粗粒化模拟研究核糖体30S小亚基的组装动力学.pdf
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1、Dynamics in the assembly of the 30S ribosomal subunitinvestigated by coarse-grained simulationsXinLiu1,andZhiyongZhang1,21School of Data Science,University of Science and Technology of China,Hefei 230027,China;2Department of Physics,University of Science and Technology of China,Hefei 230026,ChinaCor
2、respondence:ZhiyongZhang,E-mail:2023TheAuthor(s).ThisisanopenaccessarticleundertheCCBY-NC-ND4.0license(http:/creativecommons.org/licenses/by-nc-nd/4.0/).Cite This:JUSTC,2023,53(9):0906(8pp)ReadOnlineAbstract:Theribosomeisalargebiomolecularcomplexresponsibleforproteinsynthesis.InEscherichia coli(E.co
3、li),acompleteribosomeiscomposedofa30Ssmallsubunitanda50Slargesubunit.Forapproximatelyhalfacentury,the30Ssubunithasbeenakeymodelsystemforstudyingtheinvitroassemblyoftheribosome,andanassemblymaphasbeenproposed.However,structuraldetailsintheassemblyofthisproteinRNAcomplexremainelusive.Inthispaper,wecon
4、-ductedaseriesofcoarse-grainedsimulationsfollowingtheorderoftheassemblymaptoinvestigateconformationaldy-namicsduringtheassemblyprocessofthe30Ssubunit.Ithasbeenfoundthatthetertiarystructureofnaked16SrRNAisveryunstable,whichisthecaseafterbindingofearly-assemblyproteins.Themid-assemblyproteinscansignif
5、icantlyre-strictthemobilityofthe16SrRNAandmakethelatterclosetothenativestructure.Thefinalbindingofthelate-assemblyproteinswouldfullyobtainthecollectivemotionofthe16SrRNA.Inparticular,proteinsS9andS3mayhavemoreim-portantcontributionstotheassemblyofthe30SsubunitthanotherSproteins.Ourstrategyofcoarse-g
6、rainedsimulationscanbegenerallyusedtostudyassemblydynamicsoflargebiomolecularcomplexesaslongastheassemblymapisavailable.Keywords:biomolecularcomplexes;assembly;ribosome;30Ssubunit;coarse-grainedsimulation;principalcomponentanalysisCLC number:Q617Document code:A1 IntroductionTheribosomeisalargebiomol
7、ecularcomplexthatincludesproteinsandRNAsandisresponsibleforcatalyzingproteinsynthesis1.Acompleteribosomecontainsalargesubunitanda small subunit.In prokaryotes and archaea,the 50S largesubunitiscomposedofa23SrRNA,a5SrRNA,and33pro-teins,whereasthe30Ssmallsubunitconsistsofa16SrRNAand21proteins2.Theevol
8、utionofalargebiomolecularcomplexcanbeseenasalongperiodofassembly,sotheassemblycanreflectthepathwayofcomplexevolution3.Theassemblyprocessofacomplexisdynamic4,whichisrelatedtotheorderinwhichthesameordifferentcomponentscometogether3,5,6.Theas-semblyof the ribosome in vivo requires hundreds of as-sembly
9、factorstoworktogetherundercertainconditions,butthe assembly in vitro can be done without any assemblyfactors7,whichhasbeenstudiedforalongtime.Asearlyasthe1960s,theinvitrospontaneousassemblyabilityofthe30SsubunitintheE.coliribosomewasconfirmedbytheNomuralaboratory8,andsubsequently,theassemblyofthe50S
10、 subunit was confirmed by the Nierhaus laboratory9.Later,the ability of ribosomes from other bacteria to as-semble into catalytically active structures through separatenaturalRNAandproteinswasalsodemonstrated1015.The16SrRNAinthe30Sribosomalsubunithasfourinde-pendentdomains:the5domain,thecentraldomai
11、n,the3majordomain,andthe3minordomain.Eachofthefirstthreemaindomainscanbeindependentlyassembledwiththecorresponding S proteins in vitro1618.Naked 16S rRNA isveryactiveandrequirestheassemblyofSproteinstoformastableordered structure.The 30S subunit can be reconstit-utedinvitrowith16SrRNAasastartingpoin
12、tbyaddingthenecessary S proteins to exhibit their relevant biochemicalcharacteristics19.Under reconstitution conditions,severalmethodsofaddingproteinstothe16SrRNAusingcombinedbindingallowtheSproteinstobedividedintothreeclassestoformanassemblymap20.Primarybindingproteinsbinddir-ectlyandindependentlyt
