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Some cyclins rise and fall inconcentration in step with the cell cycle, increasing gradually in amount untilthey are suddenly destroyed at a particular point in the cycle. The suddendestruction of a cyclin (by targeted proteolysis) immediately shuts off its partnerCdk enzyme, and this triggers a specific step in the cell cycle.Figure3-68 The domain structureof theSrcfamily of protein kinases,mappedalongthe amino acid sequence.Forthethree-dimensionalstructureof Src.seeF i q u r e3 - 1 0 .cated by the evolutionary tree in Figure 3-66, sequence comparisons suggestthat tyrosine kinases as a group were a relatively late innovation that branihedoff from the serine/threonine kinases, with the src subfamily being only onesubgroup of the tyrosine kinases created in this way.The src protein and its relatives contain a short N-terminal region thatbecomes covalently linked to a strongly hydrophobic fatty acid, which holds thekinase at the c)'toplasmic face of the plasma membrane.
Next come two peptide-binding modules, a Src homology 3 (sH3) domain and a sH2 domain, followed by the kinase catalytic domain (Figure 3-68). These kinasesnormally existin an inactive conformation, in which a phosphorylated tyrosine near the c-terminus is bound to the SH2 domain, and the sH3 domain is bound to an internalpeptide in a way that distorts the active site of the en4/me and helps to render itinactive.Turning the kinase on involves at least two specific inputs: removal of the c-processing events that enable the cell to compute logical responsesto a complexset of conditions.ProteinsThatBindand HydrolyzeGTpAre ubiquitousceilurarRegulatorswe have described how the addition or removal of phosphate groups on a proteincan be used by a cell to control the protein's activity.
In the examples discuised soFigure3-69 The activation of a Src-typeprotein kinaseby two sequentialevents.(Adaptedfrom S.C.Harrisonet al.,Ceil112:737-7 40,2003.With permissionfromElsevier.)a c t i v a t i n gl i g a n dk i n a s ed o m a i nPHOSPHATEREMOVALLOOSENSSTRUCTUREK I N A S EC A NN O WPHOSPHORYLATET Y R O S I NTEOSELF-ACTIVATE179PROTEINFUNCTIONfar, the phosphate is transferred from an AIP molecule to an amino acid sidechain of the protein in a reaction catalyzedby a specific protein kinase. Eucaryotic cells also have another way to control protein activity by phosphate addition and removal.
In this case,the phosphate is not attached directly to the protein; instead, it is a part of the guanine nucleotide GTB which binds very tightlyto the protein. In general, proteins regulated in this way are in their active conformations with GTP bound. The loss of a phosphate group occurs when thebound GTP is hydrolyzed to GDP in a reaction catalyzed by the protein itself,and in its GDP-bound state the protein is inactive. In this way, GTP-binding proteins act as on-off switches whose activity is determined by the presence orabsence of an additional phosphate on a bound GDP molecule (Figure 3-71).GTP-binding proteins (also called GTPasesbecause of the GTP hydrolysisthey catalyze) comprise a large family of proteins that all contain variations onthe same GTP-binding globular domain. !\4ren the tightly bound GTP ishydrolyzed to GDB this domain undergoes a conformational change that inactivates it.
The three-dimensional structure of a prototypical member of this family, the monomeric GTPase called Ras, is shor.tmin Figure 3-72.The Ras protein has an important role in cell signaling (discussedin Chapter 15). In its GTP-bound form, it is active and stimulates a cascade of proteinphosphorylations in the cell. Most of the time, however, the protein is in its inactive, GDP-bound form.
It becomes active when it exchangesits GDP for a GTPmolecule in responseto extracellular signals,such as growth factors, that bind toreceptors in the plasma membrane (seeFigure 15-58).INPUTSsrc-tvpeproteinkinaseactivityturnsonto all of thefuliyonlyi{ the answersareYesabovequestionsOUTPUTFigure3-70 How a Src-tYPeProteinkinaseacts as an integrating device.Thedisruotionof the 5H3domaininteraction(green)involvesreplacingits binding tothe indicatedred linkerregionwith atighterinteractionwith an activatingligand,as illustratedin Figure3-69.ProteinsRegulatoryProteinsControlthe Activityof GTP-B|ndingWhetherGTPor GDPls Boundby DeterminingGTP-binding proteins are controlled by regulatory proteins that determinewhether GTP or GDP is bound, just as phosphorylated proteins are turned onand offby protein kinases and protein phosphatases.Thus, Rasis inactivated bya GTPase-actiuating protein (GAP),which binds to the Ras protein and inducesit to hydrolyze its bound GTP molecule to GDP-which remains tightlyboundand inorganic phosphate (PJ, which is rapidly released.The Ras protein stays inits inactive, GDP-bound conformation until it encounters a guanine nucleotideexchangefactor (GEF),which binds to GDP-Rasand causesit to releaseits GDPBecause the empty nucleotide-binding site is immediately filled by a GTPmolecule (GTPis present in large excessover GDP in cells),the GEF activatesRasby indirectly adding back the phosphate removed by GTP hydrolysis' Thus, in asense,the roles of GAP and GEF are analogous to those of a protein phosphataseand a protein kinase, respectively (Figure 3-73).FromSmallOnesCanBeGeneratedLargeProteinMovementsThe Ras protein belongs to a large superfamily of monomeric GTPases,each ofwhich consists of a single GTP-binding domain of about 200 amino acids.
