrix protein called

laminin) (see figure) It is now clear that defects in one or more

of the proteins in this complex are responsible for many of the

other forms of muscular dystrophy The glycoprotein complex is

composed of two subcomplexes, the dystroglycan complex and

the sarcoglycan complex The dystroglycan complex consists of

-dystroglycan, an extracellular protein that binds to merosin, a

laminin subunit and component of the extracellular matrix, and

-dystroglycan, a transmembrane protein that binds the C-terminal

domain of dystrophin inside the cell (see figure) The sarcoglycan complex is composed of -, -, and -sarcoglycans, all of which are

transmembrane glycoproteins Alterations of the sarcoglycan pro-teins are linked to limb-girdle muscular dystrophy and autosomal recessive muscular dystrophy Mutations in the gene for merosin, which binds to -dystroglycan, are linked to severe congenital

mus-cular dystrophy, yet another form of the disease

(b)

-DG

-DG

-SG -SG

-SG

Laminin

Hinges

Hinges N-terminus

Cytoskeletal

F-actin

Spectrin repeats

Basal lamina

-Dystroglycan–

binding domain Dystrophin

C-terminus

䊴 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 N-termini and a family of dystrophin-associated glycoproteins at their C-termini 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 dystrophin-associated membrane proteins (dystroglycans, DGs, and sarcoglycans, SGs) 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.)

H zone

H zone and I band decrease in width

Relaxed

Z line

Contracted

FIGURE 16.8 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 fila-ments slide along one another.

sliding motions in a specific direction occur because the thin and thick filaments both

have directional character Actin filaments always extend outward from the Z lines in a

uniform manner The myosin thick filaments also assemble in a directional manner The polarity of myosin thick filaments reverses at the M disc, which means that actin filaments on either side of the M disc are pulled toward the M disc during contraction

by the sliding of the myosin heads, causing net shortening of the sarcomere

Albert Szent-Györgyi’s Discovery of the Effects of Actin on Myosin The molecular events of contraction are powered by the ATPase activity of myosin Much of our pre-sent understanding of this reaction and its dependence on actin can be traced to sev-eral key discoveries by Albert Szent-Györgyi at the University of Szeged in Hungary in the early 1940s

In a series of elegant and insightful experiments, Szent-Györgyi showed the following:

• Solution viscosity is increased dramatically when solutions of myosin and actin are mixed Increased viscosity is a manifestation of the formation of an

actomyosin complex.

• The viscosity of an actomyosin solution is lowered by the addition of ATP, indi-cating that ATP decreases myosin’s affinity for actin

• Myosin ATPase activity is increased substantially by actin (For this reason,

Szent-Györgyi gave the name actin to the thin filament protein.) The ATPase turnover

number of pure myosin is 0.05/sec, but when actin is added, the turnover num-ber increases to about 10/sec, a numnum-ber more like that of intact muscle finum-bers Szent-Györgyi’s experiments demonstrated that ATP hydrolysis and the associa-tion and dissociaassocia-tion of actin and myosin are coupled It is this coupling that en-ables ATP hydrolysis to power muscle contraction

The Coupling Mechanism: ATP Hydrolysis Drives Conformation Changes in the Myosin Heads The only remaining piece of the puzzle is this: How does the close coupling of actin-myosin binding and ATP hydrolysis result in the shortening of myofibrils? Put another way, how are ATP hydrolysis and the sliding filament model related? The answer to this puzzle is shown in Figure 16.9 The free energy of ATP

ATP hydrolysis and recovery

Resting muscle

Detached

ATP

ATP

Pi, ADP

Pi ADP

Pi ADP

Switch 1 open

Switch 2 closed

Actin-cleft closed

-sheet twisted

Switch 1 open Switch 2 closed Actin-cleft closed

-sheet twisted

Switch 1 open

Switch 2 open

Actin-cleft open

-sheet untwisted

Switch 1 closed Switch 2 closed Actin-cleft half closed

-sheet untwisted

1

4

2

3

ACTIVE FIGURE 16.9 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 (Adapted from Geeves, M., and Holmes, K., 2005 The molecular mechanism of muscle contraction.