13、othe16SrRNA,whichisthoughttoinitiatethefoldingofeachofthethreemaindomains.Sec-ondarybindingproteinsrequireatleastoneprimarybindingproteinbeforebindingtothe16SrRNA,andtertiarybindingproteinsrequireatleastoneproteinfromthefirsttwostagestobeassembled.Throughinvitroassemblykineticexperi-ments,akineticas
14、semblymapwasformed,andtheSpro-teins were divided into early-,mid-,mid-late-and late-assemblyproteins.Thekineticassemblydatareflectvariousaspects of domain assembly and binding trends,indicatingthattheassemblyrangesfromthe5domaintothe3domain,whichisconsistentwiththecotranscriptionalassembly21.Article
15、http:/Received:April 18,2023;Accepted:June 08,202309061DOI:10.52396/JUSTC-2023-0064JUSTC,2023,53(9):0906Inrecentyears,variousexperimentaltechniqueshavebeenusedtostudytheassemblyofthe30Ssubunit.Electrosprayionizationmassspectrometry(MS),alongwiththelatestcryo-EM techniques,can visualize intermediates
16、 at near-atomicresolutionthroughoutthepathwayofsubunitconstructionandisthereforewidelyusedtostudytheassemblypathwayofcomplexes5.Whilesuchdatahaveproventobeaveryusefulstartingpoint,theydonotprovidedetailedinformationaboutdynamicsintheassemblyprocess.Thatis,onlythestructurecanbeobtained,andthereisnote
17、noughinformationonhowthecomplexformstheexistingstructure.Molecular dynamics(MD)simulations have long beenusedin studying the dynamic processes of large bio-molecules.They are generally atomic based on classicalmechanics as kinetic principles,and molecular mechanicalforce fields are constantly improv
18、ing22.However,all-atomMDisveryexpensiveinsimulatinglargebiomolecularcom-plexessuchastheribosome,anditwouldbedifficulttode-scribethecompleteandlongtime-scalekineticprocess23.Inrecentyears,coarse-grained(CG)modelshavebeenusedtorevealthephysicalmechanismofaseriesofprotein/nucleicacidmolecules2428becaus
19、etheircomputationalefficiencyissignificantlyhigher than that of all-atom MD.CG simula-tionshavebeencontinuouslyoptimizingandcalibratingrel-evantforcefieldsandmethodsduetotheirabilitytodisplayprocessessuchastheassemblyoflargebiomolecularcom-plexesonalongtimescale29.TherearemanyCGmodelsavailable,sucha
20、sthestructure-basedmodel30,themultiscalecoarse-grained(MS-CG)method31,theMARTINImodel32,33,andtheSAFTmodel34.Therefore,inthiswork,basedonthekineticassemblymap,westudystructuraldynamicsintheassemblyprocessofthe30SsubunitthroughCGsimulations.Thesimulationdataarecomparedwiththeexistingexperimentaldatao
21、finvitroas-sembly,whichcanrevealtheroleofthespecificSproteinsintheassemblyprocess.2 Materials and methods2.1 Simulated systemsThe 30S ribosomal subunit of E.coli is composed of 16SrRNAand21ribosomalproteins(Sproteins,namedS1S21indecreasingorderofmolecularweight).Anatomicmodelofthe30Ssubunitwastakenf
22、romRef.35,inwhichthe16SrRNAcontains1536nucleotidesand20Sproteins(S2S21)areavailable(Fig.1).Thethreemaindomains(5,central,and3major)ofthe16SrRNAmakeupthebody,platform,andhead,respectively.TheassemblyoftheSproteinsisdi-videdintofourstagesaccordingtothekineticmap(Table1).Theearly-assemblyproteinsinclud
23、eS4,S6,S11,S15,S16,S17,S18,andS20(Fig.1,magenta),themid-assemblypro-teinsincludeS7,S8,S9,S13,andS19(Fig.1,orange),themid-late-assemblyproteinsincludeS5andS12(Fig.1,blue),andthelate-assemblyproteinsincludeS2,S3,S10,S14,andS21(Fig.1,red).WebuiltthefollowingsystemstorunCGsimulations.(i)Thenaked16SrRNAi
24、sthestartingstateoftheassembly.(ii)The 30S subunit is the end state of the assembly.(iii)Betweenthestartingandendstates,weaddedtheSproteinsonebyone,followingtheordershowninTable1.2.2 The CG modelsIn the off-lattice G model of a protein,each amino acidFig.1.Structureofthe30Sribosomalsubunit.(a)Thefro
25、ntside(theinterfacewiththe50Ssubunit)and(b)theback.The16SrRNAiscoloredblack,andtheSproteinsarecoloreddifferentlyaccordingtothekineticassemblymap(Table1).Thebody,platform,head,andbeakarelabeled.Dynamicsintheassemblyofthe30Sribosomalsubunitinvestigatedbycoarse-grainedsimulationsLiuetal.09062DOI:10.523
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