H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 34
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Some motor proteins are components of macro(a)molecular assemblies, but those that move along cytoskeletalfibers are not. This latter group comprises the myosins, kinesins, and dyneins—linear motor proteins that carry attached “cargo” with them as they proceed along eithermicrofilaments or microtubules (Figure 3-22a). DNA andRNA polymerases also are linear motor proteins becausethey translocate along DNA during replication and transcription. In contrast, rotary motors revolve to cause the beatof bacterial flagella, to pack DNA into the capsid of a virus,and to synthesize ATP. The propulsive force for bacterialswimming, for instance, is generated by a rotary motor protein complex in the bacterial membrane.
Ions flow down anelectrochemical gradient through an immobile ring of proteins, the stator, which is located in the membrane. Torquegenerated by the stator rotates an inner ring of proteins andthe attached flagellum (Figure 3-22b). Similarly, in the mitochondrial ATP synthase, or F0F1 complex, a flux of ionsacross the inner mitochondrial membrane is transduced bythe F0 part into rotation of the subunit, which projects intoa surrounding ring of and subunits in the F1 part. Interactions between the subunit and the subunits directs thesynthesis of ATP (Chapter 8).From the observed activities of motor proteins, we caninfer three general properties that they possess:The ability to transduce a source of energy, either ATPor an ion gradient, into linear or rotary movement■The ability to bind and translocate along a cytoskeletalfilament, nucleic acid strand, or protein complex■■Net movement in a given directionThe motor proteins that attach to cytoskeletal fibers alsobind to and carry along cargo as they translocate.
The cargoin muscle cells and eukaryotic flagella consists of thick filaments and B tubules, respectively (see Figure 3-22a). Thesemotor proteins can also transport cargo chromosomes andmembrane-limited vesicles as they move along microtubulesor microfilaments (Figure 3-23).(b)FlagellumADPMyosinordyneinIonsActin filament or A tubuleRotor▲ FIGURE 3-22 Comparison of linear and rotary molecularmotors.
(a) In muscle and eukaryotic flagella, the head domainsof motor proteins (blue) bind to an actin thin filament (muscle) orthe A tubule of a doublet microtubule (flagella). ATP hydrolysis inthe head causes linear movement of the cytoskeletal fiber(orange) relative to the attached thick filament or B tubule of anadjacent doublet microtubule.
(b) In the rotary motor inthe bacterial membrane, the stator (blue) is immobile inthe membrane. Ion flow through the stator generates atorque that powers rotation of the rotor (orange) and theflagellum attached to it.MEDIA CONNECTIONSStatorVideo: Rotary Motor Action:FlagellumThick filament or B tubuleATP7980CHAPTER 3 • Protein Structure and FunctionTABLE 3-2Selected Molecular MotorsMotor*EnergySourceStructure/ComponentsCellular LocationMovement GeneratedLINEAR MOTORSDNA polymerase (4)ATPMultisubunit polymerase within replisomeNucleusTranslocation along DNAduring replicationRNA polymerase (4)ATPMultisubunit polymerasewithin transcriptionelongation complexNucleusTranslocation along DNAduring transcriptionRibosome (4)GTPElongation factor 2 (EF2)bound to ribosomeCytoplasm/ERmembraneTranslocation along mRNAduring translationMyosins (3, 19)ATPHeavy and light chains;head domains with ATPaseactivity and microfilamentbinding siteCytoplasmTransport of cargovesicles; contractionKinesins (20)ATPHeavy and light chains; headdomains with ATPase activityand microtubule-binding siteCytoplasmTransport of cargovesicles and chromosomesduring mitosisDyneins (20)ATPMultiple heavy, intermediate,and light chains; head domainswith ATPase activity andmicrotubule-binding siteCytoplasmTransport of cargovesicles; beating of ciliaand eukaryotic flagellaBacterial flagellarmotorH/NagradientStator and rotor proteins,flagellumPlasma membraneRotation of flagellumattached to rotorATP synthase,F0F1(8)HgradientMultiple subunits formingF0 and F1 particlesInner mitochondrialmembrane, thylakoidmembrane, bacterialplasma membraneRotation of subunitleading to ATP synthesisViral capsid motorATPConnector, proheadRNA, ATPaseCapsidRotation of connectorleading to DNA packagingROTARY MOTORS*Numbers in parentheses indicate chapters in which various motors are discussed.CargoCargo binding FIGURE 3-23 Motor protein-dependent movement ofcargo.
