LEHNINGER PRINCIPLES OF BIOCHEMISTRY - VOL 1. THE FOUNDATIONS OF BIOCHEMISTRY: STRUCTURE AND CATALYSIS - 2011

PART I. STRUCTURE AND CATALYSIS

5. PROTEIN FUNCTION

5.3. Reversible Interactions of Proteins Involving Ligands: Actin, Myosin, and Molecular Motors

Living things move. Cells move. Organelles and macromolecules within cells move. Much of this movement depends on The activity of a remarkable Class of Proteins known as Molecular Motors. Driven by chemical energy—typically derived from ATP—large assemblies of motor proteins undergo cycles of conformational change that translate into coordinated, directional motion. These movements range from the delicate division of Chromosomes in a replicating Cell to the immense force generated by a leaping marsh cat weighing up to twenty kilograms.

As you might predict, interactions between motor proteins and their tracks are mediated by the familiar ensemble of ionic, hydrophobic, and Structure/103.html">Van der Waals interactions, as well as Hydrogen Bonds within the protein binding sites. In motor proteins, however, these interactions achieve an extraordinarily high degree of spatial and temporal Organization.

Motor protein activity underlies Muscle contraction, The transport of organelles along microtubules, the rotation of bacterial flagella, and the translocation of certain proteins along DNA strands. Kinesins and dyneins move along cellular microtubules, dragging organelles or reorganizing chromosomes during Cell Division. The interaction of dynein with microtubules drives the beating of eukaryotic Cilia and flagella. Bacterial flagellar motion is powered by a complex rotary motor embedded at the Base of the flagellum (Fig. 19-39). During various Stages of DNA METABOLISM, DNA helicases, polymerases, and other proteins must move along DNA molecules (Chapter 25). In this chapter, using the classic contractile proteins of vertebrate Skeletal Muscle as a paradigm, we examine how proteins convert chemical energy into mechanical motion.

Myosin and Actin Are the Major Proteins of Muscle

The driving force of muscle contraction is the interaction between two major proteins: myosin and actin. These proteins are organized into filaments whose sliding past one another causes muscle to shorten. Together, Actin and myosin account for over 80% of total muscle protein mass.

Myosin (Mr ≈ 540,000) consists of six polypeptide chains: two heavy chains (Mr ≈ 220,000) and four light chains (Mr ≈ 20,000). The heavy chains form the structural backbone of myosin. Their C-terminal segments are arranged in extended α helices that wrap around one another to form a left-handed coiled coil, resembling the α-keratin structure (Fig. 5-27a). At the N-terminus, each heavy chain folds into a large globular domain containing the site that hydrolyzes ATP. The light chains are associated with these globular domains. When myosin is briefly treated with the protease Trypsin, the long tail region is cleaved, producing two distinct fragments termed heavy meromyosin and light meromyosin (Fig. 5-27b). Subfragment S1—which contains the globular HEAD and is commonly called the myosin "head"—is released from heavy meromyosin by Papain Digestion. The myosin head acts as the motor unit that powers muscle contraction. S1 fragments can be crystallized, and their three-dimensional structure has been resolved by Ivan Rayment and Hazel Holden (Fig. 5-27c).

Fig. 5-27. Myosin. (a) Myosin features two heavy chains (depicted in different shades of pink) whose C-termini form extended coiled-coil "tails" and whose N-termini fold into globular domains ("heads"). Two light chains (shown in blue) are associated with each myosin head. (b) Cleavage by trypsin and papain separates the head (S1 fragment) from the tail of the molecule. (c) Ribbon diagram of the myosin S1 fragment. The heavy chain is shown in gray, and the two light chains in different shades of blue (coordinates provided by Ivan Rayment).

Within muscle cells, myosin molecules aggregate to form thick filaments (Fig. 5-28a). These rod-like structures serve as the backbone of the contractile unit. In a thick filament, hundreds of myosin molecules are arranged such that their tails form a long bipolar shaft, with their globular domains protruding in a regular pattern from both ends.

Fig. 5-28. Major elements of muscle. (a) Myosin molecules assemble into bipolar structures called thick filaments. (b) F-actin is a polymer of monomeric G-actin units; two F-actin strands wind around each other to form a right-handed helix. (c) Molecular model of an actin filament (depicted in various shades of red), with one monomer bound to a myosin head (shown in blue and gray; coordinates provided by Ivan Rayment).

