MYLAN EXHIBIT - 1012 (Part 4)
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd. -
`
`

`

`Chapter 7 Protein Function
`
`227
`
`The class I MHC protein—peptide complexes on infected cells are rec-
`ognized as foreign and bound by those T,, cells with T-cell receptors having
`the appropriate binding specificity. The T-cell receptors respond only to
`peptide antigens that are complexed to class | MHC proteins. The T; cells
`have an additional receptor, CD8, also called a coreceptor, that enhances
`the binding interactions of T-cell receptors and MHCproteins (Fig. 7-22,
`middle left). The T, cells live up to the namekiller T cells by destroying the
`virally infected cell to which they are complexed through their T-cell re-
`ceptors. Cell death is brought about by a number of mechanisms, not all
`well understood. One mechanism involves the release of a protein called
`perforin, which binds to and aggregates in the plasma membraneof the
`target cell, forming molecular pores that destroy the capacity of that cell
`to regulate its interior environment. T, cells also induce a process called
`programmed cell death, or apoptosis (most commonly pronounced
`app’-a-toe’-sis), in which the cells complexed to T, cells undergo metabolic
`changes that rapidly lead to the demise of the cell.
`T. cells with the proper specificity must proliferate selectively if large
`numbers of virus-infected cells are to be destroyed. To this end, Te cells
`complexed to an infected cell generate cell-surface receptors for signaling
`proteins called interleukins. Interleukins, secreted by a variety of cells,
`stimulate the proliferation of only those T and B cells bearing the required
`interleukin receptors. Because T and B cells produceinterleukin receptors
`only when they are complexed with an antigen, the only immune system
`cells that proliferate are those few that can respond to the antigen. The
`process of producing a population of cells by stimulated reproduction of a
`particular ancestorcell is called clonal selection.
`The peptides complexed to class II MHC proteins and displayed on the
`surface of macrophages and B lymphocytes are similarly bound by the ap-
`propriate T-cell receptors of T,, cells. The T} cells also have a coreceptor,
`called CD4, that enhances the binding interactions of the T-cell receptors.
`This overall binding interaction, in concert with secondary molecularsig-
`nals that are currently being identified, activates the T,, cells. A subpopula-
`tion of activated T,, cells secrete a small signal protein called interleukin-2
`(IL-2; M, 15,000), which stimulates proliferation of nearby T, cells and Ty
`cells having the appropriate interleukin receptors. This greatly increases
`the numberof available immune system cells capable of recognizing and re-
`sponding to the antigen. Another subpopulation of activated T, cells com-
`plexed to macrophages or B lymphocytes secrete interleukin-4 (IL-4; /,
`20,000), which stimulates the proliferation of B cells that recognize the
`antigen (Fig. 7-22, bottom right). Proliferation of the responding B, Ty, and
`Ty cells continues as long as the appropriate antigen is present.
`The proliferating B cells promote the destruction of any extracellular
`viruses or bacterial cells. They first secrete large amounts of soluble anti-
`body that binds to the antigen. This bound antibody recruits a cellular sys-
`tem of about 20 proteins collectively called complement because they com-
`plement and enhancethe actionof the antibodies. The complement proteins
`disrupt the coats of many virusesor, in bacterial infections, produce holes in
`the cell walls of bacteria, causing them to swell and burst by osmotic shock.
`Unlike T cells, B cells do not undergo selection in the thymusto elimi-
`nate those producing antibodies that recognize host (self) proteins. How-
`ever, B cells do not contribute significantly to an immune response unless
`they are stimulated to proliferate by T,, cells. The T, cells do undergo se-
`lection in the thymus, leaving no T, cells capable of stimulating B cells that
`produce antibodies potentially dangerous to the host.
`The Ty cells themselves participate only indirectly in the destruction of
`infected cells and pathogens, buttheir role is critical to the entire immune
`
`
`
`