Overthe course of evolution, this domain has also become joined to larger proteinswith additional domains, creating a large family of GTP-binding proteins. Family members include the receptor-associated trimeric G proteins involved incell signaling (discussedin Chapter 15), proteins regulating the traffic of vesicles between intracellular compartments (discussed in Chapter 13), and proteins that bind to transfer RNA and are required as assembly factors for proteinACTIVENACTIVENACTIVEACTIVEFigure3-7 1 GTP-bindingproteinsasmolecularswitches.The activityof aprotein(alsocalledaGTP-bindinggenerallyrequiresthe presenceGTPase)of a tightlyboundGTPmolecule(switch'bn").Hydrolysisof this GTPmoleculeproducesGDPand inorganicphosphate(Pi),and it causesthe proteinto convertto a different,usuallyinactive,conformation(switch'bff").As shownhere,resettingthe switch requiresthea slowtightlybound GDPto dissociate,step that is greatlyacceleratedby specificaoncethe GDPhasdissociated,signals;moleculeof GTPis quicklyrebound.180Chapter3: ProteinsFigure3-72 The structureof the Rasprotein in its GTP-boundform.
<GAAC>ThismonomericGTPaseillustratesthestructureof a GTP-bindingdomain,whichis presentin a largefamilyof GTP-bindingproteins.Theredregionschangetheirconformationwhen the GTPmoleculeishydrolyzedto GDPand inorganicphosphateby the protein;the GDPremainsboundto the protein,whiletheinorganicphosphateis released.Thespecialroleof the "switchhelix"inproteinsrelatedto Rasis explainednext(seeFigure3-75).synthesis on the ribosome (discussedin chapter 6). In each case,an importantbiological activity is controlled by a change in the protein's conformation that iscaused by GTP hydrolysis in a Ras-like domain.The EF-Tu protein provides a good example of how this family of proteinsworks.
EF-Tu is an abundant molecule that servesas an elongation factor (hencethe EF) in protein synthesis, loading each aminoacyl tRNA molecule onto theribosome. The tRNA molecule forms a tight complex with the GTp-bound formof EF-Tu (Figure 3-74). In this complex, the amino acid attached to the IRNA isimproperly positioned for protein slmthesis. The IRNA can transfer its aminoacid only after the GTP bound to EF-Tu is hydrolyzed on the ribosome, allowingthe EF-Tu to dissociate.
Since the GTp hydrolysis is triggered by a proper fit ofthe IRNA to the mRNA molecule on the ribosome, the EF-Tu serves as a factorthat discriminates between correct and incorrect mRNA-IRNA pairings (seeFigure 6-67 for a further discussion of this function of EF-Tu).By comparing the three-dimensional structure of EF-Tu in its GTp-boundand GDP-bound forms, we can see how the repositioning of the IRNA occurs.The dissociation of the inorganic phosphate group (pJ, which follows the reaction GTP -+ GDP + Pi, causes a shift of a few tenths of a nanometer at the GTpbinding site, just as it does in the Rasprotein.
This tiny movement, equivalent torN. I'IiGNALrf-I,tGPPAPP PAPPGPP Psrcrunlour l\...',/\?S I G N A L I N GB Y P H O S P H O R Y L A T E DPROTEINS I G N A L I NBGY G T P - B I N D I NPGR O T E I NFigure3-73 A comparisonof the twomajor intracellularsignalingmechanismsin eucaryoticcells.In bothcasesra signalingproteinis activatedbythe additionofa phosphategroupandinactivatedby the removalof thisphosphate.To emphasizethe similaritiesin the two pathways,ATPand GTParedrawnas APPPand GPPP,and ADPandGDPasAPPand GPBrespectively.Asshownin Figure3-64,the additionof aphosphateto a proteincanalsobeinhibitorv.181PROTEINFUNCTIONThethreeFigure3-74An aminoacylboundto EF-Tu.tRNAmoleculeproteinarecolored3-75.to matchFiguredifferently,domainsof theEF-Tuproteinexistsprotein;inhowever,a verysimilarThisisa bacterial(Coordinatesetby P.Nissendeterminedwhereit iscalledEF-1.eucaryotes,fromAAA5.)270:1464-1472,1995.Withpermissional.,Sciencea few times the diameter of a hydrogen atom, causes a conformational changeto propagate along a crucial piece of a helix, called Ihe switch helix, in the Raslike domain of the protein.
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