Molecular Motors Biochemistry by Reginald Garrett and Charles Grisham Chapter 16 Molecular Motors Biochemistry by Reginald Garrett and Charles Grisham

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?

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?

Figure 16. 2 (a) The structure of the tubulin  heterodimer 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 GTP-dependent treadmilling process Figure 16.3 A model of the GTP-dependent treadmilling process. Both a- and b-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

Figure 16. 5 The structure of an axoneme 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 A-tubule of each outer pair possesses an outer dynein arm and an inner dynein arm. The larger-diameter 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 B-tubule 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 a and b (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 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.

Highways for "molecular motors" 16.2 – What Are the Molecular Motors That Orchestrate the Mechanochemistry of Microtubules? 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)

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)

The structures of vinblastine, vincristine, colchicine, and taxol.

16.3 – How Do Molecular Motors Unwind DNA? 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

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 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

What are t-tubules and SR for 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

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)

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. 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. (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 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

Figure 16.16  (a) An electron micrograph of a myosin molecule and a corresponding schematic drawing. The tail is a coiled coil of intertwined a-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 seven-residue 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, a-helices will coil!)

Figure 16.17 An axial view of the two-stranded, a-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

Figure 16. 18 The packing of myosin molecules in a thick filament 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

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

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 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 - , ,  - all transmembrane - defects linked to limb-girdle MD and autosomal recessive MD

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

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 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

Figure 16. 20 The mechanism of skeletal muscle contraction 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

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 288:88-95.)

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+ 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

Figure 16. 23 Ca2+ is the trigger signal for muscle contraction 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

The "foot structure" in terminal cisternae of SR 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!

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!

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

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

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 a-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

Smooth Muscle Effectors Useful drugs Epinephrine (as Primatene) is an over-the-counter 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

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!

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 55:905-914)