B. Alberts, A. Johnson, J. Lewis и др. - Molecular Biology of The Cell (6th edition) (1120996), страница 61
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(Adapted from D.R. Knighton et al., Science 253:407–414, 1991.)MBoC6 m6.92/3.65160Chapter 3: ProteinsF-box protein(substrate-binding arm)adaptorprotein 2target proteinE2 ubiquitinconjugatingenzymeAPC/Cubiquitinadaptorprotein 1TARGET PROTEINBINDS(A)scaffold protein(cullin)substrate-bindingtwo of manypossiblesubstrate-bindingarms(B)polyubiquitylatedprotein targetedfor destruction(C)ubiquitin ligaseIn this manner, specific proteins are targeted for rapid destruction in responseto specific signals, thereby helping to drive the cell cycle (discussed in Chapter 17).The timing of the destruction often involves creating a specific pattern of phosphorylation on the target protein that is requiredfor its recognition by the F-boxMBoC6 m3.79/3.66subunit. It also requires the activation of an SCF ubiquitin ligase that carries theappropriate substrate-binding arm.
Many of these arms (the F-box subunits) areinterchangeable in the protein complex (see Figure 3–71B), and there are morethan 70 human genes that encode them.As emphasized previously, once a successful protein has evolved, its geneticinformation tends to be duplicated to produce a family of related proteins. Thus,for example, not only are there many F-box proteins—making possible the recognition of different sets of target proteins—but there is also a family of scaffolds(known as cullins) that give rise to a family of SCF-like ubiquitin ligases.A protein machine like the SCF ubiquitin ligase, with its interchangeable parts,makes economical use of the genetic information in cells.
It also creates opportunities for “rapid” evolution, inasmuch as new functions can evolve for the entirecomplex simply by producing an alternative version of one of its subunits.Ubiquitin ligases form a diverse family of protein complexes. Some of thesecomplexes are far larger and more complicated than SCF, but their underlyingenzymatic function remains the same (Figure 3–71D).A GTP-Binding Protein Shows How Large Protein MovementsCan Be GeneratedDetailed structures obtained for one of the GTP-binding protein family members,the EF-Tu protein, provide a good example of how allosteric changes in proteinconformations can produce large movements by amplifying a small, local conformational change.
As will be discussed in Chapter 6, EF-Tu is an abundant molecule that serves as an elongation factor (hence the EF) in protein synthesis, loading each aminoacyl-tRNA molecule onto the ribosome. EF-Tu contains a Ras-likedomain (see Figure 3–67), and the tRNA molecule forms a tight complex with itsGTP-bound form. This tRNA molecule can transfer its amino acid to the growingE2-bindingSCF(D)10 nmFigure 3–71 The structure and mode ofaction of an SCF ubiquitin ligase.
(A) Thestructure of the five-protein ubiquitin ligasecomplex that includes an E2 ubiquitinconjugating enzyme. Four proteins form theE3 portion. The protein denoted here asadaptor protein 1 is the Rbx1/Hrt1 protein,adaptor protein 2 is the Skp1 protein, andthe cullin is the Cul1 protein. One of themany different F-box proteins completesthe complex. (B) Comparison of the samecomplex with two different substrate-bindingarms, the F-box proteins Skp2 (top) andβ-trCP1 (bottom), respectively. (C) Thebinding and ubiquitylation of a target proteinby the SCF ubiquitin ligase. If, as indicated,a chain of ubiquitin molecules is added tothe same lysine of the target protein, thatprotein is marked for rapid destruction by theproteasome. (D) Comparison of SCF (bottom)with a low-resolution electron microscopystructure of a ubiquitin ligase called theanaphase-promoting complex (APC/C; top)at the same scale.
The APC/C is a large,15-protein complex. As discussed in Chapter17, its ubiquitylations control the late stagesof mitosis. It is distantly related to SCF andcontains a cullin subunit (green) that lies alongthe side of the complex at right, only partlyvisible in this view. E2 proteins are not shownhere, but their binding sites are indicated inorange, along with substrate-binding sitesin purple. (A and B, adapted from G. Wuet al., Mol. Cell 11:1445–1456, 2003. Withpermission from Elsevier; D, adapted fromP. da Fonseca et al., Nature 470:274–278,2011. With permission from MacmillanPublishers Ltd.)PROTEIN FUNCTION161GTPNH2site oftRNAbindingdomain1release oftRNAGPswitch helixdomain2HOOC(A)(B)PPGGTP hydrolysisPPboundGDPGTPbindingsiteswitchhelixdomain3Figure 3–72 The large conformational change in EF-Tu caused by GTP hydrolysis.
