Lodish H. - Molecular Cell Biology (5ed, Freeman, 2003) (794361), страница 35
Текст из файла (страница 35)
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.
In the absence ofbound nucleotide, a myosin head binds actin tightly in a “rigor”state. Step 1 : Binding of ATP opens the cleft in the myosinhead, disrupting the actin-binding site and weakening theinteraction with actin. Step 2 : Freed of actin, the myosin headhydrolyzes ATP, causing a conformational change in the head thatmoves it to a new position, closer to the () end of the actinfilament, where it rebinds to the filament. Step 3 : As phosphate(Pi) dissociates from the ATP-binding pocket, the myosin headundergoes a second conformational change—the power stroke—which restores myosin to its rigor conformation.
Because myosinis bound to actin, this conformational change exerts a force thatcauses myosin to move the actin filament. Step 4 : Release ofADP completes the cycle. [Adapted from R. D. Vale and R. A. Milligan,2002, Science 288:88.]Head pivots andbinds a newactin subunitFocus Animation: Myosin Crossbridge CycleMEDIA CONNECTIONSADP•PiKEY CONCEPTS OF SECTION 3.4Molecular Motors and the Mechanical Work of CellsPi releaseMotor proteins are mechanochemical enzymes that convert energy released by ATP hydrolysis into either linearor rotary movement (see Figure 3-22).■3PiADPHead pivots andmoves filament(power stroke)Linear motor proteins (myosins, kinesins, and dyneins)move along cytoskeletal fibers carrying bound cargo,which includes vesicles, chromosomes, thick filaments inmuscle, and microtubules in eukaryotic flagella.■Myosin II consists of two heavy chains and several lightchains.
Each heavy chain has a head (motor) domain,which is an actin-activated ATPase; a neck domain, whichis associated with light chains; and a long rodlike tail domain that organizes the dimeric molecule and binds to thickfilaments in muscle cells (see Figure 3-24).■ADP release4ADPsmall conformational change in the head domain that isamplified into a large movement of the neck region. Thesmall conformational change in the head domain is localized to a “switch” region consisting of the nucleotide- andactin-binding sites.
A “converter” region at the base of thehead acts like a fulcrum that causes the leverlike neck tobend and rotate.Homologous switch, converter, and lever arm structuresin kinesin are responsible for the movement of kinesin motorproteins along microtubules.
The structural basis for dyneinmovement is unknown because the three-dimensional structure of dynein has not been determined.Movement of myosin relative to an actin filament resultsfrom the attachment of the myosin head to an actin filament, rotation of the neck region, and detachment in acyclical ATP-dependent process (see Figure 3-25). The samegeneral mechanism is thought to account for all myosinand kinesin-mediated movement.■3.5 Common Mechanismsfor Regulating Protein FunctionMost processes in cells do not take place independently ofone another or at a constant rate. Instead, the catalytic activity of enzymes or the assembly of a macromolecular complex is so regulated that the amount of reaction product orthe appearance of the complex is just sufficient to meet theneeds of the cell.
As a result, the steady-state concentrations3.5 • Common Mechanisms for Regulating Protein Functionof substrates and products will vary, depending on cellularconditions. The flow of material in an enzymatic pathway iscontrolled by several mechanisms, some of which also regulate the functions of nonenzymatic proteins.One of the most important mechanisms for regulatingprotein function entails allostery. Broadly speaking, allosteryrefers to any change in a protein’s tertiary or quaternarystructure or both induced by the binding of a ligand, whichmay be an activator, inhibitor, substrate, or all three. Allosteric regulation is particularly prevalent in multimeric enzymes and other proteins.
We first explore several ways inwhich allostery influences protein function and then considerother mechanisms for regulating proteins.Cooperative Binding Increases a Protein’sResponse to Small Changes in LigandConcentrationIn many cases, especially when a protein binds several molecules of one ligand, the binding is graded; that is, the binding of one ligand molecule affects the binding of subsequentligand molecules. This type of allostery, often called cooper-% Saturation10050P50 = 260204060p O2 (torr)p O2 in capillariesof active muscles80100p O2 in alveoliof lungs▲ EXPERIMENTAL FIGURE 3-26 Sequential binding ofoxygen to hemoglobin exhibits positive cooperativity. Eachhemoglobin molecule has four oxygen-binding sites; at saturationall the sites are loaded with oxygen. The oxygen concentration iscommonly measured as the partial pressure (pO2).
P50 is the pO2at which half the oxygen-binding sites at a given hemoglobinconcentration are occupied; it is equivalent to the Km for anenzymatic reaction. The large change in the amount of oxygenbound over a small range of pO2 values permits efficientunloading of oxygen in peripheral tissues such as muscle. Thesigmoidal shape of a plot of percent saturation versus ligandconcentration is indicative of cooperative binding. In the absenceof cooperative binding, a binding curve is a hyperbola, similar tothe simple kinetic curves in Figure 3-19. [Adapted from L. Stryer,Biochemistry, 4th ed., 1995, W. H.
Freeman and Company.]83ativity, permits many multisubunit proteins to respond moreefficiently to small changes in ligand concentration thanwould otherwise be possible. In positive cooperativity, sequential binding is enhanced; in negative cooperativity,sequential binding is inhibited.Hemoglobin presents a classic example of positive cooperative binding. Each of the four subunits in hemoglobincontains one heme molecule, which consists of an iron atomheld within a porphyrin ring (see Figure 8-16a). The hemegroups are the oxygen-binding components of hemoglobin(see Figure 3-10). The binding of oxygen to the heme molecule in one of the four hemoglobin subunits induces a localconformational change whose effect spreads to the othersubunits, lowering the Km for the binding of additional oxygen molecules and yielding a sigmoidal oxygen-binding curve(Figure 3-26).
![](https://s.studizba.com/z.php?f=/templates/si/i/noavatar.png&w=100&h=100&t=1"){kind=avatar}