`

`228
`
`Part || Structure and Catalysis
`
`response. This is dramatically illustrated by the epidemic produced by HIV
`(human immunodeficiency virus), the virus that causes AIDS (acquired im-
`mune deficiency syndrome). The primary targets of HIV infection are Ty
`cells. Elimination of these cells progressively incapacitates the entire im-
`mune system.
`Once antigen is depleted,activated immunecells generally die in a mat-
`ter of days by programmed cell death. However, a few of the stimulated B
`and T cells mature into memory cells. These are long-lived cells that do
`notparticipate directly in the primary immune response when the antigen
`is first encountered. Instead they become permanentresidentsof the blood,
`ready to respond to a reappearance of the same antigen. Memory cells,
`when subsequently challenged by the antigen, can mount a secondary im-
`mune response that is generally much more rapid and vigorous than the
`primary response because of prior clonal expansion. By this mechanism,
`vertebrates once exposed to a virus or other pathogen can respond quickly
`to the pathogen when exposed again. This is the basis of the long-term im-
`munity conferred by vaccines and the natural immunity to repeated infec-
`tions by the samestrain of a virus.
`
`figure 7-23
`The structure of immunoglobulin G. (a) Pairs of heavy
`and light chains combine to form a Y-shaped molecule.
`Two antigen-binding sites are formed by the combination
`of variable domains from onelight (V,) and one heavy
`(V4) chain. Cleavage with papain separates the Fab and
`Fe portions of the protein in the hinge region. The Fe
`portion of the molecule also contains bound carbo-
`hydrate. (b) A ribbon model of the first cornplete IgG
`molecule to be crystallized and structurally analyzed.
`Although the molecule contains two identical heavy
`chains (two shades of blue) and two identical light chains
`(two shadesof red), it crystallized in the asymmetric con-
`formation shown. Conformational flexibility may be impor-
`tant to the function of immunoglobulins.
`
`Antigen-
`binding
`site
`/
`Papain
`hays /
`cleavage
`
`sites=Vy f Ne
`
`Antibodies Have Two Identical Antigen-Binding Sites
`Immunoglobulin G (IgG) is the major class of antibody molecule and one
`of the most abundantproteins in the blood serum. IgG has four polypeptide
`chains: two large ones, called heavy chains, and twolight chains, linked by
`noncovalent and disulfide bonds into a complex of M, 150,000. The heavy
`chains of an IgG molecule interact at one end, then branch to interact sep-
`arately with the light chains, forming a Y-shaped molecule (Fig. 7-23). At
`the “hinges” separating the base of an IgG molecule fromits branches, the
`immunoglobulin can be cleaved with proteases. Cleavage with the protease
`papain liberates the basal fragment, called Fe becauseit usually crystallizes
`readily, and the two branches, which are called Fab, the antigen-binding
`
`fragments, Each branchhas a single antigen-bindingsite.
`
`
`
`
`
`Cyl
`
`A
`
`fee : Aee C%
`
`ous
`
`cog”
`
`C = constant domain
`V = variable domain
`
`H, L = heavy,light chains
`
`(a)
`
`

`

`The fundamental structure of immunoglobulins wasfirst established by
`Gerald Edelman and Rodney Porter. Each chain is made up of identifiable
`domains; some are constant in sequence andstructure from one IgG to the
`next, others are variable. The constant domains have a characteristic struc-
`ture known as the immunoglobulin fold, a well-conserved structural mo-
`tif in the all-@ class. There are three of these constant domains in each
`heavy chain and onein each light chain. The heavy and light chains also
`have one variable domain each, in which most of the variability in amino
`acid residue sequenceis found. The variable domains associate to create
`the antigen-binding site (Fig. 7-24).
`
`Chapter 7 Protein Function
`
`229
`
`figure 7-24
`Binding of IgG to an antigen. To generate an optimal
`fit for the antigen, the binding sites of IgG often undergo
`slight conformational changes. Such inducedfit is
`common to manyprotein-ligand interactions.
`
`~ooc
`
`coo
`
`Antibody
`
`Antigen-antibody complex
`
`In many vertebrates, IgG is only oneoffive classes of immunoglobulins.
`Fachclass has a characteristic type of heavy chain, denoted a, 6, e, y, and
`uw for IgA, IgD, IgE, IgG, and IgM, respectively. Two types of light chain,
`x and X, occur in all classes of immunoglobulins. The overall structures of
`IgD and IgE are similar to that of IgG. IgM occurs in either a monomeric,
`membrane-bound form or a secreted form that is a cross-linked pentamer
`of this basic structure (Fig. 7-25). IgA, found principally in secretions such
`as saliva, tears, and milk, can be a monomer, dimer, or trimer. [gM is the first
`antibody to be made by B lymphocytes and is the major antibody in the
`early stages of a primary immuneresponse. Some B cells soon begin to pro-
`duce IgD (with the same antigen-binding site as the IgM produced by the
`same cell), but the unique function of IgD is less clear.
`
`pu Heavy
`
` Boe
`chains
`
`figure 7-25
`IgM pentamer of immunoglobulin units. The pentamer
`is cross-linked with disulfide bonds. The J chain is a
`polypeptide of M, 20,000 found in both IgA and IgM.
`
`
`
`

