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Hoe onderskei aminoasiel-tRNA-sinteses tussen soortgelyke aminosure?

Hoe onderskei aminoasiel-tRNA-sinteses tussen soortgelyke aminosure?


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Hoe herken aminoasiel tRNA-sinteses die regte aminosuur vir hul tRNA? Wat is die strukturele rede agter die selektiewe erkenning? Ek vind dit moeilik om te sien hoe byvoorbeeld leusien en isoleusien selektief herken kan word deur verskillende bindingsakke wanneer hulle soortgelyke hidrofobisiteit, molekulêre volume ens.


Aminoasiel-tRNA-sitetases is hoogs spesifiek vir hul ooreenstemmende aminosuur. Eerstens, die aktiveringsplek, waar die aminosuur bind, vorm 'n komplekse netwerk van intermolekulêre interaksies. Byvoorbeeld, treonien, gekataliseer deur treoniel-tRNA-sintetase, is baie soortgelyk aan valien en serien. Valien het 'n metielgroep in plaas van die hidroksiegroep van treonien, en serien, aan die ander kant, het geen bykomende metielsygroep nie. Uniek aan treoniel-tRNA-sintetase, bevat dit 'n Zn2+ ioon wat 'n interaksiestruktuur vertoon wat spesifiek vir treonien is. Nietemin kan die treoniel-tRNA-sintetase steeds verkeerd gelaai word met serien omdat die sinkioonplek nie spesifiek genoeg is nie, wat lei tot 1% verkeerd gelaaide tRNA's. Vir hierdie doel het aminoasiel-tRNA-sintetases 'n tweede, redigering webwerf wat die gelaaide tRNA proeflees. Die redigeerplek kan serien herken en dit van die tRNA afsply.

Figuur: Interaksienetwerk van die aktiveringsplek van die treoniel-tRNA-sintetase in A. pernix. Die threonien-AMP word in groen getoon.

Bronne:

a Berg JM, Tymoczko JL, Stryer L. Biochemie. 5de uitgawe. New York: W H Freeman; 2002. Afdeling 29.2, Aminoasiel-oordrag RNA-sintetases Lees die genetiese kode. Beskikbaar vanaf: https://www.ncbi.nlm.nih.gov/books/NBK22356/

b Nagaoka, Yoshiyuki, et al. "EVOLUSIE EN tRNA-HERKENNING VAN THREONYL-tRNA-SINTETASE VANAF 'N EKSTERME TERMOFIELIESE ARGEAON, Aeropyrum pernix K1." Viva Origino 1.31 (2003), http://www.origin-life.gr.jp/3101/3101062/3101062.html


TRNA Aminoasilasie

Die tweede stap in tRNA-aminoasilering is oordrag van die geaktiveerde aminosuur vanaf die aa-AMP na die A76-ribose 2'-OH (vir klas I-ensieme) of 3'-OH (vir die meeste klas II-ensieme). Meganisties sal die nukleofilisiteit van hierdie hidroksielgroepe aansienlik verbeter word deur deprotonering, wat 'n vereiste vir 'n aktiewe terreinbasis met 'n p voorstel. Ka naby neutraal gepas geposisioneer binne die aktiewe terreine van die AARS. Hierdie selfde groep kan daarna optree om 'n proton aan die AMP-verlaatgroep te skenk na bindingsklowing. Geen duidelike kandidate het egter na vore gekom vir gekonserveerde residue wat as algemene basis kan optree selfs wanneer aktiewe terreinstrukture bewaar word en volgordemotiewe vir elke AARS-klas geïdentifiseer is nie. Alternatiewelik kan substraat-ondersteunde katalise, wat óf die fosfaatgroep in die aa-AMP óf die α-amien van die aminosuur (in die aa-AMP) as die vereiste basis gebruik, 'n alternatiewe meganistiese scenario bied en klasonafhanklik wees.

Een moontlikheid vir 'n potensiële aktiewe terreinbasis is opgemerk vanuit die drieledige kompleks tussen klas I E coli GlnRS, sy verwante tRNA Gln, en die glutaminiel-adenilaat-analoog 5'-O-[N-(l-glutaminiel)-sulfamoyl]adenosien (QSI), wat 'n begrawe glutamaat (Glu34) vir hierdie rol voorgestel het [79]. Die Glu34-karboksilaat is 2,7 Å weg van 'n kristallografiese water aangrensend aan die tRNA 2'-OH. Die voorgestelde protonoordragaflos sal staatmaak op Glu34-gemedieerde waterdeprotonering, gevolg deur deprotonasie van die 2'-OH met gepaardgaande aanval op die karbonielgroep van die ensiemgebonde glutaminieladenilaat. Die oorgangstoestand van hierdie reaksie sal 'n tetraëdriese koolstoftussenproduk bevat wat vermoedelik deur die guanidino-groep van Arg260 gestabiliseer moet word [79]. Hierdie voorgestelde meganisme vir aminosuuroordrag het die vroeëre voorstel, gebaseer op die GlnRS:tRNA Gln:ATP-struktuur, weerspreek dat 'n nie-oorbruggende suurstof in Gln-AMP die A76 2'-OH sal deprotoneer [55]. Gegewe die baie lae pKa (1.5-2) van die fosfaat suurstof en dat Gln in die struktuur gemodelleer is, was die latere voorspelling van Glu34 as die algemene basis aantreklik [79]. In 'n poging om die meganistiese kwessie van ensiem- of substraat-gekataliseerde deprotonasie van die 2'-OH-groep op te los, het Perona en medewerkers beide Glu34 en die nabygeleë Glu73 gemuteer en reaksietempo's geëvalueer deur bestendige-toestand en voor-bestendige-toestand benaderings [80] . 'n Glu34Gln-mutasie het onbeduidende verandering getoon kchem, die enkelomset-aminoasilasietempo (alhoewel hierdie substitusie 'n verhoogde Kd vir die Gln-substraat). Die ooreenstemmende Glu73Gln-vervanging het afgeneem kchem met 10 3-voudig relatief tot wild-tipe GlnRS hierdie daling is toegeskryf aan verlies van kontakte met aangrensende residue wat die tRNA 3'-end in die aktiewe plek posisioneer. Gevolglik is daar tot die gevolgtrekking gekom dat nie een van die Glu-residu deelneem aan die bevordering van A76 2'-OH-deprotonasie nie [80].

Sonder 'n oënskynlike algemene basis om tRNA-aminoasilasie in óf klas I óf II ensieme te bevorder, is aandag gevestig op die moontlikheid van substraat-ondersteunde katalise. Die nie-oorbruggende fosfaatoksianion het 'n interessante moontlikheid gebied ten spyte van sy lae pKa, veral omdat die blKa waardes van die AMP-reaksieproduk is hoër by 3.8 en 6.2 (The Merck Index, Rahway, NJ). Gevolglik is 'n gesamentlike meganisme aantreklik, sodanig dat die adenilaat-nie-oorbruggende suurstof die 2'-OH deprotoneer, die gevolglike suurstofnukleofiel die adenilaatkarbonielkoolstof aanval en die aangrensende CO-binding breek (Fig. 4). Hierdie substraat-ondersteunde strategie sal waarskynlik algemeen wees vir ensieme van beide klasse, en kan 'n voorvaderlike meganisme weerspieël wat vooraf die opkoms van die twee afsonderlike strukturele klasse bestaan ​​het.

Fig. 4 . 'n Algemene meganisme vir aa-AMP-deelname aan tRNA-aminoasilering. Afgelei van meganismes wat voorheen voorgestel is [33,81]. Ado, adenosien R, aminosuursyketting verwant aan die gegewe AARS.