Advances in Protein Chemistry 71:161–193.)Test yourself on the concepts in this figure at www.cengage.com/login.

hydrolysis is translated into conformation changes in the myosin head, so

dissocia-tion of myosin and actin, hydrolysis of ATP, and rebinding of myosin and actin

oc-cur with stepwise movement of the myosin head along the actin filament The

con-formation changes in the myosin head are driven by the binding and hydrolysis of

ATP

As shown in the cycle in Figure 16.9, the myosin heads—with the hydrolysis

prod-ucts ADP and Pibound—are mainly dissociated from the actin filaments in resting

muscle When the signal to contract is presented (see following discussion), the

myosin heads move out from the thick filaments to bind to actin on the thin

fila-ments (step 1) Actin binding closes the cleft in the myosin head, which causes a

twist in the large -sheet The twist causes switch 1 and the P-loop to “open,” both

of them moving away from the bound ADP and Pi(Figure 16.10) The -sheet twist

also straightens the kink in the relay helix These conformation changes result in

the top-of-power stroke state shown in Figure 16.9, but this state is transient The

power stroke occurs almost immediately, accompanied by dissociation of Pi and

then ADP The power stroke consists of a 60° rotation of the converter domain and

the long -helical neck into the down position relative to the myosin head (Figure

16.9)—a movement that results in a 100-Å movement of the end of the neck in the

direction of contraction

The end of the power stroke is termed the rigor-like state because without access

to additional ATP, the actin–myosin pair would be locked together, unable to

dis-sociate However, binding of another ATP causes dissociation of myosin from actin,

CRITICAL DEVELOPMENTS IN BIOCHEMISTRY

Molecular “Tweezers” of Light Take the Measure of a Muscle Fiber’s Force

The optical trapping experiment involves the attachment of

myosin molecules to silica beads that are immobilized on a

mi-croscope coverslip (see accompanying figure) Actin filaments

are then prepared such that a polystyrene bead is attached to

each end of the filament These beads can be “caught” and held

in place in solution by a pair of “optical traps”—two

high-intensity infrared laser beams, one focused on the polystyrene

bead at one end of the actin filament and the other focused on

the bead at the other end of the actin filament The force acting

on each bead in such a trap is proportional to the position of

the bead in the “trap,” so displacement and forces acting on the

bead (and thus on the actin filament) can both be measured

When the “trapped” actin filament is brought close to the

myosin-coated silica bead, one or a few myosin molecules may

interact with sites on the actin and ATP-induced interactions of

individual myosin molecules with the trapped actin filament can

be measured and quantitated Such optical trapping

experi-ments have shown that a single cycle or turnover of a single myosin

molecule along an actin filament involves an average movement of 4 to

11 nm (40–110 Å) and generates an average force of 1.7 to 4  10 12

newton (1.7–4 piconewtons [pN]).

The magnitudes of the movements observed in the optical

trap-ping experiments are consistent with the movements predicted by

the cryoelectron microscopy imaging data Can the movements

and forces detected in a single contraction cycle by optical

trap-ping also be related to the energy available from hydrolysis of a

sin-gle ATP molecule? The energy required for a contraction cycle is

defined by the “work” accomplished by contraction, and work (w)

is defined as force (F) times distance (d):

w  F  d

For a movement of 4 nm against a force of 1.7 pN, we have

w (1.7 pN)  (4 nm)  0.68  1020J

For a movement of 11 nm against a force of 4 pN, the energy re-quirement is larger:

w (4 pN)  (11 nm)  4.4  1020J

If the cellular free energy of hydrolysis of ATP is taken as 50 kJ/ mol, the free energy available from the hydrolysis of a single ATP molecule is

G  (50 kJ/mol)/(6.02  1023molecules/mol) 8.3  1020J

Thus, the free energy of hydrolysis of a single ATP molecule is sufficient to drive the observed movements against the forces that have been measured.