`

`230
`
`Part Il Structure and Catalysis
`
`The IgG described aboveis the major antibody in secondary immunere-
`sponses, which are initiated by memory B cells. As part of the organism's
`ongoing immunity to antigens already encountered and dealt with, IgG is
`the most abundant immunoglobulin in the blood. When IgG binds to an in-
`vading bacterium or virus, it not only activates the complement system, but
`also activates certain leukocytes such as macrophagesto engulf and destroy
`the invader. Yet anotherclass of receptors onthecell surface of macrophages
`recognizes and binds the Fc region of IgG. When these Fc receptors bind an
`antibody-pathogen complex,
`the macrophage engulfs the complex by
`phagocytosis (Fig. 7-26).
`
`figure 7-26
`Phagocytosis of an antibody-bound virus by a
`macrophage. The Fc regions of the antibodies bind to Fc
`receptors on the surface of the macrophage, triggering
`the macrophage to engulf and destroy the virus.
`
`virus
`
`
`
`Phagocytosis
`>
`
`Macrophage
`
`IgG-coated
`
`Fe region
`of IgG
`
`
`
`IgG Fe
`receptor
`
`IgE plays an importantrole in the allergic response,interacting with ba-
`sophils (phagocytic leukocytes) in the blood and histamine-secreting cells
`called mast cells that are widely distributed in tissues. This immunoglobu-
`lin binds, throughits Fe region, to special Fc receptors on the basophils or
`mast cells. In this form, IgE serves as a kind of receptor for antigen.If anti-
`gen is bound,the cells are induced to secrete histamine and otherbiologi-
`cally active amines that cause dilation and increased permeability of blood
`vessels. These effects on the blood vessels are thought to facilitate the
`movement of immune system cells and proteins to sites of inflammation.
`They also produce the symptoms normally associated with allergies. Pollen
`or other allergens are recognizedas foreign, triggering an immune response
`normally reserved for pathogens.
`
`Antibodies Bind Tightly and Specifically to Antigen
`The binding specificity of an antibody is determined by the amino acid
`residuesin the variable domainsof its heavy and light chains. Many residues
`in these domains are variable, but not equally so. Some, particularly those
`lining the antigen-bindingsite, are hypervariable—especially likely to differ.
`Specificity is conferred by chemical complementarity between the antigen
`and its specific bindingsite, in terms of shape andthe location of charged,
`nonpolar, and hydrogen-bonding groups. For example, a bindingsite with a
`negatively charged group may bind an antigen with a positive charge in the
`complementary position. In many instances, complementarity is achieved
`interactively as the structures of antigen and binding site are influenced by
`each other during the approach of the ligand. Conformational changes in
`the antibody and/or the antigen then occur that allow the complementary
`groups tointeract fully. This is an example of induced fit (Fig. 7-27).
`
`