Bewyse wat 'n substraat-ondersteunde gesamentlike meganisme vir tRNA-aminoasilasie ondersteun soos wat in Fig. 4 getoon word, is beskikbaar vir beide klasse AARS'e. Francklyn en kollegas het hul E coli HisRS: His-AMP-struktuur met die tRNA His A76 gemodelleer in die aktiewe terrein om 'n gedetailleerde, gesamentlike meganisme vir tRNA-aminoasilering voor te stel [33]. In hierdie model is vier sleutelresidu's in die nabyheid van beide die gebonde His-AMP en die terminale ribose: Arg259, Arg113, Gln127 en Glu83. Die twee Arg-residu is onderskeidelik in wisselwerking met die adenilaat Sp en Rp nie-oorbruggende suurstof, terwyl Gln die α-karboniel van die adenilaat kontak. Arg259His-, Glu83Gln- en Glu83Ala-variante het almal aansienlik verlaagde aminosuuroordragtempo's getoon wat aan defekte in katalise of A76-posisionering toegeskryf kon word. Om hierdie onsekerheid aan te spreek, is fosforotioaat analoë van die adenilaat by beide Sp en Rp posisies bekendgestel om die rol van His-AMP in katalise te ondersoek. Vir wild-tipe HisRS het die ATPαSp substitusie gelei tot 'n 10 000-voudige verlies aan aktiwiteit in vergelyking met slegs 'n 50-voudige verlies vir die Rp-vervanging. Soortgelyke neigings is waargeneem met die Arg259His en Glu83Gln variant ensieme. Dus, die histidiel-adenilaat nie-oorbruggende Sp suurstof is die waarskynlike algemene basis vir A76 3'-OH deprotonasie, en histidien oordrag word voorgestel as 'n gesamentlike, substraat-ondersteunde reaksie [33]. Hierdie meganisme word ondersteun deur digtheid funksionele teorie (DFT) berekeninge [81].

Eksperimentele bewyse dui ook daarop dat klas I AARS'e staatmaak op aa-AMP-ondersteunde katalise deur 'n nie-oorbruggende suurstof te gebruik om deprotonasie te bevorder, in hierdie geval van die 2'-OH op die 3'-end van die tRNA-substraat. Die nadelige impak van swaelvervanging vir óf nie-oorbruggende suurstof in die α-fosfaat van ATP is so vroeg as 1982 vir MetRS-aktiwiteit erken: een stereoisomeer (genoem ATPαSA) veroorsaak 'n

180-voudige inval Vmaks, terwyl die ander (ATPαSB) ensiemaktiwiteit heeltemal uitgeskakel het [82]. Met die meganisme getoon in Fig. 4 in gedagte, lyk dit waarskynlik dat die swael in ATPαSB die nie-oorbruggende suurstof vervang het wat krities sou wees vir deprotonasie van die aanvallende 2'-hidroksielgroep. In 2000 het First en kollegas resultate van gedetailleerde voor-bestendige-toestand kinetika gebruik om 'n 6-ledige ringoorgangstoestand voor te stel met die Tyr-AMP nie-oorbruggende suurstof op die fosfaat wat geposisioneer is om die 2'-OH op tRNA Tyr te deprotoneer [83 ] . En in 2017 het Aboelnga en Gauld die tRNA-aminoasilasiemeganismes van die E coli GlnRS en T. maritima ND-GluRS. Molekulêre dinamika-simulasies, gekombineer met QM- en QM/MM-berekeninge, het gelei tot 'n meganistiese voorstel soortgelyk aan dié wat in Fig. 4 getoon word. In hierdie gevalle is 'n geordende watermolekule tussen die nie-oorbruggende fosfaat suurstof in die aa-AMP en die 2'-OH in die tRNA geposisioneer. Dus sal die oorgangstoestand deur 'n 8-lede ring voortgaan en die vereiste herlei van protonoordragte sal voortgaan via aa-AMP-gemedieerde deprotonasie van hierdie watermolekule met onmiddellike reprotonering deur die proton van die 2'-OH te gebruik. Onmiddellike nukleofiele aanval by die α-fosfaat sal die siklus voltooi [84]. Of water aan protonoordrag vir alle klas I AARS'e deelneem of nie, moet nog bepaal word.

'n Moontlike uitsondering op die algemene geval van aa-AMP-gesteunde deprotonering van die A76-hidroksiel is die klas II ThrRS. Vergelyking van E coli ThrRS:AMP en ThrRS:tRNA Thr:AMP strukture dui aan dat 'n gekonserveerde His-residu (His309) heroriënteer op tRNA-binding om die A76 2'-OH [34] te kontak. Mutasie van His309 het aminoasilasie verminder, en hierdie verlies is toegeskryf aan die oordragstap, met 'n afname in ktrans van > 200-voudig. Die skrywers argumenteer dat die A76 2'-OH ook aktief is in die oordragstap, aangesien substitusie met 2'-deoksie of 2'-fluoro afgeneem het, maar nie afgeskaf het nie, ktrans met verliese van 10 2 –10 3 relatief tot die inheemse tRNA. Deelname deur die adenilaat nie-oorbruggende suurstof in deprotonasie van die A76 3'-OH is verdiskonteer, aangesien fosforotioaatvervangings nie aminosuuroordrag beduidend beïnvloed het nie [34]. Dubbelmutantsiklusanalise tussen die His309Ala en 2'-deoksie of 2'-fluoro substitusies dui termodinamiese koppeling aan, wat lei tot 'n katalitiese model vir Thr-tRNA Thr sintese wat proton-relais van His309 na A76 2'-OH na die nukleofiele A76 roep ′-OH (Fig. 5 A). Daaropvolgende DFT-berekeninge het alternatiewelik 'n moontlike rol vir die substraat treonien-aminogroep as die algemene basis voorgestel. ThrRS maak staat op 'n aktiewe plek Zn 2 + kofaktor vir aminosuur seleksie en aktivering deur te koördineer na die α-amien en β-hidroksie groepe in Thr (en nie-verwante Ser) (word in meer besonderhede later in hierdie hoofstuk beskryf). Die nabyheid van hierdie Zn 2 + ioon en die berekende labiliteit van die binding tussen die Zn 2 + en die α-amien bevorder die basaliteit van die amien (Fig. 5 B) [85]. Hierdie nuwe meganisme is nie eksperimenteel getoets nie.

Fig. 5 . tRNA aminoasilasie meganismes voorgestel vir ThrRS. (A) His309 dien as 'n katalitiese basis om 'n herlei van protonoordragte te inisieer wat die 3'-OH as 'n nukleofiel aktiveer [32]. (B) Die α-amien in die Thr-AMP substraat dien as die basis wat die 3'-OH deprotoneer. Thr-tRNA Thr (regs) is die produk wat uit enige roete sou ontstaan ​​[83].


Sink-ioon-gemedieerde aminosuur-onderskeiding deur treoniel-tRNA sintetase

Akkurate vertaling van die genetiese kode hang af van die vermoë van aminoasiel-tRNA-sintetases om tussen soortgelyke aminosure te onderskei. Om die basis van aminosuurherkenning te ondersoek en om die rol wat die sinkioon teenwoordig in die aktiewe plek van treoniel-tRNA sintetase speel te verstaan, het ons die kristalstrukture van komplekse van 'n aktiewe afgeknotte vorm van die ensiem met 'n treoniel bepaal. adenilaat analoog of treonien. Die sinkioon is direk betrokke by treonienherkenning en vorm 'n pentakoördinaat-tussenproduk met beide die aminogroep en die syketting-hidroksiel. Aminosuuraktiveringseksperimente toon dat die ensiem geen aktivering van isosteriese valien toon nie, en aktiveer serien teen 'n tempo 1 000 maal minder as dié van verwante treonien. Hierdie studie demonstreer dat die sinkioon nie streng katalities of struktureel is nie en stel voor hoe die sinkioon verseker dat slegs aminosure wat 'n hidroksielgroep besit wat aan die β-posisie geheg is, geaktiveer word.


Resultate

Datastel

Gebaseer op alle beskikbare strukture in die PDB, is 424 (189 Klas I, 235 Klas II) driedimensionele strukture van aaRS'e wat saamgekristalliseer is met hul ooreenstemmende aminosuurligande ontleed. Die geselekteerde data dek aaRS'e van 56 verskillende spesies in totaal, 180 van eukariote, 213 van bakterieë, en 31 van archaea (SI Aanhangsel Fig. S1). In totaal is 70 menslike strukture deel van die datastel. Elke proteïenketting wat 'n proteïen-ligand-kompleks van 'n katalitiese aaRS-domein bevat, is oorweeg. Data was beskikbaar vir elk van die 20 aaRSs, plus die nie-standaard aaRSs pirrolisiel-tRNA sintetase (PylRS) en fosfoseriel-tRNA sintetase (SepRS). Ongelukkig kon Klas I LysRS nie vir ontleding oorweeg word nie. Die enkele struktuur van hierdie ensiem van Pyrococcus horikoshii (PDB-ID: 1irx), wat deel is van die datastel, bevat geen mede-gekristalliseerde aminosuurligand nie. Die aantal proteïen-ligand komplekse beskikbaar vir elke aaRS word gegee in SI Bylaag Fig. S2. Vir twaalf aaRSs was proteïen-ligand komplekse beskikbaar in beide pre-aktivering en post-aktivering reaksie toestande, dit wil sê saamgekristalliseer met óf aminosuur óf aminoasiel ligand (SI Aanhangsel Fig. S3). Uit alle geanaliseerde strukture is 240 in pre-aktivering en 184 in post-aktivering toestand. Uit die post-aktiveringskomplekse is 72 adenosienmonofosfaat (AMP) esters en 112 is nie-hidroliseerbare analoë, hoofsaaklik sulfamoylderivate.