Actin Polystyrene beads

䊱 Movements of single myosin molecules along an actin filament can be

measured by means of an optical trap consisting of laser beams focused

on polystyrene beads attached to the ends of actin molecules (Adapted from Finer, J T., et al., 1994 Single myosin molecule mechanics: Piconewton forces and nanometre

steps Nature 368:113–119 See also Block, S M., 1995 Macromolecular physiology Nature 378:132–133.)

as first noticed by Szent-Györgyi This dissociation occurs with “opening” of switch

2, in which the switch 2 segment (the lower part of strand 5 and a short following loop) moves out of the plane of the seven-stranded -sheet (Figure 16.10)

ATP forms a strong interaction with switch 1 in the myosin active site, inducing switch 1 to close (moving toward the - and -phosphates of bound ATP),

presum-ably also causing switch 2 to close (with the lower part of strand 5 moving back into the plane of the seven-stranded -sheet) Switch 2 closing induces formation of the

kink in the relay helix, causing a 60° rotation of the converter domain and neck he-lix into the up position relative to the myosin head This movement completes the cycle of contraction as it “primes” the motor, preparing it for the next power stroke This cycle is repeated at rates up to 5/sec in a typical skeletal muscle contraction The conformational changes occurring in this cycle are the secret of the energy coupling that allows ATP binding and hydrolysis to drive muscle contraction

16.3 What Are the Molecular Motors That Orchestrate

the Mechanochemistry of Microtubules?

Filaments of the Cytoskeleton Are Highways That Move Cellular Cargo

Most eukaryotic cells contain elaborate networks of protein fibers collectively

termed the cytoskeleton The cytoskeleton is a dynamic, three-dimensional

struc-ture (Figure 16.11) that fills the cytoplasm and functions to:

• Establish cell shape

• Provide mechanical strength

• Facilitate cell movement

• Support intracellular transport of organelles and other cellular cargo

• Guide chromosome separation during mitosis and meiosis Three types of fibers comprise the cytoskeleton: microfilaments of actin (with a diameter of 3 to 6 nm), microtubules made from tubulin (20 to 25 nm diameter), and intermediate filaments formed from a variety of proteins (about 10 nm diame-ter) All of these have dynamic properties that facilitate the movement of organelles and other molecular cargo through the cell

Intermediate filaments provide a supporting network that allows cells to resist mechanical stress and deformation Intermediate filaments are dynamic, and short filament segments (termed “squiggles” by Robert Goldman and his co-workers) can

be transported across cells by motor proteins riding on microfilaments and micro-tubules Polymeric actin microfilaments serve at least two functions in cells: They form networks just beneath the plasma membrane that link transmembrane pro-teins to cytoplasmic propro-teins, and they provide transcellular tracks on which or-ganelles can be transported by myosin-like proteins Microtubules are the best un-derstood components of the cytoskeleton, and they are the focus of this section

Microtubulesare hollow, cylindrical structures, approximately 30 nm in diameter,

formed from tubulin, a dimeric protein composed of two similar 55-kD subunits

known as -tubulin and -tubulin Eva Nogales, Sharon Wolf, and Kenneth Downing

have determined the structure of the bovine tubulin -dimer to 3.7 Å resolution

(Fig-ure 16.12a) Tubulin dimers polymerize as shown in Fig(Fig-ure 16.12b to form micro-tubules, which are essentially helical structures, with 13 tubulin monomer “residues” per turn Microtubules grown in vitro are dynamic structures that are constantly being assembled and disassembled Because all tubulin dimers in a microtubule are oriented similarly, microtubules are polar structures The end of the microtubule at which

growth occurs is the plus end, and the other is the minus end Microtubules in vitro carry out a GTP-dependent process called treadmilling, in which tubulin dimers are