The second major muscle protein is actin, which is abundant in virtually all Eukaryotic cells. In muscle, monomeric actin molecules—G-actin (globular actin; Mr = 42,000)—are polymerized into long, fibrous chains known as F-actin (filamentous actin). Thin filaments (Fig. 5-28c) are composed of F-actin along with the regulatory proteins troponin and Tropomyosin. Thin filaments grow by the sequential addition of actin monomers to one end. Furthermore, each actin monomer binds an ATP molecule and hydrolyzes it to ADP, meaning that every actin monomer within the filament exists as an ADP complex. Consequently, ATP Hydrolysis by actin occurs exclusively during filament assembly; ATP does not deliver its energy directly at the moment of contraction. Each actin monomer in the thin filament can bind specifically and tightly to a single myosin head (Fig. 5-28c).

Other Proteins Organize Thin and Thick Filaments into Ordered Arrays

Skeletal muscle is composed of parallel bundles of muscle fibers. Each fiber is a single, extraordinarily large, multinucleated cell ranging from 20 to 100 µm in diameter. These cells arise during development through the fusion of numerous precursor cells and often extend the entire length of the muscle. A single fiber contains about 1,000 myofibrils (each roughly 2 µm in diameter), which in turn contain massive arrays of regularly packed thin and thick filaments complexed with accessory proteins (Fig. 5-29). Each myofibril is surrounded by a network of flattened membranous vesicles called the sarcoplasmic reticulum. Under the Electron microscope, muscle fibers display alternating regions of high and low electron density known as the A and I bands (Fig. 5-29b, c). These banding patterns result from the precise overlap of thin and thick filaments. The I band corresponds to the region containing exclusively thin filaments. The darker A band encompasses the full length of the thick filaments, including the zones where thick and thin filaments overlap. The I band is bisected by the Z disc, a dense Protein Structure perpendicular to the axis of the thin filaments that serves as an anchor for their attachment. Similarly, the A band is bisected by the M line, or M disc, a region of high electron density at the center of the thick filaments. The entire contractile unit—consisting of a central stack of thick filaments flanked by interdigitating thin filaments—is called a sarcomere. This interdigitated arrangement allows thin and thick filaments to slide past one another (via the sliding-filament mechanism described below), resulting in the progressive shortening of each sarcomere (Fig. 5-30).

Fig. 5-29. Structure of skeletal muscle. (a) A muscle fiber is a single elongated multinucleated cell formed by the fusion of many precursor cells. Within the fiber, numerous myofibrils (only six are shown here for clarity) are enclosed by the sarcoplasmic reticulum membrane. Electron Microscopy reveals alternating light and dark bands formed by the myofibrillar filaments. During contraction, the I band narrows and adjacent Z discs move closer together. (b) Relaxed muscle. (c) Contracted muscle.

Fig. 5-30. Muscle contraction. Thick filaments are bipolar structures formed by the association of numerous myosin molecules. (a) Muscle contraction occurs as thin and thick filaments slide past each other, drawing the Z discs of adjacent I bands closer together. (b) Thin and thick filaments interdigitate such that each thick filament is surrounded by six thin filaments.

Thin actin filaments are anchored at one end to the Z disc. Proteins such as α-actinin, desmin, and vimentin participate in forming this attachment. In addition, thin filaments contain nebulin, a giant protein (comprising roughly 7,000 amino acid residues) thought to adopt an extended α-helical conformation that spans the entire length of the filament. Similarly, the M line organizes thick filaments with the help of structural proteins such as paramyosin, C-protein, and M-protein. Another class of proteins, the titins, represents the largest single-polypeptide chains known (human cardiac titin consists of 26,926 amino acid residues). Titin links the thick filaments to the Z disc, providing an additional level of structural organization. Nebulin and titin are believed to act as "molecular rulers" that regulate the length and thickness of thin and thick filaments, respectively. Titin extends from the Z disc to the M line, Setting the resting length of the sarcomere and preventing overextension of the muscle. Sarcomere length varies across different tissue types, a diversity linked, in part, to the expression of multiple titin isoforms in vertebrates.

Myosin Thick Filaments Slide Along Actin Thin Filaments

The interaction between actin and myosin, like all Protein-Ligand interactions, is driven by weak noncovalent forces. In the absence of bound ATP, the myosin head binds tightly to actin (Fig. 5-31). When myosin binds and hydrolyzes an ATP molecule to ADP and inorganic phosphate, it undergoes a cyclic series of conformational changes during which it releases one F-actin subunit and binds to the next one along the filament.

Fig. 5-31. Molecular Mechanism of muscle contraction. Conformational Changes in the myosin "head" coupled to the ATP hydrolysis cycle lead to the dissociation of myosin from its complex with one actin subunit and its binding to another located further along the thin filament. This is how thick filaments slide past thin filaments (see Fig. 5-30).