Interaksie kenmerke

Die frekwensies van waargenome nie-kovalente bindingsplek-interaksies ten opsigte van die aaRS-klas en die tipe interaksie word in Tabel 1 getoon. Oor die algemeen is hidrofobiese interaksies die mees algemene interaksies vir Klas I aaRS'e met 'n frekwensie van 44.60% t.o.v. die totale aantal interaksies, terwyl waterstofbindings die meeste waargeneem word in Klas II aaRS'e met 59.23% frekwensie. Vyf (waterstofbindings, hidrofobiese interaksies, soutbrûe, (pi ) -stapeling en metaalkomplekse) interaksietipes is in aaRS'e waargeneem. Geen (pi ) -kation interaksies is waargeneem wat betrokke was by aminosuurbinding nie. Waterbrûe is uitgesluit van die interaksie-analise. Sommige aaRS-strukture wat in die PDB gedeponeer word, word opgelos, insluitend water, maar ander strukture bevat nie watermolekules nie. In hierdie gevalle kan geen waterbrûe met PLIP opgespoor word nie, ten spyte daarvan dat dit bestaan in vivo, wat tot 'n eksperimentele vooroordeel sou lei. Nietemin is dit bekend dat watermolekules belangrike interaksies vir ligandherkenning bemiddel 48 en hul rol moet nie onderskat word nie.

Aminosuurherkenning

Die annotasie van nie-kovalente proteïen-ligand-interaksies het toegelaat om interaksievoorkeure van elke aaRS op die vlak van individuele atome van hul aminosuurligande te karakteriseer. Hierdie ontleding beklemtoon die voorkeurmetodes van binding vir elk van die 22 aminosuurligande. Figuur 2 toon die interaksies wat voorkom vir elke aaRS gebaseer op die analise met PLIP. Elke interaksie word geannoteer met sy besetting, dit wil sê die relatiewe frekwensie van voorkoms ten opsigte van die totale aantal strukture vir hierdie aaRS. Bindingsplekkenmerke word op hierdie punt afgeskeep en alle interaksies word getoon met betrekking tot die aminosuurligand.

Die herkenning van individuele aminosure deur aaRS'e wat na hul ligande gekarteer is. Die ligande word gegroepeer volgens fisies-chemiese eienskappe 49 en aaRS-klas. Verskillende tipes nie-kovalente proteïen-ligand interaksies is met PLIP 46 bepaal en toegewys aan individuele atome van die ligand deur gebruik te maak van subgraaf isomorfisme opsporing 50. Ruggraatatome van die ligand word uitgebeeld as sirkels sonder gevulde binnekant. Die relatiewe besetting van elke interaksie ten opsigte van die totale aantal ondersoekde strukture (getal tussen hakies vir elke aaRS) word deur sirkeldiagramme gegee. Interaksies met 'n besetting onder 0,1 word verwaarloos. Interaksies waarvoor 'n unieke kartering aan 'n individuele atoom nie moontlik is nie as gevolg van dubbelsinnige isomorfisme, bv. vir die syketting van valien, is aan veelvuldige atome toegewys. (pi ) -stapeling-interaksies word in donkergroen getoon en verwys na alle atome van die aromatiese ringstrukture in TyrRSs, TrpRSs en PheRSs. Sommige aaRS'e voorkom die verkeerde lading van hul tRNA's via foutkorreksiemeganismes ("redigering") 51 . Die aaRS'e wat foutkorreksie uitvoer, is vetgedruk.

Klas I

In die algemeen tree Klas I aaRS'e hoofsaaklik in wisselwerking via waterstofbindings en hidrofobiese interaksies met die ligand. Die ruggraatatome van alle Klas I-ligande het waterstofbinding met die primêre amiengroep. Die besetting van hierdie interaksie is hoog deur alle Klas I aaRS'e, wat 'n deurslaggewende rol van hierdie interaksie vir ligandfiksasie aandui. Daarbenewens is die suurstofatoom van die ligand se karboksielgroep betrokke by waterstofbinding, behalwe vir glutaminiel-tRNA sintetase (GlnRS), isoleucyl-tRNA sintetase (IleRS), en valyl-tRNA sintetase (ValRS). Dieselfde atoom vorm bykomende soutbrûe in leucyl-tRNA sintetase (LeuRS), arginiel-tRNA sintetase (ArgRS), metioniel-tRNA sintetase (MetRS), en glutamyl-tRNA sintetase (GluRS). Die sykettings van die alifatiese aminosure leusien, isoleusien en valien word uitsluitlik gebind deur hidrofobiese interaksies. ArgRS en GluRS vorm soutbrûe tussen bindingsplekresidue en die gelaaide karboksiel- en guanidiengroepe van die ligand, onderskeidelik. Glutamien word deur GlnRS gebind via gekonserveerde waterstofbindings aan die amiedgroep en hidrofobiese interaksies met beta- en delta-koolstofatome. Die twee aromatiese aminosure tirosien en triptofaan word herken deur (pi )-stapeling-interaksies en uitgebreide hidrofobiese kontaknetwerke. Triptofaan word verkieslik vanaf die een kant van sy indoolgroep op posisies een, ses en sewe gebind. Die swaelatoom van die sisteïniel-tRNA sintetase (CysRS) ligand vorm 'n metaalkompleks met 'n sinkioon in beide strukture. MetRS'e bind hul ligand met 'n hoogs gekonserveerde hidrofobiese interaksie met die beta-koolstofatoom.

Klas II

Klas II aaRS'e werk konsekwent met die ruggraatatome van die ligand via waterstofbindings en soutbrûe. Die primêre amiengroep vorm waterstofbindings met hoë besetting en is betrokke by metaalkompleksvorming in treoniel-tRNA-sintetases (ThrRSs) en seriel-tRNA-sintetases (SerRSs). Die karboksiel suurstofatome van die ligande word gebind deur 'n kombinasie van waterstofbinding en elektrostatiese soutbrug interaksies. Die algehele ruggraatinteraksiepatroon is hoogs bewaar binne Klas II aaRS'e. Nadere ondersoek het aan die lig gebring dat 'n voorheen beskryfde strukturele motief van twee arginienresidu 43, verantwoordelik vir ATP-fiksasie, blykbaar betrokke is by die stabilisering van die aminosuurkarboksielgroep met sy N-terminale arginienresidu. Die gelaaide aminosuurligande in histidiel-tRNA sintetase (HisRS) en LysRS vorm hoogs gekonserveerde waterstofbindings met die bindingsplekresidue. Ander spesifisiteit-verlenende interaksies sluit in (pi ) -stapelingsinteraksies en hidrofobiese kontakte waargeneem vir fenielalanien-tRNA-sintetase (PheRS), metaalkompleksvorming vir ThrRS en SerRS met sink, en soutbrûe sowel as waterstofbindings vir aspartyl-tRNA sintetase (AspRS). Die aminosure alanien en prolien word gebind deur alaniel-tRNA sintetases (AlaRSs) en prolyl-tRNA sintetases (ProRSs) via hidrofobiese interaksies. Geen spesifisiteit-verlenende interaksies kan beskryf word vir die kleinste aminosuur glisien as gevolg van die afwesigheid van 'n syketting nie. Gevolglik kan glisiel-tRNA sintetase (GlyRS) slegs interaksies met die ruggraatatome van die ligand vorm. Verder bemiddel asparaginiel-tRNA-sintetases (AsnRS'e) hoogs gekonserveerde waterstofbindings met die amiedgroep van hul asparagienligand. Die nie-standaard aminosuur pirrolisien word deur PylRS gebind via verskeie waterstofbindings en hidrofobiese interaksies met die pirroliengroep. SepRSs gebruik hoofsaaklik soutbrug-interaksies om die fosfaatgroep van die fosfoserienligand te fikseer.