`

`Chapter 7
`
`Protein Function
`
`231
`
`
`
`Conformation with
`no antigen bound
`(a)
`
`Antigen bound
`(hidden)
`(b)
`
`Antigen bound
`(shown)
`(c)
`
`A typical antibody-antigen interaction is quite strong, characterized by
`K, values as low as 10°"” m (recall that a lower K, corresponds to a stronger
`binding interaction). The Ky reflects the energy derived from the various
`ionic, hydrogen-bonding, hydrophobic, and van der Waals interactions that
`stabilize the binding. The binding energy required to produce a Ky of 107" m
`is about 65 kJ/mol.
`A complex of a peptide derived from HIV (a model antigen) and an Fab
`molecule illustrates some of these properties (Fig. 7-27). The changes in
`structure observed on antigen binding are particularly striking in this example.
`
`figure 7-27
`Induced fit in the binding of an antigen to IgG. The
`molecule, shown in surface contour, is the Fab fragment
`of an IgG. The antigen this IgG binds is a small peptide
`derived from HIV. Two residues from the heavy chain
`(blue) and one from the light chain (pink) are colored to
`provide visual points of reference. (a) View of the Fab
`fragment looking down on the antigen-binding site.
`(b) The same view, but here the Fab fragmentis in the
`“bound” conformation; the antigen has been omitted
`from the image to provide an unobstructed view of the
`altered binding site. Note how the binding cavity has
`enlarged and several groups have shifted position.
`(c) The same view as in (b), but with the antigen pictured
`in the binding site as a red stick structure.
`
`The Antibody-Antigen Interaction Is the Basis for a Variety
`of Important Analytical Procedures
`The extraordinary binding affinity and specificity of antibodies makes them
`valuable analytical reagents. Two types of antibody preparations are in use:
`polyclonal and monoclonal. Polyclonal antibodies are those produced by
`many different B lymphocytes responding to one antigen, such as a protein
`injected into an animal. Cells in the population of B lymphocytes produce
`antibodies that bind specific, different epitopes within the antigen. Thus,
`polyclonal preparations contain a mixture of antibodies that recognize dif-
`ferent parts of the protein. Monoclonal antibodies, in contrast, are syn-
`thesized by a population ofidentical B cells (a clone) grown in cell culture.
`These antibodies are homogeneous,all recognizing the same epitope. The
`techniques for producing monoclonal antibodies were developed by
`Georges Kohler and Cesar Milstein.
`The specificity of antibodies has practical uses. A selected antibody can
`be covalently attached to a resin and used in a chromatography column of
`the type shown in Figure 5—18c. When a mixture of proteinsis addedto the
`column,the antibody will specifically bind its target protein and retain it on
`the column while other proteins are washed through. The target protein can
`then be eluted from theresin by a salt solution or some other agent. This is
`a powerful tool for protein purification.
`In another versatile analytical technique, an antibody is attached to a
`radioactive label or some other reagent that makes it easy to detect. When
`the antibody binds the target protein, the label reveals the presence of the
`protein in a solution or its location in a gel or evenaliving cell. Several
`Cesar Milstein
`variations of this procedure are illustrated in Figure 7-28.
`
`
`Georges Kohler
`
`