The head domains of myosin, dynein, and kinesin motorproteins bind to a cytoskeletal fiber (microfilaments ormicrotubules), and the tail domain attaches to one of varioustypes of cargo—in this case, a membrane-limited vesicle.Hydrolysis of ATP in the head domain causes the head domain to“walk” along the track in one direction by a repeating cycle ofconformational changes.TailNeckMotorproteinATP hydrolysisFiber bindingHeadCytoskeletal fiber3.4 • Molecular Motors and the Mechanical Work of Cells81(b) Head domain(a) Myosin IITailHead NeckNucleotidebinding siteRegulatorylight chainEssentiallight chainHeavy chainsRegulatorylight chainActinbindingsiteEssentiallight chainHeavy chain▲ FIGURE 3-24 Structure of myosin II.
(a) Myosin II is adimeric protein composed of two identical heavy chains (white)and four light chains (blue and green). Each of the head domainstransduces the energy from ATP hydrolysis into movement. Twolight chains are associated with the neck domain of each heavychain. The coiled-coil sequence of the tail domain organizesmyosin II into a dimer. (b) Three-dimensional model of a singlehead domain shows that it has a curved, elongated shape and isbisected by a large cleft. The nucleotide-binding pocket lies onone side of this cleft, and the actin-binding site lies on the otherside near the tip of the head.
Wrapped around the shaft of the helical neck are the two light chains. These chains stiffen theneck so that it can act as a lever arm for the head. Shown hereis the ADP-bound conformation.All Myosins Have Head, Neck, and Tail Domainswith Distinct Functionshead, wrapped around the neck like C-clamps. In this position, the light chains stiffen the neck region and are thereforeable to regulate the activity of the head domain.To further illustrate the properties of motor proteins, we consider myosin II, which moves along actin filaments in musclecells during contraction. Other types of myosin can transportvesicles along actin filaments in the cytoskeleton.
Myosin IIand other members of the myosin superfamily are composedof one or two heavy chains and several light chains. Theheavy chains are organized into three structurally and functionally different types of domains (Figure 3-24a).The two globular head domains are specialized ATPasesthat couple the hydrolysis of ATP with motion. A critical feature of the myosin ATPase activity is that it is actin activated.In the absence of actin, solutions of myosin slowly convertATP into ADP and phosphate. However, when myosin iscomplexed with actin, the rate of myosin ATPase activity isfour to five times as fast as it is in the absence of actin.
Theactin-activation step ensures that the myosin ATPase operates at its maximal rate only when the myosin head domain is bound to actin. Adjacent to the head domain lies the-helical neck region, which is associated with the lightchains. These light chains are crucial for converting smallconformational changes in the head into large movementsof the molecule and for regulating the activity of the head domain. The rodlike tail domain contains the binding sites thatdetermine the specific activities of a particular myosin.The results of studies of myosin fragments produced byproteolysis helped elucidate the functions of the domains.X-ray crystallographic analysis of the S1 fragment of myosinII, which consists of the head and neck domains, revealed itsshape, the positions of the light chains, and the locations ofthe ATP-binding and actin-binding sites.
The elongatedmyosin head is attached at one end to the -helical neck (Figure 3-24b). Two light-chain molecules lie at the base of theConformational Changes in the Myosin HeadCouple ATP Hydrolysis to MovementThe results of studies of muscle contraction provided the firstevidence that myosin heads slide or walk along actin filaments.
Unraveling the mechanism of muscle contractionwas greatly aided by the development of in vitro motility assays and single-molecule force measurements. On the basisof information obtained with these techniques and the threedimensional structure of the myosin head, researchers developed a general model for how myosin harnesses the energyreleased by ATP hydrolysis to move along an actin filament.Because all myosins are thought to use the same mechanismto generate movement, we will ignore whether the myosintail is bound to a vesicle or is part of a thick filament as it isin muscle. One assumption in this model is that the hydrolysis of a single ATP molecule is coupled to each step taken bya myosin molecule along an actin filament.
Evidence supporting this assumption is discussed in Chapter 19.As shown in Figure 3-25, myosin undergoes a series ofevents during each step of movement. In the course of onecycle, myosin must exist in at least three conformationalstates: an ATP state unbound to actin, an ADP-P i statebound to actin, and a state after the power-generatingstroke has been completed. The major question is how thenucleotide-binding pocket and the distant actin-binding siteare mutually influenced and how changes at these sites areconverted into force. The results of structural studies ofmyosin in the presence of nucleotides and nucleotideanalogs that mimic the various steps in the cycle indicatethat the binding and hydrolysis of a nucleotide cause a82CHAPTER 3 • Protein Structure and FunctionThick filamentATP-bindingsiteMyosin headActin thin filamentNucleotidebinding1ATPHead dissociatesfrom filamentHydrolysis2 FIGURE 3-25 Operational model for the coupling of ATPhydrolysis to movement of myosin along an actin filament.Shown here is the cycle for a myosin II head that is part of athick filament in muscle, but other myosins that attach to othercargo (e.g., the membrane of a vesicle) are thought to operateaccording to the same cyclical mechanism.
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