Bewaarde interaksiepatrone

Klas I aaRS'e toon 'n sterk bewaring van waterstofbindings met die primêre amiengroep van die aminosuurligand met 83.16% van alle strukture wat hierdie interaksie vorm. Interaksies met die karboksielgroep is minder behoue ​​met 'n frekwensie van onderskeidelik 32.65% vir waterstofbindings en 28.57% vir soutbrûe. In hierdie konteks is die soutbruggies met die karboksielgroep 'n vorm van ekstra sterk waterstofbinding 52 . Interaksiepatrone met die ruggraatatome van die aminosuurligand is opvallend konsekwent binne Klas II aaRS'e. Hierdie klas vorm waterstofbindings met die primêre amiengroep in 92,15% van alle strukture. Daarbenewens kom waterstofbindings met die suurstofatoom van die karboksielgroep in 65.70% van alle strukture voor en soutbrûe met dieselfde atoom word in 39.26% van alle Klas II-proteïen-ligand-komplekse gevorm.

Soortgelyke herkenning vereis redigeermeganismes

Daar is bekend dat verskeie aaRS'e voor- of na-oordrag redigering uitvoer (sien die werk van Perona en Gruic-Sovulj 51 vir 'n gedetailleerde bespreking van redigeermeganismes) ten einde behoorlike kartering van aminosure aan hul verwante tRNA's te verseker. Die ooreenkoms van interaksievoorkeure wat in Fig. 2 uitgebeeld word, dui daarop dat groepe baie soortgelyke aminosure redigeermeganismes benodig vir hul korrekte hantering. Veral die drie alifatiese aminosure isoleucine, leucine en valine word gebind via onspesifieke en swak hidrofobiese interaksies, wat die noodsaaklikheid van redigeringsmeganismes wat vir hul aaRSs 53 waargeneem word, staaf en dat substraathidrofobisiteit nie heeltemal vir spesifisiteit 54 kan verantwoordelik wees nie. Onderskeid tussen daardie drie soortgelyke aminosure word voorgestel om via die "dubbelsif" 52 meganisme te gebeur. By voorbeeld vir IleRS word aminosure groter as isoleusien uitgesluit met die "eerste sif" by die aminoasilasieplek, terwyl kleiner aminosure (soos valien en leusien) uitgesorteer word deur die redigeringsdomein, wat as 'n fyner "sif" funksioneer. Spesifisiteit kan dus bewerkstellig word deur steriese seleksie gebaseer op sykettinglengte en -vorm by die redigeerplek 53 . 'n Soortgelyke neiging kan waargeneem word, bv. vir AlaRS 55 om alanien van serien of glisien te onderskei.

Bindplekgeometrie en holtevolume

Ons het bindingsplekgeometrie en holtevolume ondersoek om hul potensiële bydrae tot aminosuurherkenning te kwantifiseer. Bekende redigeringsmeganismes in aaRS'e is gefokus op die voorkoming of regstelling van tRNA-mislading binne een aaRS-klas (intra-klas), bv. die aminosure isoleucine, leucine en valine behoort aan Klas I. GluRSs en AspRSs het egter 'n hoogs soortgelyke interaksiepatroon van waterstofbindings en soutbrûe met die karboksielgroep en swak hidrofobiese interaksies. Beide aaRS'e gebruik nie redigering nie en word deur verskillende aaRS-klasse hanteer. In hierdie geval kan die geometrie en grootte van die bindingsplek optree as 'n bykomende laag van selektiwiteit 'n meganisme wat ook deur ValRS 53,56 ontgin word. Om die bydrae van bindingsplek-geometrie te kwantifiseer, is sewe strukture van GluRS en ses strukture van AspRS gesuperponeer met betrekking tot hul algemene adenien-onderbou met behulp van die Fit3D 57-sagteware. Aangesien hierdie superimposisie slegs vir proteïen-ligand komplekse bereken kan word wat soos die post-reaksie toestand lyk, is slegs 'n subset van die strukture gebruik. Die resultate toon dat die ligande van GluRSs en AspRSs georiënteer is na verskillende kante van 'n vlak gedefinieer deur hul gemeenskaplike adenien substruktuur (Fig. 3A). Daar is 'n beduidende verskil (Mann-Whitney U bl<0.01) in ligandoriëntasie, beskryf deur die torsiehoek tussen fosfaat en die aminosuursubstruktuur van die ligand (Fig. 3B). Klas I GluRS'e het 'n torsiehoek van 54.64 ± 7.12 (^) , terwyl die torsiehoek van Klas II AspRS'e −65.02 ± 7.40 (^) is. Verder is die volume van die spesifisiteit-verlenende deel van die bindingsplek (sien Fig. 1) beraam met die POVME 58 algoritme. Dit verskil aansienlik (Mann-Whitney U bl<0.01) tussen GluRS (147.00 ± 22.31 Å (^3) ) en AspRS (73.34 ± 17.12 Å (^3) ). Hierdie neiging kan onderskeidelik vir alle Klas I- en Klas II-strukture waargeneem word. 'n Ontleding van alle verteenwoordigende strukture vir Klas I en Klas II aaRS'e toon dat Klas I bindingsplekke aansienlik is (Mann-Whitney U bl<0.01) gemiddeld groter (Fig. 3C). Terwyl Klas I-bindingsholtes 'n gemiddelde volume van 143.40 ± 39.62 Å (^3) het, is Klas II-bindingsplekke gemiddeld 90.36 ± 32.09 Å (^3) in volume.

Bindingsgeometrie en bindingsholte-volume-analise. (A) Bindingsgeometrie van GluRS'e en AspRS'e. Aminoasielligande van Klas I GluRS'e en Klas II AspRS'e in post-aktivering toestand in lyn met Fit3D 57 met betrekking tot hul adenien substruktuur. Die middelpunte van nie-kovalente interaksies 46 met bindingsplekresidue word as klein sfere uitgebeeld. Blou is waterstofbinding, geel is soutbrug, en grys is hidrofobiese interaksie. (B) Verspreiding van torsiehoeke tussen die fosfaat- en aminosuursubstruktuur van die ligand. Die oriëntasie van die ligand in die bindingsplek verskil aansienlik (Mann–Whitney U (p<0.01) ) tussen GluRSs en AspRSs. (C) Die volume van die spesifisiteit-verlenende eenheid van die bindingsplek, geskat met die POVME-algoritme 58, verskil aansienlik tussen Klas I en Klas II aaRS'e (Mann-Whitney U (p<0.01) ).