`

`232
`
`Part Il
`
`Structure and Catalysis
`
`An ELISA (enzyme-linked immunosorbent assay) allows for rapid
`screening and quantification of the presence of an antigen in a sample (Fig.
`7-28b). Proteins in a sample are adsorbed to an inert surface, usually a
`96-well polystyrene plate. The surface is washed with a solution of an inex-
`pensive nonspecific protein (often casein from nonfat dry milk powder) to
`block proteins in subsequentsteps from also adsorbing to these surfaces. The
`surface is then treated with a solution containing the primary antibody—
`an antibody against the protein of interest. Unbound antibody is washed
`away and the surfaceis treated with a solution containing antibodies against
`the primary antibody. These secondary antibodies have been linked to an
`enzyme that catalyzes a reaction that forms a colored product. After un-
`bound secondary antibody is washed away, the substrate of the antibody-
`linked enzyme is added. Product formation (monitored as color intensity)
`is proportional to the concentration of the proteinof interest in the sample.
`In an immunoblot assay (Fig. 7-28c), proteins that have been sepa-
`rated by gel electrophoresis are transferred electrophoretically to a nitro-
`cellulose membrane. The membrane is blocked (as described above for
`ELISA), then treated successively with primary antibody, secondary anti-
`body linked to enzyme, and substrate. A colored precipitate forms only
`along the band containing the protein of interest. The immunoblot allows
`
`figure 7-28
`Antibody techniques. The specific reaction of an anti-
`body with its antigen is the basis of several techniques
`that identify and quantify a specific protein in a complex
`sample. (a) A schematic representation of the general
`method. (b) An ELISA testing for the presence of herpes
`simplex virus (HSV) antibodies in blood samples. Wells
`were coated with an HSV antigen, to which antibodies
`against HSV in a patient's blood will bind. The second
`antibody is anti-human IgG linked to horseradish peroxi-
`dase. Blood samples with greater amounts of HSV anti-
`body turn brighter yellow. (c) An immunoblot. Lanes 1 to
`3 are from an SDSgel; samples from successive stagesin
`the purification of a protein kinase have been separated
`and stained with Coomassie blue. Lanes 4 to 6 show the
`same samples, but these were electrophoretically trans-
`ferred to a nitrocellulose membraneafter separation on
`an SDS gel. The membrane was then “probed” with anti-
`body against the protein kinase. The numbers between
`the gel and the immunoblot indicate M, (x 1073).
`
`
`of specific antigen.
`
`
`
`@ Coat surface with sampleJ& &
`(antigens).
`
`(2) Block unoccupied sites
`with nonspecific protein.
`
`8) Incubate with primary
`antibody against specific
`antigen.
`
`(4) Incubate with
`antibody-enzyme complex
`that binds primary antibody.
`
`® Add substrate.
`
`©) Formationof colored
`¢
`product indicates presence %
`
`(a)
`
`-215-
`
`-14.4 -
`
`ELISA assay
`(b)
`
`SDSgel
`
`Immunoblot
`
`(c)
`
`

`

`Chapter 7 Protein Function
`
`233
`
`the detection of a minor component in a sample and provides an approxi-
`mation of its molecular weight.
`We will encounter other aspects of antibodies in later chapters. They
`are extremely important in medicine and can tell us much about the struc-
`ture of proteins and the action of genes.
`
`Two supercoiled,Amino
`a nenee
`ao
`
`(a)
`
`z
`
`Myosin
`
`-
`
`Protein Interactions Modulated by Chemical Energy:
`Actin, Myosin, and Molecular Motors
`Organisms move. Cells move. Organelles and macromolecules within cells
`move. Most of these movements arise from the activity of the fascinating
`class of protein-based molecular motors. Fueled by chemical energy, usually
`derived from ATP, large aggregates of motor proteins undergo cyclic con-
`formational changes that accumulate into a unified, directional force—the
`tiny force that pulls apart chromosomes in a dividing cell, and the immense
`“Tail
` 150 nm
`force that levers a pouncing, quarter-ton jungle cat into the air.
`:
`The interactions among motor proteins, as you might predict, feature
`complementary arrangements of ionic, hydrogen-bonding, hydrophobic, ys terminus
`and van der Waals interactions at protein binding sites. In motor proteins,
`,
`however, these interactions achieve exceptionally high levels of spatial and
`temporal organization.
`Motor proteins underlie the contraction of muscles, the migration ofor-
`ganelles along microtubules, the rotation of bacterial flagella, and the move-
`ment of some proteins along DNA. As we noted in Chapter2, proteins called
`kinesins and dyneins move along microtubules in cells, pulling along or-
`ganelles or reorganizing chromosomesduring cell division (see Fig. 2-19).
`An interaction of dynein with microtubules brings about the motion of eu-
`karyotic flagella andcilia. Flagellar motion in bacteria involves a complex
`rotational motor at the base of the flagellum (see Fig. 19-32). Helicases,
`polymerases, and other proteins move along DNA as they carry out their
`functions in DNA metabolism (Chapter 25). Here, we focus on the well-
`studied example of the contractile proteins of vertebrate skeletal muscle as
`a paradigm for how proteins translate chemical energy into motion.
`
`
`
`
`Lath
`
`Light
`meromyosin
`
`=
`=
`
`Hayy
`meromyosin
`+ Bee
`
`Ko53o =(eas
`LL! S
`
`(b)
`
`$2
`
`The Major Proteins of Muscle Are Myosin and Actin
`
`Thecontractileforceofmuscleisgeneratedbytheinteractionoftwoproteins,
`
`myosin and actin. These proteins are arranged in filaments that undergo
`transient interactions and slide past each other to bring about contraction.
`Together, actin and myosin make up over 80% of the protein mass of muscle.
`Myosin (, 540,000) has six subunits: two heavy chains (M, 220,000)
`and four light chains (M, 20,000). The heavy chains account for much of
`the overall structure. At their carboxyl termini, they are arranged as ex-
`tended @ helices, wrapped around each other in a fibrous, left-handed
`coiled coil similar to that of a-keratin (Fig. 7-29a). At its amino termini,
`each heavy chain hasa large globular domain containing a site where ATP
`is hydrolyzed. The light chains are associated with the globular domains.
`
`
`
`
`
` figure 7-29
`
`Myosin. (a) Myosin has two heavy chains(in two shadesof
`pink), the carboxyl termini forming an extended coiled coil
`(tail) and the amino termini having globular domains (heads).
`Twolight chains (blue) are associated with each myosin
`head. (b) Cleavage with trypsin and papain separates the
`myosin heads (S1 fragments) from the tails. (c) Ribbon rep-
`resentation of the myosin S1 fragment. The heavy chain is
`in gray, the twolight chains in two shadesof blue.
`
`