Switch 2 open

Switch 2 closed

Helix W

Relay helix

Switch 1

Switch 2 P-loop

Converter

SH2 helix SH1 helix

ATP

( a) Post-rigor state

( b) Pre-power stroke state

7

6

5 3 2

7

6

5 3 2

Relay helix kink

䊴 FIGURE 16.10 Details of the switch domains, the relay helix and the converter domain are shown for (a) the post-rigor state and (b) the pre-power stroke state of skeletal muscle myosin ATP hydrolysis drives these conformation

changes Actin binding induces a twist in the large -sheet of the myosin head, causing the switch 1 and P-loop

segments to “open.”(Adapted from Geeves, M., and Holmes, K., 2005.The molecular mechanism of muscle contraction Advances in

Protein Chemistry 71:161–193 Figure provided by Kenneth Holmes, Max Plank Institute for Medical Research, Heidelberg.)

added to the plus end at about the same rate at which dimers are removed from the

minus end (Figure 16.13)

Although composed only of 55-kD tubulin subunits, microtubules can grow

suffi-ciently large to span a eukaryotic cell or to form large structures such as cilia and

fla-gella Inside cells, networks of microtubules play many functions, including formation

of the mitotic spindle that segregates chromosomes during cell division, the

move-ment of organelles and various vesicular structures through the cell, and the variation

and maintenance of cell shape In most cells, microtubules are oriented with their

mi-nus ends toward the centrosome and their plus ends toward the cell periphery This

consistent orientation is important for mechanisms of intracellular transport

(a)

(d)

Mitochondrion

Ribosome

Intermediate filaments Microfilaments

Microtubule

Endoplasmic reticulum

Plasma membrane

(g)

FIGURE 16.11 Micrographs and electron micrographs of cytoskeletal

ele-ments, cilia, and flagella: (a) microtubules (shown in red); (b) rat sperm tail

microtubules (cross section); (c) Stylonychia, a ciliated protozoan

(under-going division); (d) cytoskeleton of a eukaryotic cell (microtubules are

green; nucleus is blue); (e) Pseudomonas fluorescens (aerobic soil

bac-terium), showing flagella; (f) nasal cilia; and (g) schematic drawing of

ele-ments of the cytoskeleton.

Three Classes of Motor Proteins Move Intracellular Cargo

Three principal classes of motor proteins move organelles and other cellular cargo

on cytoskeletal filament highways in both eukaryotic and prokaryotic cells In addi-tion to the myosins, most cells contain kinesins and dyneins Humans possess

40 genes for myosins, 45 for kinesins, and at least 14 for dyneins (Table 16.1) The large number of genes in each class reflects specialized structures required for a va-riety of functions This diversity notwithstanding, these three classes of motor pro-teins share remarkable similarities of structure and function, as we shall see

Kinesin 1,also called conventional kinesin, is a tetramer consisting of a dimer of heavy chains (110 kD) associated with two light chains (65 kD) The heavy chains contain an N-terminal motor domain, a long coiled-coil stalk with a central hinge, and a globular C-terminal tail domain where the light chains bind (Figure 16.14) The motor domain binds to tubulin in microtubules, and the globular tail domain associates with the intended cellular cargo, for example, an organelle, an mRNA molecule, or an intermediate filament In different kinesin families, the motor

Tubulin heterodimer (8 nm)

Protofilament

24 nm

(b)





FIGURE 16.12 (a) The structure of the tubulin -heterodimer (pdb id  1JFF) (b) Microtubules may be viewed

as consisting of 13 parallel, staggered protofilaments of alternating -tubulin and -tubulin subunits.The

se-quences 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.

-Tubulin

-Tubulin

GDP

GTP

(a)

Plus end

(growing end)

Minus end

Dimers off

Dimers on





ACTIVE FIGURE 16.13 A model of

the GTP-dependent 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

begin-ning to be understood in detail Test yourself on the

concepts in this figure at www.cengage.com/login.

Number of Genes

Giardia lamblia (protozoan parasite) 25 10 0

Saccharomyces cerevisiae (yeast) 25 1 5

Drosophila melanogaster (fruit fly) 25 13 13

Caenorhabditis elegans (roundworm) 20 2 17

Arabidopsis thaliana (flowering plant) 61 0 17

TABLE 16.1 Genes for Molecular Motors