Interaksiepatrone van individuele aaRS'e

Benewens die ondersoek van interaksievoorkeure vanuit die ligand-oogpunt, is die bindingsplekke van elke aaRS ontleed ten opsigte van die residue wat interaksies met die aminosuurligand vorm. Omdat elke aaRS gerugsteun word deur veelvuldige proteïene van diverse organismes met aansienlik uiteenlopende volgordes, het ons 'n berekeningsabstraksie ontwerp om die leser in staat te stel om aminosure van individuele proteïene af te lei via 'n struktuurgedrewe veelvuldige volgorde-belynings (MSA's) (sien "Metodes" afdeling) . Oorspronklike rynommers vir elke posisie kan afgelei word met die karteringtabelle wat saam met hierdie manuskrip gepubliseer is (sien Databeskikbaarheid). Elke ry in die tabel stem ooreen met die kunsmatige volgordeposisie, terwyl elke kolom die oorspronklike posisie vir elke struktuur in ons datastel gee soos gedefinieer deur die PDB. Figuur 4A toon 'n volgorde logo 59 voorstelling van bindingsplek interaksies vir AlaRS. Elke gekleurde posisie in die volgorde-logo verteenwoordig interaksies wat by hierdie posisie plaasvind. Hoogs bewaarde interaksies kan waargeneem word by hernommerde posisie 135. Die ooreenstemmende waterstofbinding en soutbrug interaksies word gevorm met die ruggraatatome van die ligand. Aan die proteïenkant word hierdie interaksie bemiddel deur 'n gekonserveerde arginienresidu wat ooreenstem met die N-terminale oorblyfsel van die voorheen beskryfde Arginine Tweezers-motief 43. Nog 'n prominente interaksie word gevorm deur valien by hernommerde posisie 293. Hierdie oorblyfsel tree in wisselwerking met die beta-koolstofatoom van die alanienligand via hidrofobiese interaksies. In sommige strukture word hierdie hidrofobiese interaksie gekomplementeer deur 'n alanienresidu by hernommerde posisie 325. Asparaginsuur by hernommerde posisie 323 is hoogs behoue ​​in AlaRSs en blyk betrokke te wees by aminosuurfiksasie via waterstofbinding van die primêre amiengroep. Oor die algemeen is die spesifisiteit-verlenende interaksies met die klein syketting van alanien hidrofobiese kontakte. 'n Voorbeeld vir aminosuurherkenning in AlaRS'e word in Fig. 4B gegee. Die struktuur van bakteriese Escherichia coli AlaRS vorm die hele reeks waargenome interaksies. Volgordelogo's van die oorblywende aaRS'e word in SI Aanhangsel Fig. S4–S24. Gebaseer op die interaksies tussen bindingsplekresidue en die ligand, is 'n kwalitatiewe opsomming van spesifisiteitverlenende meganismes en sleutelresidu saamgestel (Tabel 2). Verder is die ligandgrootte en telling van waargenome interaksies nagegaan vir afhanklikheid. Daar is 'n swak maar beduidende positiewe korrelasie tussen die gemiddelde aantal interaksie bindingsplekresidue vir elke aaRS en die aantal nie-waterstofatome van die aminosuurligand (Pearson r=0.32, bl<0.01). Dit dui aan dat die aantal gevormde interaksies oor die algemeen toeneem met ligandgrootte. Kleiner aminosure het egter nie noodwendig 'n minder komplekse herkenningspatroon nie. ThrRS'e bind byvoorbeeld hul aminosuurligand met gemiddeld meer as 'n dosyn bindingsplekreste, terwyl ValRS'e gemiddeld vyf bindingsplekresidu gebruik. Die hidroksielgroep van treonien laat toe dat 'n uitgebreide reeks nie-kovalente interaksies gevorm word met bindingsplekresidue in vergelyking met valien, waar slegs hidrofobiese kontakte gevestig kan word. Verspreidings van interaksie bindingsplek residue vir elke aaRS word gegee in SI Bylaag Fig. S25.

Interaksiepatrone van AlaRS. (A) Volgorde-logo 59 van verteenwoordigende rye vir AlaRS'e. Nie-kovalente interaksies met die aminosuurligand wat op sekere posisies voorkom, word deur gekleurde sirkels aangedui. Gevulde sirkels is interaksies met die sykettingatome, terwyl hol sirkels interaksies met enige van die ruggraatatome van die aminosuurligand is. Blou is waterstofbinding, geel is soutbrug, grys is hidrofobiese interaksie. (B) Uitbeelding van interaksies in die bindingsplek (bloustokmodel) van 'n AlaRS van Escherichia coli (PDB:3hxz ketting A) met sy ligand (oranje stok model). Hier word waterstofbindings (soliede blou lyne) en hidrofobiese interaksies (gestreepte grys lyne) gevestig. Die volgorde posisies van die interaksie residue word gegee in ooreenstemming met die MSA (swart) sowel as die oorspronklike struktuur (rooi). Figuur geskep met PyMol 60. Dubbelbindings word deur parallelle lynsegmente aangedui, aromatiese bindings deur sirkelvormige stippellyne.

Kwantitatiewe vergelyking van ligandherkenning

Om voorsiening te maak vir 'n kwantitatiewe analise en vergelyking van ligandherkenning tussen verskeie aaRS'e, is interaksie- en bindingsplekkenmerke as binêre vektore, sogenaamde interaksievingerafdrukke, voorgestel (sien "Metodes" afdeling). Gebaseer op hierdie vingerafdrukke, is die Jaccard-afstand vir elke paar strukture bereken om die ongelykheid in ligandherkenning voor te stel. Vervolgens is die Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) algoritme 61 gebruik vir dimensionaliteitsvermindering en inbedding van die hoëdimensionele vingerafdrukke in twee dimensies vir visualisering. Hierdie inbedding word beskou as die herkenningsruimte van aaRSs. Die tweedimensionele visualisering van hierdie herkenningsruimte (Fig. 5) kan gesien word as 'n kaart wat die ooreenkoms in ligandherkenning oor alle aaRS'e beskryf. Daardeur stem elke datapunt ooreen met 'n enkele aminosuurbindingsplek wat gekenmerk is deur interaksie en bindingsplekkenmerke. In general, a similar recognition mechanism between two aaRSs can be assumed if they are located close to each other in this map. The more distant two aaRSs are from each other, the less similar their amino acid recognition. However, it has to be noted that the applied dimension reduction does not perfectly conserve distances. Figure 5A shows the embedding results for all aaRSs in the dataset colored according to the aaRS classes. A Principal Component Analysis (PCA) of the same data is given in SI Appendix Fig. S26. For each aaRS the average position of all data points in the embedding space was calculated and is shown as one-letter code label. Figure 5B shows the same data colored according to the physicochemical properties of the amino acid ligand, i.e. positive (lysine, arginine, and histidine), aromatic (phenylalanine, tyrosine, and tryptophan), negative (aspartic acid and glutamic acid), polar (asparagine, cysteine, glutamine, proline, serine, and threonine), and unpolar (glycine, alanine, isoleucine, leucine, methionine, and valine).

Recognition space analysis of all aaRSs. (A) Embedding 61 space of interaction fingerprints for all aaRS structures in the dataset. Scaling is in arbitrary units. The data points are colored according to the aaRS class. One letter code labels are given for each aaRS based on the averaged coordinates in the embedding space. An asterisk indicates the non-standard amino acids phosphoserine (J*) and pyrrolysine (O*). (B) Embedding space of interaction fingerprints for all aaRS structures in the dataset except phosphoserine and pyrrolysine. Scaling is in arbitrary units. One-letter codes of amino acid ligands are used to identify each aaRS. Every data point represents an individual protein-ligand complex. The color of the data points encodes the physicochemical properties 49 of the ligand.

Klas I

In terms of amino acid binding both aaRS classes seem to employ different overall mechanism they separate almost perfectly in the embedding space. Especially aromatic amino acid recognition in Class I tryptophanyl-tRNA synthetases (TrpRSs) and tyrosyl-tRNA synthetases (TyrRSs) is distinct from Class II aaRSs and forms two outgroups in the embedding space. Remarkably, two different recognition mechanisms exist for TrpRSs, indicated by two clusters approximately at positions (−2.0,6.0) and (1.0,8.5) of the embedding space, respectively. The cluster at position (−2.0,6.0) is formed by structures from bacteria and archaea, while the cluster at position (1.0,8.5) is formed by eukaryotes and archaea and is in proximity to TyrRSs. Closer investigation of two representatives from these clusters shows two distinct forms of amino acid recognition for TrpRSs. Human aaRSs employ a tyrosine residue in order to bind the amine group of the indole ring, while prokaryotes employ different residues (SI Appendix Fig. S27). The Class I aaRSs that are closest to Class II are GluRSs and CysRSs. A cluster of high density is formed by Class I IleRS, MetRS, and ValRS, which handle aliphatic amino acids. This indicates closely related recognition mechanisms and difficult discrimination between these amino acids.

Klas II

For Class II aaRSs the recognition space is less structured. Nonetheless, clusters are formed that coincide with individual Class II aaRSs, e.g. a distinct recognition mechanism in AlaRSs. The aaRSs handling the small and polar amino acids threonine, serine, and proline are closely neighbored in the embedding space. Recognition of GlyRSs seems to be diverse GlyRSs are not grouped in the embedding space. However, the recognition of glycine, which has no side chain, is limited by definition and thus the fingerprinting approach might fail to capture subtle recognition features. AspRSs and AsnRSs are located next to each other in the embedding space. Their recognition mechanisms seem to be very similar as the only difference between these two amino acids is the carboxylate and amide group, respectively.