`

`234
`
`Part ||
`
`Structure and Catalysis
`
`figure 7-30
`The major components of muscle. (a) Myosin aggre-
`gates to form a bipolar structure called a thick filament.
`(b) F-actin is a filamentous assemblage of G-actin
`monomers that polymerize two by two, giving the appear-
`anceof twofilaments spiraling about one anotherin a
`right-handed fashion. An electron micrograph and a
`model of F-actin are shown. (c) Space-filling model of an
`actin filament (red) with one myosin head (gray and two
`shades of blue) bound to an actin monomerwithin the
`filament.
`
`
`
`When myosin is treated briefly with the protease trypsin, much of the fi-
`broustail is cleaved off, dividing the protein into componentscalled light
`and heavy meromyosin (Fig. 7-29b). The globular domain, called myosin
`subfragment 1, or S1, or simply the myosin head group, is liberated from
`heavy meromyosin by cleavage with papain. The S1 fragment produced by
`this procedureis the motor domain that makes muscle contraction possible.
`S1 fragments can be crystallized, and their structure has been determined,
`The overall structure of the S1 fragment as determined by Ivan Rayment
`and Hazel Holdenis shown in Figure 7-29c.
`In muscle cells, molecules of myosin aggregate to form structures called
`thick filaments (Fig. 7-30a). These rodlike structures serve as the core of
`the contractile unit. Within a thick filament, several hundred myosin mole-
`cules are arranged with their fibrous “tails” associated to form a long bipo-
`lar structure. The globular domains project from either end of this struc-
`ture, in regular stacked arrays.
`The second major muscle protein, actin, is abundantin almost all eu-
`karyotic cells. In muscle, molecules of monomeric actin, called G-actin
`(globular actin; M, 42,000), associate to form a long polymer called F-actin
`(filamentousactin). The thin filament (Fig. 7-30b) consists of F-actin,
`along with the proteins troponin and tropomyosin. The filamentous parts of
`thin filaments assemble as successive monomeric actin molecules add to
`one end. On addition, each monomerbinds ATP, then hydrolyzes it to ADP,
`so all actin molecules in the filament are complexed to ADP. However, this
`ATP hydrolysis by actin functions only in the assembly ofthe filaments; it
`does not contribute directly to the energy expended in muscle contraction.
`Each actin monomerin the thin filament can bindtightly and specifically to
`one myosin head group (Fig. 7—30c).
`
`Myosin head
`
`Actin filament
`
`(c)
`
`
`
`
`G-actin
`subunits
`
`(b)
`
`