This cycle consists of four main stages (Fig. 5-31). In stage 1, an ATP molecule binds to myosin, the Actomyosin complex dissociates, and actin is released. In stage 2, the ATP molecule is hydrolyzed, inducing conformational Changes in the protein that shift it into a higher-energy state; As a result, the myosin head pivots and changes its orientation relative to the actin filament. A weak bond then forms between the myosin head and the next actin monomer unit located closer to the Z disc. In Stage 3, the phosphate group generated during ATP hydrolysis dissociates from myosin, which triggers another conformational change in the myosin molecule leading to a tighter interaction within the actomyosin complex. Next, in stage 4, the myosin head returns to its initial state, causing the myosin tail to shift relative to the actin filament toward the Z disc. Finally, the ADP molecule is released. Each such cycle performs work equivalent to 3-4 nN, displacing the thick filament relative to the thin filament by 5-10 nm.

A thick filament contains numerous myosin heads, and at any given moment only a small fraction of them (likely 1-3%) are bound to thin filaments. This prevents the thick filament from slipping backward at the moment an individual myosin head releases the actin subunit to which it was attached. In this manner, thick filaments are actively propelled along thin filaments. This process, coordinated across all sarcomere units of a muscle fiber, results in muscle contraction.

The interaction between actin and myosin must be regulated so that muscle contraction occurs only in response to an appropriate Nervous system signal. This regulation is mediated by two proteins—tropomyosin and troponin (Fig. 5-32). Tropomyosin binds to thin filaments and blocks the binding sites for the myosin heads. Troponin is a Ca²⁺-binding protein. A Nerve Impulse triggers the release of Ca²⁺ from the sarcoplasmic reticulum. Ca²⁺ ions bind to troponin (yet another example of protein-ligand interaction), inducing conformational changes in the troponin-tropomyosin complex that expose the myosin-binding site on the thin filament, thereby initiating muscle contraction.

Fig. 5-32. Regulation of Muscle contraction by tropomyosin and troponin. Tropomyosin and troponin are associated with F-actin in thin muscle filaments. In relaxed muscle, both proteins wrap around the actin filament, blocking the myosin-binding sites. Tropomyosin is an α-helical protein consisting of two Peptides that form a coiled coil (sharing the same structural motif as α-keratin; Fig. 4-10). It forms head-to-tail polymers that wind around the two actin chains. Troponin attaches to the actin/tropomyosin complex at regular intervals of 38.5 nm. Troponin consists of three distinct subunits: I, C, and T. Troponin I prevents the myosin head from binding to actin; troponin C contains a binding site for Calcium Ions; and troponin T anchors the entire troponin complex to tropomyosin. When the muscle receives a signal to contract, calcium ions are released from the sarcoplasmic reticulum (Fig. 5-29a) and bind to troponin C. This induces conformational changes in troponin C, which alters THE POSITION OF troponin I and tropomyosin, relieving the inhibition by troponin I and enabling muscle contraction.

During skeletal Muscle Function, proteins perform their two canonical roles—binding and catalysis. The interaction of actin with myosin and IMMUNOGLOBULINS with Antigens represent classic instances of protein-ligand interactions. These processes are reversible, leaving the participating molecules unchanged upon completion. However, the ATP bound to myosin undergoes hydrolysis to yield ADP and inorganic phosphate. Thus, myosin is not merely an actin-binding protein, but also an enzyme (an ATPase). The catalytic activity of Enzymes is discussed in detail in the following chapter.

Summary of Section 5.3 Energy-Dependent Protein Interactions: Actin, Muscle, and Molecular Motors

■ In motor proteins, protein-ligand interaction achieves an exceptionally high degree of spatial and temporal organization. Muscle contraction results from the fine-tuned interaction between myosin and actin, coupled with myosin-catalyzed ATP hydrolysis.

■ Myosin is composed of two heavy chains and four light chains that form a long helical domain (the "tail") and a globular domain (the "head"). Myosin molecules assemble into thick filaments capable of sliding along thin filaments, which are based on actin. Myosin-catalyzed ATP hydrolysis drives a series of conformational changes in the myosin head, causing myosin to dissociate from its complex with one actin subunit and bind to another located further along the thin filament. In this way, myosin filaments glide past thin actin filaments.

■ Muscle contraction is stimulated by the release of Ca²⁺ from the sarcoplasmic reticulum. Ca²⁺ ions bind to the protein troponin, inducing conformational changes in the troponin-tropomyosin complex and initiating the cyclical interaction between actin and myosin.