Mechanisms that drive specificity

In order to quantify the influence of different aspects of binding site evolution on amino acid recognition by aaRSs, different interaction fingerprint designs were compared against each other. Each design includes varying levels of information and combinations thereof: the sequence composition of the enzyme’s binding site (Seq), non-covalent interactions formed between side chains of the enzyme’s binding site and the amino acid ligand (Int), whether pre- or post-transfer correction (i.e. “editing”) is conducted (Ed), and the overall volume of the enzyme’s binding cavity (Vol). To assess the segregation power of each fingerprint variant, the mean silhouette coefficient 62 , a quantification for the error in clustering methods, over all data points was calculated. This score allows to assess to which extent the recognition of one aaRS differs from other aaRSs and how similar it is within its own group. Perfect discrimination between all amino acids would give a value close to one, while a totally random assignment corresponds to a value of zero. Negative values indicate that the recognition of a different aaRS is rated to be more similar than the recognition of the same aaRS. Figure 6 shows the results of this comparison. When using fingerprints describing the sequence composition of the enzyme’s binding site (Seq (_ ext ) ), the mean silhouette coefficient over all samples is −0.0510, which indicates many overlapping data points and unspecific recognition. By including non-covalent interactions (Seq, Int) the value increases to 0.1361. If pre- or post-transfer correction mechanisms are considered (Seq, Int, Ed), the silhouette coefficient improves further to 0.2731. Adding information about the binding cavity volume (Seq, Int, Ed, Vol) slightly increases the quality of the embedding to 0.2757. The silhouette coefficients for error correction and volume-based fingerprints were calculated as baseline comparison. If only pre- or post-transfer correction mechanisms (Ed) are considered the mean silhouette coefficient amounts to −0.3027. For binding cavity volume (Vol) the mean silhouette coefficient is −0.4682.

Relation to physicochemical properties of the ligands

In order to investigate whether the fingerprinting approach is a simple encoding of the physicochemical properties of the amino acids, the results were related to experimentally determined phase transfer free energies for the side chains of amino acids from water ( (Delta G_) ) and vapor ( (Delta G_) ) to cyclohexane 3,63 . These energies are descriptors for the size and polarity of amino acid side chains and underlie both, the rules of protein folding and the genetic code 64 . The Spearman’s rank correlation between pairwise distances for each aaRS in the recognition space and physicochemical property space is weak with ( ho ) =0.2564 and bl (<0.01) (see SI Appendix Fig. S28). This indicates that the fingerprinting approach used in this study is a true high-dimensional representation of the complex binding mechanisms of amino acid recognition in aaRSs. This assumption is supported by a PCA (SI Appendix Fig. S26) of the fingerprint data, where the first two principal components account for only 9.24% and 8.44% of the covered variance, respectively.

Comparison of different fingerprint designs that include the sequence composition of the enzyme’s binding site (Seq), non-covalent interactions formed between side chains of the enzyme’s binding site and the amino acid ligand (Int), pre- or post-transfer correction (i.e. “editing”) mechanisms (Ed), and volume of the enzyme’s binding cavity (Vol). Simple sequence-based fingerprints (Seq (_ ext ) ) are a 20-dimensional representation of binding site composition. The line plot shows the silhouette coefficient 62 for each embedding. Points represent mean values, error bars are calculated based on all silhouette coefficients for each data point.


Bespreking

In this study, we characterize the translocation of threonine, α-aminobutyrate, and cysteine during editing by ValRS. For all three amino acids, the translocation rates are similar to their respective overall editing rates (2.7–3.5 s −1 ). The rate of translocation is considerably slower than the maximum rate of hydrolysis of exogenously added misacylated tRNA Val (20–40 s −1 ) (18, 19). Thus, once the misactivated amino acid is translocated to the site for editing, the chemical step for hydrolysis is relatively instantaneous. Consequently, under normal circumstances where a noncognate amino acid is mixed with tRNA Val , translocation is rate-limiting for editing.

The close similarities in the translocation rate constants measured for threonine and cysteine establish that the editing domain is not designed to select a specific misactivated amino acid. This possibility is supported by our observation that α-aminobutyrate also is efficiently translocated during editing, even though it is an unnatural amino acid in E coli (hence, the existence of evolutionary pressure on the E coli editing domain to select α-aminobutyrate as a substrate is not obvious). Instead, to carry out its role as the “fine sieve” in the double-sieve-editing model, the editing domain probably is structured to accept all misactivated amino acids and to strictly prevent the entrance of the activated cognate amino acid.

The translocation of misactivated amino acid during ValRS editing is thought to occur primarily by the posttransfer pathway, that is, by the deacylation of mischarged tRNA Val (20). A possible molecular mechanism for posttransfer editing in IleRS has been inferred from the structure of IleRS complexed to tRNA Ile (9). Specifically, the last three nucleotides of the tRNA (74–76) are proposed to adopt two conformations in the IleRS⋅tRNA Ile complex. The hairpinned conformation projects the A 76 nucleotide into the active site of the enzyme, where it can be aminoacylated, whereas the stacked conformation places the A 76 nucleotide in the editing domain, where an incorrect amino acid (attached to A 76 ) can be hydrolyzed. The switch from the hairpinned to the stacked conformation could therefore provide the mechanism for translocation during editing.

A similar mechanism might be responsible for the translocation of misactivated amino acids during editing by ValRS. In such a mechanism, the rate of translocation will depend primarily on the rate of the tRNA switching from the hairpinned to the stacked conformation, and not on the side chain of the amino acid attached to the tRNA. This prediction is consistent with our observation that threonine, α-aminobutyrate, and cysteine are translocated with similar efficiencies. It also implies that there is no physical contact between the amino acid side chain and the surface of the enzyme that lies between the synthetic active site and the editing site. If the side chain of an aminoacyl group made contact with the enzyme during translocation, then the difference between the more polar threonine and cysteine, on the one hand, and the hydrophobic α-aminobutyrate, on the other, would probably be reflected in different rates of translocation. Thus, we infer that the amino acid side chain points out, away from the surface of the protein during translocation.


How do aminoacyl-tRNA synthases distinguish between similar amino acids? - Biologie

a School of Biology, Biomedical Sciences Research Complex, University of St Andrews, St Andrews, Fife KY16 9ST, UK
E-pos: [email protected]

b Arab Academy for Science, Technology, and Maritime Transport (AASTMT), Cairo Campus, Egypt

c Institute of Quantitative Biology, Biochemistry and Biotechnology, University of Edinburgh, Waddington 1 Building, King's Buildings, Edinburgh, UK

Abstrak

Cyclodipeptide synthases (CDPSs) produce a variety of cyclic dipeptide products by utilising two aminoacylated tRNA substrates. We sought to investigate the minimal requirements for substrate usage in this class of enzymes as the relationship between CDPSs and their substrates remains elusive. Here, we investigated the Bacillus thermoamylovorans enzyme, BtCDPS, which synthesises cyclo(L-Leu–L-Leu). We systematically tested where specificity arises and, in the process, uncovered small molecules (activated amino esters) that will suffice as substrates, although catalytically poor. We solved the structure of BtCDPS to 1.7 Å and combining crystallography, enzymatic assays and substrate docking experiments propose a model for how the minimal substrates interact with the enzyme. This work is the first report of a CDPS enzyme utilizing a molecule other than aa-tRNA as a substrate providing insights into substrate requirements and setting the stage for the design of improved simpler substrates.


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Quality control of the translation machinery

Faithful translation of the mRNA codons into protein is essential for cellular physiology. The fidelity of the translation machinery firstly depends on the specific coupling of amino acids to their cognate tRNA species, which is catalyzed by aminoacyl-tRNA synthetases (aaRSs) (Fig 4a and 4b). aaRS is capable of discriminating its cognate substrates from structurally analogous tRNAs and amino acids [39]. Subsequently, eukaryotic elongation factor 1A (eEF-1A) or prokaryotic EF-Tu delivers the aminoacyl-tRNA to the ribosome A site for elongation of nascent peptide chain after proper codon–anticodon recognition [40]. Thus, aaRSs are cardinal in protecting protein synthesis against misacylation [39], but their specificity is not absolute. Byvoorbeeld, in E. coli, four types of misacylated-tRNA—including Cys-tRNA Pro , Ser-tRNA Thr , Glu-tRNA Gln , and Asp-tRNA Asn —do not evoke a correctional reaction [41]. In both mice and bacteria, serine is prone to be misacylated by alanyl-tRNA synthetases (AlaRSs) [42]. In mycobacteria, an increase in the substitution of glutamic acid→glutamine and aspartic acid→asparagine by translational misincorporation has been linked to phenotypic resistance to rifampicin treatment [43]. Thus, beneficial mistranslation in both prokaryotes and eukaryotes may exist and improve their survival or facilitate drug resistance [43–45]. Apart from misdecoding, misacylation of amino acids to tRNA molecules is another important source of mistranslated proteins, despite the presence of mechanisms preventing such events.