`

`Chapter 7
`
`Protein Function
`
`235
`
`Additional Proteins Organize the Thin and Thick Filaments
`into Ordered Structures
`Skeletal muscle consists of parallel bundles of muscle fibers, each fiber a
`single, very large, muleinuciested eel, 20 to age aon im diameter, formed
`from many cells fused together and often spanning the length of the mus-
`cle. Hach fiber, in turn, contains about 1,000 myofibrils, 2 »m in diameter,
`each consisting of a vast number of regularly arrayed thick and thin fila-
`ments complexed to other proteins (Fig. 7-31). A system of flat membra-
`nousvesicles called the sarcoplasmic reticulum surrounds each myofib-
`ril. Examined under
`the electron microscope, muscle fibers
`reveal
`5
`:
`P ty
`alternating regions of high and low electron density, called the A and I
`bands (Fig. 7-31b,c). The A and I bandsarise from the arrangement of
`
`figure 7-31
`Structure of skeletal muscle. (a) Muscle fibers consist of
`ingle, elongated, multinucleatedcells that arise from the
`fusion of many precursorcells. Within the fibers are many
`myofibrils (only six are shown here for simplicity) sur-
`founded by the membranoussarcoplasmic reticulum.
`_‘"he organizationof thick andthin filaments in the
`Finer Biss a Sis annetate Yee
`contracts, the | bands narrow and the Z disks come
`closer together, as seen in electron micrographsof
`relaxed (b) and contracted (ce) muscle.
`
`(a)
`
`Bundle of
`muscle fibers
`
`Myofibrils
`
`Capillaries
`
`"a
`Muscle Fiber
`
`
`
`Sarcoplasmic
`:
`reticulum
`
`Sarcomere
`
`I band
`
`A band
`
`
`
`
`
`
`
`Z disk
`
`M line
`
`1.8 «m
`
`Z disk
`
`M line
`
`Z disk
`
`

`

`236
`
`Part || Structure and Catalysis
`
`thick and thin filaments, which are aligned and partially overlapping. The I
`band is the region of the bundle that in cross section would contain only
`thin filaments. The darker A band stretches the length of the thick filament
`and includes the region where parallel thick and thin filaments overlap. Bi-
`secting the I band is a thin structure called the Z disk, perpendicular to the
`thin filaments and serving as an anchor to which the thin filaments are at-
`tached. The A bandtoo is bisected by a thin line, the M line or M disk, a
`region of high electron density in the middle of the thick filaments. The en-
`tire contractile unit, consisting of bundles of thick filaments interleaved at
`either end with bundles of thin filaments, is called the sarcomere. The
`arrangement of interleaved bundles allows the thick and thin filaments to
`slide past each other (by a mechanismdiscussed below), causing a pro-
`gressive shortening of each sarcomere (Fig. 7-32).
`The thin actin filaments are attached at one end to the Z disk in a reg-
`ular pattern. The assembly includes the minor muscle proteins e-actinin,
`desmin, and vimentin. Thin filaments also contain a large protein called
`nebulin (~7,000 amino acid residues), thought to be structured as an
`a helix long enough to span the length of the filament. The Mline similarly
`
`band
` Z disk
`
`figure 7-32
`Muscle contraction. Thick filaments are bipolar struc-
`tures created by the association of many myosin mole-
`cules. (a) Muscle contraction occurs by the sliding of the
`thick and thin filaments past each other so that the Z
`disks in neighboring | bands approach each other.
`(b) The thick and thin filaments are interleaved such that
`eachthick filament is surrounded bysix thin filaments.
`
`band
`
`Thin
`filament
`
`B— Thick
`filament
`
`A
`bandee—"*K,
`=
`
`I
`
`Contracted
`
`(a)
`
`(b)
`
`