(A and B) The three-dimensionalstructure of EF-Tu with GTP bound. The domain at the top has a structure similar to the Ras protein, and its red α helix is theswitch helix, which moves after GTP hydrolysis. (C) The change in the conformation of the switch helix in domain 1 allowsdomains 2 and 3 to rotate as a single unit by about 90 degrees toward the viewer, which releases the tRNA that was bound tothis structure (see also Figure 3–73). (A, adapted from H. Berchtold et al., Nature 365:126–132, 1993. With permission fromMacmillan Publishers Ltd. B, courtesy of Mathias Sprinzl and Rolf Hilgenfeld. PDB code: 1EFT.)polypeptide chain only after the GTP bound to EF-Tu is hydrolyzed, dissociatingMBoC6m3.75/3.67the EF-Tu.
Since this GTP hydrolysis is triggered bya properfit of the tRNA to themRNA molecule on the ribosome, the EF-Tu serves as a factor that discriminatesbetween correct and incorrect mRNA–tRNA pairings (see Figure 6–65).By comparing the three-dimensional structure of EF-Tu in its GTP-bound andGDP-bound forms, we can see how the repositioning of the tRNA occurs. Thedissociation of the inorganic phosphate group (Pi), which follows the reactionGTP → GDP + Pi, causes a shift of a few tenths of a nanometer at the GTP-bindingsite, just as it does in the Ras protein. This tiny movement, equivalent to a fewtimes the diameter of a hydrogen atom, causes a conformational change to propagate along a crucial piece of α helix, called the switch helix, in the Ras-like domainof the protein.
The switch helix seems to serve as a latch that adheres to a specificsite in another domain of the molecule, holding the protein in a “shut” conformation. The conformational change triggered by GTP hydrolysis causes the switchhelix to detach, allowing separate domains of the protein to swing apart, througha distance of about 4 nm (Figure 3–72). This releases the bound tRNA molecule,allowing its attached amino acid to be used (Figure 3–73).Notice in this example how cells have exploited a simple chemical change thatoccurs on the surface of a small protein domain to create a movement 50 timeslarger.
Dramatic shape changes of this type also cause the very large movementsthat occur in motor proteins, as we discuss next.Motor Proteins Produce Large Movements in CellsWe have seen that conformational changes in proteins have a central role inenzyme regulation and cell signaling. We now discuss proteins whose majorfunction is to move other molecules.
These motor proteins generate the forcesresponsible for muscle contraction and the crawling and swimming of cells.Motor proteins also power smaller-scale intracellular movements: they help tomove chromosomes to opposite ends of the cell during mitosis (discussed inChapter 17), to move organelles along molecular tracks within the cell (discussedin Chapter 16), and to move enzymes along a DNA strand during the synthesis ofa new DNA molecule (discussed in Chapter 5). All these fundamental processesdepend on proteins with moving parts that operate as force-generating machines.How do these machines work? In other words, how do cells use shape changesin proteins to generate directed movements? If, for example, a protein is requiredto walk along a narrow thread such as a DNA molecule, it can do this by undergoing a series of conformational changes, such as those shown in Figure 3–74.But with nothing to drive these changes in an orderly sequence, they are perfectlyEF-Tuamino acid linkedto tRNAGTPtRNAFigure 3–73 An aminoacyl tRNA moleculebound to EF-Tu.
Note how the boundprotein blocks the use of the tRNA-linkedamino acid (green) for protein synthesis untilGTP hydrolysis triggers the conformationalchangesMBoC6shownm3.74/3.69in Figure 3–72C,dissociating the protein-tRNA complex.EF-Tu is a bacterial protein; however, a verysimilar protein exists in eukaryotes, whereit is called EF-1 (Movie 3.12).
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