(a) aa-tRNAs are synthesized by sampling from the amino acid pool and tRNA pool and require catalysis by aaRSs. This process may accidently introduce misacylated aa-tRNAs, because the types of tRNAs and amino acids are difficult to be distinguished by involved aminoacyl synthetase because of analogous structures. (b) During elongation, tRNA wobbling will increase translation efficiency. Misincorporation can also be introduced because of tRNA misdecoding (amino acid misincorporation caused by excessive wobble decoding), especially when certain codon-paired tRNA species are missing. Finally, the fidelity of translation machinery will be impaired and produce mutated proteome, including RNA and DNA polymerases, aaRSs, and accessories. (c) Mistranslation of RdRP in RNA viruses will augment generation of a mutated virome (quasispecies) and facilitate viral evolution and adaption. (d) Similarly, mistranslation of cellular DNA replication-related enzymes and relative proteins amplifies mutagenesis in the genome and contributes to cancer development. aaRS, aminoacyl-tRNA synthetase aa-tRNA, aminoacyl-tRNA RdRP, RNA-dependent RNA polymerase tRNA, transfer RNA.

How could tRNA wobbling guarantee faithful decoding by the codon–anticodon duplex? During elongation, eEF-1A or EF-Tu delivers amino acid–coupled tRNA to the ribosome A site [40]. Subsequently, the ribosome rechecks the codon–anticodon duplex that involves the highly conserved G530, A1492, and A1493 of 16S RNA via stabilization of the first two Watson-Crick pairs of the duplex [31, 46]. A correct confirmation of the codon–anticodon duplex will induce a conformational domain closure in the ribosome and result in the formation of the appropriate peptide bond and elongate the nascent protein [47]. Analysis of X-ray structures suggests that the positions 1 and 2 of the A codon are obligatory Watson-Crick base pairs. In prokaryotes, when U•G and G•U wobbles at the first or second codon–anticodon position, the decoding center forces this pair to adopt the geometry close to that of a canonical C•G pair [40]. Using nuclear magnetic resonance (NMR) relaxation dispersion, it has recently been revealed that dG•dT misincorporation during replication is likely mediated via tautomerization and ionization [37]. As discussed, these Watson-Crick-like mismatches may further contribute to tRNA wobbling and consequently misdecoding [5]. Although the hydrogen bond is the major force to form codon–anticodon pairs [1], the van der Waals forces, steric complementarity, and shape acceptance may concurrently contribute to the codon–anticodon recognition essentially for quality control [3, 40].


Not an inside job: non-coded amino acids compromise the genetic code

The sophistication of the editing mechanisms that prevent gene translation errors indicates that amino acid misincorporation is generally a problem to be avoided. Mistranslation is considered invariably deleterious and often caused by confusion between similar proteogenic amino acids. These views are being challenged. The evidence linking misincorporation of dietary non-proteogenic amino acids to human disease continues to grow, and a report in this issue of The EMBO Journal demonstrates the importance of preventing non-proteogenic amino acid misincorporation for cellular homeostasis (Cvetesic etਊl, 2014).

Sien ook: N Cvetesic et al (August 2014)

The genetic code holds the key to translate 64 codons into 20-odd amino acids. The enzymes that aminoacylate tRNAs, aminoacyl-tRNA synthetases (ARS), are the keepers of the code as they create the molecular link between amino acids and triplet information in the tRNA. ARS form two families of enzymes with a peculiar symmetric organization that clusters them in groups that recognize chemically similar amino acids. These two families possibly emerged from an ancestral complex of two proteins around a single tRNA molecule that evolved to increase the number of cognate substrates as the genetic code grew to its extant size. This expansion in cognate substrates logically involved the gradual incorporation of relatively similar side chains to those that were previously used (Ribas de Pouplana & Schimmel, 2001).

The extent to which some proteogenic amino acids are similar to each other𠅊s well as the structural organization of the ARS themselves𠅎xplain the difficulty in discriminating between certain residues during tRNA aminoacylation. To make matters worse, several nonprotein amino acids, which are ubiquitous in many cellular metabolic pathways, can also be mistakenly incorporated into proteins through ARS recognition errors that also require editing reactions to be corrected (Jakubowski, 2012).

Linus Pauling was the first to note that the chemical proximity between some side chains makes it impossible for ARS to discriminate between them with a tolerable error rate (Pauling, 1958). Hence the necessity of editing activities to remove incorrectly charged amino acids was postulated. A “second sieve” model for aminoacylation editing was proposed by Alan Fersht, and later proven to exist in several ARS (reviewed in Yadavalli & Ibba, 2012).

Valine, isoleucine, and leucine are good examples of amino acids requiring proofreading due to their chemical similarity. The discovery of a common editing domain shared by the ARS cognate to these three residues reinforced the notion that misincorporations would mostly involve related proteogenic amino acids, and that such errors always need to be corrected. However, mistranslation need not be limited to proteogenic amino acids and, in some cases, it may offer adaptive advantages to cells.

In this issue of The EMBO Journal Gruic-Sovulj and colleagues elegantly demonstrate that the editing domain of leucyl-tRNA synthetase (LeuRS) is not designed to fend off the misincorporation of isoleucine, as was previously thought. Earlier reports that suggested otherwise were marred by an unsuspected contamination of leucine in commercial preparations of isoleucine. Once the contaminating cognate amino acid is removed from the reaction the authors clearly show, by kinetic, structural, thermodynamic and in vivo approaches, that isoleucine is in fact a very poor substrate for LeuRS, which gets discriminated early in the reaction cycle and is not incorporated (substantially) to tRNA (Cvetesic etਊl, 2014). This clearly obviates a need for isoleucine editing (Fig ​ (Fig1 1 ).

Contrary to previous belief isoleucine is very effectively discriminated by the synthetic active site of LeuRS, and is not activated nor transferred to tRNALeu. Norvaline is easily charged to tRNA, and requires a posterior docking into the editing domain of the enzyme to prevent its incorporation into proteins.

Norvaline, a non proteogenic amino acid that in microaerobic conditions accumulates in the cytosol of E.਌oli (Soini etਊl, 2008) is, on the other hand, an excellent analog of leucine and is readily mischarged to tRNA Leu by LeuRS. However, accumulation of norvaline-tRNA Leu is prevented by the editing domain of LeuRS (Cvetesic etਊl, 2012).

A beautiful physiologic explanation to this biochemistry is offered in the paper when the authors show that E.਌oli grown under aerobic conditions do not require editing by LeuRS, whereas this activity becomes essential when intracellular concentrations of norvaline increase as a result of growth in microaerobic conditions.

Norvaline thus joins the ranks of non-proteogenic amino acids that can be misincorporated into proteins and cause toxicity. Indeed, recent reports have established links between several types of human neurodegeneration and the ingestion of non-proteogenic amino acids. For example, beta-methylamino-L-alanine is an amino acid analog taken up in the diet, and mischarged by seryl-tRNA synthetases respectively due to its similarity to serine (Dunlop etਊl, 2013). It is still unclear why the nervous system is more affected by this insult than other tissues.

Opposite to the previous examples, a body of literature is also starting to accumulate that reports on cellular strategies that utilize mistranslation to improve biologic fitness. For example, the adaptive nature of random variations in the proteome caused by amino acid misincorporation has been demonstrated in Candida albicans. The proteome of this pathogenic fungus undergoes generalized serine to leucine substitutions as an adaptive strategy that increases the virulence of this species (Moura etਊl, 2010).

The existence of an adaptive mistranslation has been confirmed in bacteria and human cells, and we now know that the mis-methiolation of proteins is a strategy used across the phylogenetic tree to minimize the damage caused by oxidative stress (Pan, 2013). Thus, amino acid misincorporation needs not be a deleterious mistake, but can sometimes be seen as a beneficial relaxation in translation fidelity that increases the fitness of the organism.


