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Most of the proteins in each family have evolved to performsomewhat different functions, as for the enzymes elastase and chymotrypsinillustrated previously in Figure 3-12. These are sometimes called paralogsto dtstinguish them from the corresponding proteins in different organisms(orthologs,such as mouse and human elastase).As described in Chapter 8, becauseofthe powerful techniques ofx-ray crystallography and nuclear magnetic resonance (NMR), we now know the threedimensional shapes,or conformations,of more than 20,000proteins.
<GGCC>By carefully comparing the conformations of these proteins, structural biologists (that is, experts on the structure of biological molecules) have concludedthat there are a limited number of ways in which protein domains fold up innature-maybe as few as 2000. The structures for about 800 of these proteinfolds have thus far been determined. These known folds tend to be those mostrepresented in the universe of protein structures: for example, 50 folds accountfor nearly three-fourths of the domain families with predicted structures.A complete catalog of the most significant protein folds that exist in living organismswould therefore seem to be within our reach.SearchesCanldentifyCloseRelativesSequenceThe present database of knor,nmprotein sequencescontains more than ten milIion entries, and it is growing very rapidly as more and more genomes aresequenced-revealing huge numbers of new genes that encode proteins.
Powerful computer search programs are available that allow us to compare eachnewly discoveredprotein with this entire database,looking for possible relatives.Many proteins whose geneshave evolved from a common ancestral gene can beidentified by the discovery of statistically significant similarities in amino acidsequences.With such a large number of proteins in the database,the search programsfind many nonsignificant matches, resulting in a background noise level thatmakes it very difficult to pick out all but the closest relatives. Generally speaking, one requires a 30To identity in sequence to consider that two proteinsmatch. However, we know the function of many short signature sequences("fingerprints"), and these are widely used to find more distant relationships(Figure 3-r4).Protein comparisons are important because related structures often implyrelated functions.
Many years of experimentation can be saved by discoveringthat a new protein has an amino acid sequence similarity with a protein ofknown function. Such sequence relationships, for example, first indicated thatcertain genes that cause mammalian cells to become cancerous are proteinkinases. In the same way, many of the proteins that control pattern formationduring the embryonic development of the fruit fly Drosophilawere quickly recognized to be gene regulatory proteins.IIYITGKITRRESERL,LGTFt,V!1liSI!-WYFGKTBRRESERI,LLNAENPRE?FLVRESETTKGAYCLSVSDFDNAKGL Y LSV D+++ +G W+F + R+E+++LLL ENP GTFLVR SEENPES.IF1VRp,SEHNPNGYSL SVKDWEDGRGY WFFENVLRREADKS1STLAE11020s i g n a t u r es e q u e n c e shUMANs e q u e n c em a t c h e sDr osop h i IaFigure3-14The useof short signaturesequencesto find related Proteinofdomains.Thetwo shortsequences15 and 9 amino acidsshown(green)canfor abe usedto searchlargedatabasesproteindomainthat isfound in manyproteins,the SH2domain.Here,the first50 aminoacidsof the SH2domainof100aminoacidsis comParedfor thehuman and DrosophilaSrcprotein (seeFigure3-10).In the computer-generateclsequencecomparison(yellowrow),exactmatchesbetweenthe humanandDrosophilaproteinsare noted by the onefor the aminoacid;theletterabbreviationoositionswith a similarbut nonidenticalaminoacidaredenotedbY+, andare blank.In this diagram,nonmatcheswhereverone or both proteinscontainanexactmatch to a positionin the greenareboth alignedsequencessequences,coloredred.140Chapter3: ProteinsSomeProteinDomainsFormPartsof ManyDifferentproteinsAs previously stated, most proteins are composed of a seriesof protein domains,in which different regions of the polypeptide chain have folded independentlyto form compact structures.
such multidomain proteins are believed to haveoriginated from the accidental joining of the DNA sequencesthat encode eachdomain, creating a new gene. Novel binding surfaceshave often been created atthe juxtaposition of domains, and many of the functional sites where proteinsbind to small molecules are found to be located there.
In an evolutioniry process called domain shuffIing, many large proteins have evolved through thejoining of preeisting domains in new combinations (Figure 3-f 5).A subset of protein domains have been especially mobile during evolution;these seem to have particularly versatile structures and are sometimes referredto as protein modules.The structure of one such module, the SH2 domain, wasillustrated in Panel3-2 (pp. r32-r33). Some other abundant protein domains areillustrated in Figure 3-16.Each of the domains shor.rmhas a stable core structure formed from strandsof B sheet, from which less-orderedloops of polypeptide chain protru de (green).The loops are ideally situated to form binding sites for other molecules, ai mostclearly demonstrated for the immunoglobulin fold, which forms the basis forantibody molecules (seeFigure 3-41). Most likely, such B-sheet-baseddomainshave achieved their evolutionary success because they provide a convenientframework for the generation of new binding sites for ligands through smallchanges to their protruding loops.EGFHzNCOOHtt"tl,tf"tt't.oo"HzNUROKINASECOOHFACTORIXH2NHzNcoOHPLASMINOGENcooHFigure3-15 Domainshuffling.Anextensiveshufflingof blocksof proteinsequence(proteindomains)hasoccurredduringproteinevolution.Thoseportionsof a proteindenotedby the sameshapeand colorin this diagramareevolutionarilyrelated.Serineproteaseslike chymotrypsinareformed from twodomains(brown).Inthe threeotherproteasesshown,whicharehighlyregulatedand morespecialized,thesetwo proteasedomainsare connectedtoone or moredomainsthat aresimilartodomainsfound in epidermalgrowthfactor (EGF;green),to a calcium-bindingprotein(yellow),or to a "kringle"domain(blue)that containsthree internaldisulfidebridges.Chymotrypsinisillustratedin Fioure3-12.fr-t.I,\t'ii ldrcomplement controlmoduleimmunoglobulinmodulefibronectintype 1 modulefibronectintype 3 modulegroMh factormoduleFigure3-16 The three-dimensionalstructuresof some protein modules.Intheseribbondiagrams,B-sheetstrandsareshownas arrows,and the N- andC-terminiare indicatedby redspheres.(Adaptedfrom M.
