16.2 What Is the Molecular Mechanism of Muscle Contraction? 483 1 to 2 m in diameter. Myofibrils are linear arrays of cylindrical sarcomeres, the 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 Ca 2ϩ , and the release of Ca 2ϩ 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 Ca 2ϩ ions from the SR. These Ca 2ϩ ions 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 I band I bandA band Z line Z lineH zone 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. Courtesy of Hugh Huxley, Brandeis University 484 Chapter 16 Molecular Motors 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, Ca 2ϩ ions 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 – NH 3 + NH 3 + (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 ϭ 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 ϭ 1J6Z). FIGURE 16.4 A molecular model of an actin polymer, based on the actin monomer structure shown in Figure 16.3 (pdb id ϭ 1A5X). 16.2 What Is the Molecular Mechanism of Muscle Contraction? 485 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 ϫ 7) residue and 196 (28 ϫ 7) residue levels, specialized amino acid sequence patterns promote packing of large numbers of myosin tails in offset or staggered arrays (Figure 16.7). a d d a b c c b e g f f g e 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, mechanical 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 AAAϩ 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). Myosin heads Bare zone 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. ᮣ P-loops of histidine permease (pdb id ϭ 1B0U, orange) and HprK protein kinase (pdb id ϭ 1KKL, green). ATP is shown in light blue. Go to CengageNOW at www.cengage.com/login and click BiochemistryInteractive to explore myosin. 486 Chapter 16 Molecular Motors 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/ hing e 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 dystro p hin structure are indicated. Hinge 1 Hinge 2 Hinge 3 Hinge 4 1N N 23 456789101112 1234 1234 C 56789 1011121314151617181920 C 13 14 15 16 17 18 19 20 21 C22 23 24 Dystrophin 16.2 What Is the Molecular Mechanism of Muscle Contraction? 487 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 Dystro p hin C-terminus ᮤ 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.) A band I band 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. 488 Chapter 16 Molecular Motors 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 number more like that of intact muscle fibers. Szent-Györgyi’s experiments demonstrated that ATP hydrolysis and the associa- tion and dissociation 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 Attached Power ATP hydrolysis and recovery Resting muscle Rigor-like Top-of-power stroke Post rigor Pre-power stroke Detached ATP ATP P i , ADP P i ADP P i ADP Switch 1 open Switch 2 closed A ctin-cleft closed -sheet twisted Switch 1 open Switch 2 closed Actin-cleft closed -sheet twisted Switch 1 open Switch 2 open A ctin-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. 16.2 What Is the Molecular Mechanism of Muscle Contraction? 489 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 P i bound—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 P i (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 P i 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 ϫ 10 Ϫ12 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 ϭ F и d For a movement of 4 nm against a force of 1.7 pN, we have w ϭ (1.7 pN) и (4 nm) ϭ 0.68 ϫ 10 Ϫ20 J For a movement of 11 nm against a force of 4 pN, the energy re- quirement is larger: w ϭ (4 pN) и (11 nm) ϭ 4.4 ϫ 10 Ϫ20 J If the cellular free energy of hydrolysis of ATP is taken as Ϫ50 kJ/ mol, the free energy available from the hydrolysis of a single ATP molecule is ⌬G ϭ (Ϫ50 kJ/mol)/(6.02 ϫ 10 23 molecules/mol) ϭ 8.3 ϫ 10 Ϫ20 J 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 Myosin Silica bead Optical trap Optical trap ᮡ 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.) 490 Chapter 16 Molecular Motors 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 proteins, 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. Microtubules are 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 Figure 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 ᮤ 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.) 16.3 What Are the Molecular Motors That Orchestrate the Mechanochemistry of Microtubules? 491 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) (e) (f) (d) (b) (c) © Dr. Gopal Murti/Visuals Unlimited David Phillips/Visuals Unlimited Eric Grave/Phototake © Thomas Deerinck/Visuals Unlimited Dr. Tony Brain/Custom Medical Stock © Veronika Burmeister/Visuals Unlimited 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. 492 Chapter 16 Molecular Motors 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 ϭ 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 Ca 2ϩ 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 Genome Kinesins Dyneins Myosins 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 Homo sapiens (human) 45 14–16 40 TABLE 16.1 Genes for Molecular Motors