The return of pretransfer editing in protein synthesis

The accuracy with which the genetic information contained in protein-coding genes is faithfully translated into the corresponding sequence of amino acids has long fascinated biologists. Before the mechanisms of transcription and protein synthesis had been uncovered in the exquisite molecular detail we know today, some of the inherent problems of faithful gene expression were obvious. Crick's seminal adaptor hypothesis (1) predicted the existence of many then-unknown components of translation, including the aminoacyl-tRNA synthetases. The aminoacyl-tRNA synthetases in effect define the genetic code by catalyzing a 2-step reaction that pairs amino acids with their cognate tRNAs to provide substrates for ribosomal protein synthesis. In the first step, an amino acid is condensed with ATP to form an aminoacyl-adenylate. In the second reaction, the aminoacyl group is transferred to the 3′ end of the tRNA. The aminoacyl-tRNA synthetases also provide a critical safeguard to maintain fidelity during translation of the genetic code by discriminating against and, when necessary, editing noncognate amino acids. Crick was quick to point out that specificity would be of paramount importance to the synthetases, because their function in protein synthesis would require them to precisely distinguish similar amino acids such as isoleucine and valine. Linus Pauling (2), who reasoned that small differences in binding energy between aliphatic amino acids would not provide the level of discrimination necessary for faithful protein synthesis, had also noted this particular problem in molecular recognition. This discrepancy, between the specificity achievable during recognition and the accuracy required for translation, was resolved with the discovery of editing.

It was observed that although isoleucyl-tRNA synthetase did indeed recognize and activate valine, the intermediate valyl-adenylate was subsequently hydrolyzed in a tRNA-dependent reaction (3). The net result is that although isoleucyl-tRNA synthetase can use both isoleucine and valine, only the cognate product isoleucyl-tRNA Ile accumulates. Numerous studies of isoleucyl-tRNA and other synthetases have provided a general picture of the structure and mechanism of editing (Fig. 1). It had long been known that editing could occur either before (pretransfer) or after (posttransfer) amino acids are covalently attached to tRNA, an essential component of the protein synthesis machinery. In both cases the net result is the same, synthesis and release of noncognate aminoacyl-tRNA is prevented and translational accuracy is maintained. In recent years the biological relevance of the pretransfer route had come under question, and the prevailing dogma was that, with a few notable exceptions, editing was essentially a posttransfer process. In this issue of PNAS, Martinis and coworkers (4) now show that posttransfer editing can mask pretransfer editing activity, a finding with far-reaching implications for both quality control and the evolution of protein synthesis.

Posttransfer editing can mask pretransfer editing activity.

Pretransfer and posttransfer editing of noncognate amino acids by aminoacyl-tRNA synthetases. The amino acid (AA) is activated in an ATP-dependent reaction at the active site (AS) to form an enzyme-bound aminoacyl-adenylate (AA-AMP). In pretransfer editing, AA-AMP is hydrolyzed directly, thereby preventing the synthesis of a noncognate aminoacyl-tRNA. In posttransfer editing, AA-AMP is a substrate for esterification of the 3′ end of the tRNA, which then translocates to the editing site (ES) where the AA is removed. PPi, pyrophosphate.

The posttransfer reaction either occurs in cis, before the noncognate aminoacyl-tRNA is released, or in trans catalyzed by synthetases or specific hydrolases (5). A large number of biochemical and structural studies have clearly shown that this reaction occurs in a second active site that is distinct from the ancient catalytic core. Less frequently, the noncognate aminoacyl-adenylate will be hydrolyzed before tRNA esterifcation in a so-called pretransfer reaction (6). However, the relationship between these 2 pretransfer and posttransfer editing pathways has remained unclear in most systems. In particular, the mechanism and physiological relevance of pretransfer editing has continued to be contentious. Many of the points of dispute arise from the inherent instability of aminoacyl-adenylates, which makes their study difficult in vitro and almost impossible in vivo. In addition, much of the controversy surrounding the pretransfer pathway had come from difficulties in envisioning the molecular mechanism by which an unstable aminoacyl-adenylate could translocate some 30 Å from the active site before hydrolysis. Indeed, several examples of pretransfer editing by synthetases that lack a separate editing domain have been reported (7 ⇓ –9).

Leucyl-tRNA synthetase (LeuRS) presents an ideal model system for studying the relationship between, and relative significance of, pretransfer and posttransfer editing. In addition to the well-documented modularity of the conserved CP1 editing domain (10 ⇓ –12), different LeuRSs show widely divergent editing mechanisms. Byvoorbeeld, Escherichia coli LeuRS is known to use a posttransfer editing mechanism to deacylate tRNAs charged with a variety of noncognate amino acids, including isoleucine, valine, norvaline, and methionine. Most notably for the present study, although it has been proposed that the yeast enzyme predominantly relies on pretransfer editing, the E coli enzyme shows no such activity and solely uses posttransfer editing (13). In their study, Martinis and coworkers (4) set out to exploit the known properties of the E coli enzyme to probe the relationship between pretransfer and posttransfer editing in more detail.

Previous studies had suggested that particular residues are needed for the effective transfer of editing substrates from the synthetic active site to the hydrolytic editing site in the CP1 module of LeuRS (14). To study this postulated translocation pathway Martinis and coworkers (4) used a combination of LeuRS mutants defective in posttransfer editing and CP1 deletion mutants of both E coli (ecΔCP1) and yeast mitochondrial (ymΔCP1) LeuRSs. The ΔCP1 deletion mutants were found to retain significant cognate aminoacylation activity (Leu-tRNA Leu synthesis), although they did show some loss compared with the wild type. This modest loss in leucylation by the ΔCP1 mutants was not unexpected and was consistent with suggestions that the CP1 domain stabilizes enzyme–tRNA interactions. When tested for hydrolysis in trans of noncognate Ile-tRNA Leu , both of the ΔCP1 deletion mutants display negligible levels of deacylation, confirming that, as expected, these mutants retain little or no posttransfer editing activity. Martinis and coworkers next performed a final control and tested the ability of the mutants to synthesize mischarged tRNAs, a typical property of enzymes in which posttransfer editing has been disrupted. Whereas the editing-site mutant T252Y showed robust Ile-tRNA Ile synthesis as predicted, the ecΔCP1 and ymΔCP1 mutants were incapable of mischarging tRNA Leu with Ile. Pyrophosphate exchange assays performed to test the misactivation of Ile and other noncognate substrates showed that the ecΔCP1 mutant misactivated Ile to a level comparable with that of wild type. The ability of ecΔCP1 to misactivate Ile, together with its inability to form Ile-tRNA, suggested the existence of a dormant pretransfer editing activity, which is only apparent when the posttransfer editing CP1 domain is removed. Finally, by using inactive tRNAs modified to lack the site for amino acid attachment, pretransfer editing activity was shown to be strongly stimulated by tRNA despite the absence of the CP1 domain.

In unveiling pretransfer editing activity in E coli LeuRS, an enzyme believed to solely depend on posttransfer editing for quality control, Martinis and coworkers (4) raise a number of questions about the origin and function of editing. The most immediate result of their study is to force a reassessment of tRNA-dependent pretransfer editing. Their work clearly shows that translocation to a distinct editing domain is not a prerequisite for tRNA-dependent pretransfer editing as had previously been proposed, which, in turn, raises the question as to how tRNA facilitates editing without itself being aminoacylated. Presumably this would involve increasing the rate of hydrolysis within the active site itself and/or accelerating adenylate dissociation as a prelude to spontaneous hydrolysis, both of which were recently shown to contribute to tRNA-independent editing by prolyl-tRNA synthetase (6). However this particular pretransfer editing mechanism is eventually resolved, the observation that the extant E coli LeuRS:tRNA Leu pair contains the functional remnants of a rudimentary ribonucleoprotein has some interesting evolutionary implications. The tRNA dependence of this hidden pretransfer editing activity is consistent with the coevolution of tRNAs and quality-control mechanisms, helping to explain how structurally similar amino acids were added to the genetic code without compromising the fidelity of translation.

The biggest remaining mystery of this study is why the E coli LeuRS has retained a “hidden” capacity for pretransfer editing that would appear to be redundant given that it already has robust posttransfer activity. Editing is not ubiquitous to synthetases as illustrated by the fact that some enzymes, including human mitochondrial LeuRS, have lost their proofreading capacity and rely instead on accurate substrate recognition (15 ⇓ –17). In this context, the retention of 2 distinct editing pathways is all the more surprising and would seem to suggest that the pretransfer reaction of E coli LeuRS may actually be required for an activity other than translational quality control. No matter what this activity might turn out to be, to paraphrase Mark Twain, reports of the death of pretransfer editing are greatly exaggerated.


Kyk die video: Formation of Aminoacyl tRNA. Sub. Español (Oktober 2022).