Baron,D.G.Normanandl.D.Campbell,TrendsBiochem.Sci.16:i3-17,1991,with permissionfrom Elsevier,andD.J.Leahyet al.,Science258:987-99i, 1992,with permissionfrom AAAS.)141THESHAPEAND STRUCTUREOF PROTEINScan be readily linked in series to form extended structures-either with themselves or with other in-line domains (Figure 3-f7). Stiff extended structurescomposed of a series of domains are especially common in extracellular matrixmolecules and in the extracellular portions of cell-surface receptor proteins'Other modules, including the SH2 domain and the kringle domain illustrated inFigure 3-16, are of a "plug-in" typ", with their N- and C-termini close together.After genomic rearrangements,such modules are usually accommodated as aninsertion into a loop region of a second protein.A comparison of the relative frequency of domain utilization in differenteucaryotes reveals that, for many common domains, such as protein kinases,this frequency is similar in organisms as diverse as yeast, plants, worms, flies,and humans (Figure 3-f 8).
But there are some notable exceptions, such as theMajor Histocompatibility Complex (MHC) antigen-recognition domain (seeFigure 25-52) that is present in 57 copies in humans, but absent in the otherfour organisms just mentioned. Such domains presumably have specializedfunctions that are not shared with the other eucaryotes,being strongly selectedfor during evolution so as to give rise to the multiple copies observed. Similarly,a domain like SH2 that shows an unusual increase in its numbers in highereucaryotes might be assumed to be especially useful for multicellularity (compare the multicellular organisms with yeast in Figure 3-18).CertainPairsof DomainsAreFoundTogetherin ManyProteinsWe can construct a large table displaying domain usagefor each organism whosegenome sequence is knor,r,rr.For example, the human genome is estimated tocontain about 1000 immunoglobulin domains, 500 protein kinase domains, 250DNA-binding homeodomains, 300 SH3 domains, and 120 SH2 domains.
Important additional information can be derived by comparing the frequencies andarrangements of domains in the more than 100 eucaryotic, bacterial, andarchaeal genomes that have been completely sequenced. For example, we findthat more than two-thirds of proteins consist of two or more domains, and thatthe same pairs of domains occur repeatedly in the same relative arrangement ina protein. Although half of all domain families are common to archaea,bacteria,and eucaryotes,only about 5 percent of the two-domain combinations are similarly shared.This pattern suggeststhat most proteins containing especiallyuseful two-domain combinations arose relatively late in evolution.The 200 most abundant two-domain combinations occur in about onefourth of all of the proteins with recognizable domains in the complete data set.It would therefore be very useful to determine the precise three-dimensionalstructure for at least one protein from each common two-domain combination,so as to reveal how the domains interact in that type of protein structure.(A)(B)Figure3-17 An extendedstructureformed from a seriesof in-line proteinmodules.Fourfibronectintype 3 modules(seeFigure3-16)from the extracellularmatrixmoleculefibronectinare illustratedmodels.in (A)ribbonand (B)space-filling(Adaptedfrom D.J.Leahy,l.
Aukhil and1996.WithCell84:155-164,H.P.Erickson,from Elsevier.)oermission4642co38oca34E 30E26oc6zz18loo1410aa0602.f tr'.c*Jeucaryoticprotein kinaseI."d ot\-\"s^a"\csD N A - b i n d i n gh o m e o d o m a i n./ f'S H 2d o m a i nFigure3-18 The relativefrequenciesofthree protein domains in five eucaryoticorganisms.The approximatepercentagesgivenhavebeendeterminedby dividingthe numberof copiesof eachdomainbythe total numberof distinctproteinsthought to be encodedbY eachorganism,asdeterminedfrom thesequenceof its genome.Thus,for 5H2= 0.005.domainsin humans,120/24,000142Chapter3: ProteinsTheHumanGenomeEncodesa ComplexSetof proteins,RevealingMuchThatRemainsUnknownThe result of sequencing the human genome has been surprising, because itreveals that our chromosomes contain only about 25,000 genes.Based on genenumber alone, we would appear to be no more complex than the tiny mustardweed, Arabidopsis, and only about l.3-fold more complex than a nematodeworm.
The genome sequencesalso reveal that vertebrates have inherited nearlyall of their protein domains from invertebrates-with only 7 percent of identified human domains being vertebrate-specific.Each of our proteins is on average more complicated, however (Figure3-19). Domain shuffling during vertebrate evolution has given rise to manynovel combinations of protein domains, with the result that there are nearlytwice as many combinations of domains found in human proteins as in a wormor a fly.
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