`

`Chapter 7 Protein Function
`
`237
`
`
`
`filament
`
`
`
`Myosin
`thick
`filament
`
`
`‘
`ATPbinds to myosin head,
`®
`|
`causingdissociation
`
`from actin.
`
` ;
`
`@)
`
`As tightly bound ATPis hydrolyzed,
`a conformational change occurs.
`ADPand P; remain associated
`with the myosin head.
`
`i
`i
`
`
`
`
`
`
` 4
`
`
`
`@
`
`Pi
`
`Myosin head attaches
`to actin filament,
`causingreleaseofPj.
`
`
`
`(4)
`
`i
`
`ADP @ |
`
`P, release triggers a "powerstroke,"
`a conformational change in the myosin
`filaments relative to one another.
`ADPis released in the process.
`
`head that moves actin and myosin
`
`organizes the thick filaments. It contains the proteins paramyosin, C-pro-
`tein, and M-protein. Another class of proteins called titins, the largest
`known single polypeptide chains (the titin of human cardiac muscle has
`26,926 amino acid residues), link the thick filaments to the Z disk, providing
`additional organization to the overall structure. Among their structural
`functions, the proteins nebulin and titin are believed to act as “molecular
`rulers,” regulating the length of the thin and thick filaments, respectively.
`Titin extends from the Z disk to the M line, regulating the length of the sar-
`comere itself and preventing overextension of the muscle. The characteris-
`tic sarcomere length varies from one muscle tissue to the next in a verte-
`brate organism, attributed in large part to the expression of different titin
`variants.
`
`Myosin Thick Filaments Slide along Actin Thin Filaments
`The interaction between actin and myosin, like that betweenall proteins
`and ligands, involves weak bonds. When ATP is not bound to myosin, a face
`on the myosin head group bindstightly to actin (Fig. 7-33). When ATP binds
`to myosin and is hydrolyzed to ADP and phosphate, a coordinated and
`cyclic series of conformational changes occur in which myosin releases the
`F-actin subunit and binds another subunit farther along the thin filament.
`The cycle has four major steps (Fig. 7-33). @) ATP binds to myosin,
`and a cleft in the myosin molecule opens, disrupting the actin-myosin inter-
`action so that the bound actin is released. ATP is then hydrolyzed (step Q),
`causing a conformational change in the protein to a “high-energy”state that
`moves the myosin head and changesits orientation in relation to the actin
`thin filament. Myosin then binds weakly to an F-actin subunit closer to the
`Z disk than the one just released. As the phosphate product of ATP hydrol-
`ysis is released from myosinin step @), another conformational change oc-
`curs in which the myosin cleft closes, strengthening the myosin-actin bind-
`ing. This is followed quickly by the final step, (4), a “power stroke” during
`which the conformation of the myosin head returns to the original resting
`state, its orientation relative to the bound actin changing so as to pull the
`tail of the myosin toward the Z disk. ADP is then released to complete the
`cycle. Each cycle generates about 3 to 4 pN (piconewtons) of force and
`moves the thick filament 5 to 10 nm relative to the thin filament.
`Because there are many myosin headsin a thick filament, at any given
`moment some (probably 1% to 3%) are bound to the thin filaments. This
`prevents the thick filaments from slipping backward when an individual
`myosin head releases the actin subunit to which it was bound. Thethickfil-
`ament thus actively slides forward past the adjacent thin filaments. This
`process, coordinated among the many sarcomeres in a muscle fiber, brings
`about muscle contraction.
`The interaction between actin and myosin must be regulated so that
`contraction occurs only in response to appropriate signals from the nervous
`
`figure 7-33
`Molecular mechanism of muscle contraction. Conforma-
`tional changes in the myosin head that are coupled to
`stages in the ATP hydrolytic cycle cause myosin to suc-
`cessively dissociate from one actin subunit, then asso-
`ciate with another farther along the actin filament. In this
`way the myosin heads slide along the thin filaments,
`drawing the thick filament array into the thin filament
`array (see Fig. 7-32).
`
`

`

`238
`
`Part || Structure and Catalysis
`
`system. The regulation is mediated by a complex of two proteins,
`tropomyosin and troponin. Tropomyosin binds to the thin filament,
`blocking the attachment sites for the myosin head groups. Troponin is a
`Ca”*-binding protein. A nerve impulse causes release of Ca2* from the sar-
`coplasmic reticulum. The released Ca** binds to troponin (another protein-
`ligand interaction) and causes a conformational change in the tropomyosin-
`troponin complexes, exposing the myosin-binding sites on the thin
`filaments. Contraction follows.
`Working skel