Review Methionyl-tRNA synthetase *

Methionyl-tRNA synthetase (MetRS) belongs to the family of 20 enzymes essential for protein biosynthesis. It links covalently methionine with its cognate tRNA. Crystal structures solved for bacterial MetRSs have given a number of interesting insights into enzyme architecture and methionylation catalysis. A comparison of sequences of MetRSs belonging to all kingdoms of life, as well as numerous biochemical and genetic studies have revealed the presence of various additional domains appended to the catalytic core of synthetase. They are responsible for interactions with tRNA and proteins. Tertiary structure of C-terminal tRNA-binding appendices can be deduced from those determined for their homologues: tRNA binding protein 111 and endothelial monocyte-activating polypeptide II. Contacts between MetRS and other proteins could be mediated not only by noncatalytic peptides but also by structural elements present in the catalytic core, e.g. Arg-Gly-Asp (RGD) motifs. Additional activities involve MetRS in the maintenance of translational fidelity and in coordination of ribosome biogenesis with protein synthesis.

aminoacylation.The lat ter pro cess is cat alyzed by a fam ily of 20 aminoacyl-tRNA synthe tas es (AARSs).They per form a two step reac tion, in which an ac ti vated amino acid is trans ferred to the 3¢-ter mi nal adenosine of the tRNA.All aminoacyl-tRNA syn the tas es have been di vided into two classes, each consist ing of 10 en zymes [1][2][3][4].Their clas si fi cation is based on struc tural and bio chem i cal data: se quence sim i lar ity (al though very limited to short mo tifs), fold of cat a lytic do main, the way of in ter ac tion with and charg ing of tRNA.Each class has been ad di tion ally divided into sub classes the mem bers of which show higher se quence and struc tural homology.Methionyl-tRNA synthetase (MetRS) be longs to sub class Ia, to gether with isoleucyl-, valyl-, leucyl-, cysteinyl-and arginyl-tRNA syn the tas es (IleRS, ValRS, LeuRS, CysRS, and ArgRS, re spec tively).The other mem bers of class I are: glutaminyl-and glutamyl-tRNA syn the tas es (GlnRS, GluRS) be long ing to sub class Ib, and tyrosyl-tRNA synthetase (TyrRS) form ing, with tryptophanyl-tRNA synthetase (TrpRS), class Ic.
Amongst the en zymes of its class, MetRS is es pe cially in ter est ing.It rec og nizes an ini ti ator tRNA as well as the tRNA de liv er ing methionine for elon ga tion of pro tein chain.
More over, un like most of other AARSs, MetRS iso lated from a va ri ety of sources (species) shows struc tural di ver sity, con nected with the abil ity to in ter act with other pro teins to form func tional com plexes.
All class I AARSs, de spite dif fer ences in amino-acid se quence, share a com mon structural mo tif be ing their cat a lytic cen ter (Fig. 1).It is a nu cle o tide bind ing fold (called 338 M.A. Deniziak and J. Barciszewski 2001 Fig ure 1. Struc tural fea tures of MetRSs as ob served in a crys tal of T. thermophilus methionyl-tRNA synthetase (PDB ID 1A8H) [6].
Cat a lytic do main is formed by Rossmann fold and SC fold do mains where "HIGH" and "KMSKS" sig na ture mo tifs, re spec tively, are pres ent.Cor rect struc ture of ac tive site is main tained by zinc fin ger do main pres ent in con nec tive pep tide (CP) in ser tion.Anticodon bind ing do main forms an a-he lix bun dle.
Rossmann fold), con sist ing of five par al lel b strands con nected by a he li ces.Un til re cently it has been as sumed that Rossmann fold comprises both of the so-called class I sig na ture mo tifs: "HIGH" and "KMSKS", lo cated in close prox im ity and form ing struc tural el ements crit i cal for aminoacyl-adenylate for mation.Now crys tal struc tures of MetRSs from Esch e richia coli [5] and Thermus thermophilus [6] pro vided new informations con cern ing struc tural or ga ni za tion of their cat a lytic domains and re gions re spon si ble for sub strates rec og ni tion.

STRUC TURAL FEA TURES OF BAC TE RIAL MetRSs
The Rossmann fold of T. thermophilus MetRS is formed by two polypeptide segments sep a rated by an in ser tion of 117 amino ac ids, called the "con nec tive polypeptide" domain (CP do main) [6].The "HIGH" mo tif (HLGH in the case of T. thermophilus MetRS) is lo cated in a loop be tween b1 and a1 el ements, with the two histidine res i dues stacked upon each other.The "KMSKS" pep tide (KMSKT) -pre vi ously rec og nized to oc cur within the Rossmann fold was fi nally lo cal ized in a sep a rate b-a-a-b-a struc ture, named SC fold (stem con tact fold) [6], be tween the Rossmann fold and C-terminal a-he lix-bundle.The SC fold is re spon si ble for in ter ac tions of MetRS with tRNA Met anticodon (Fig. 1).It is con served very well in other syn the tas es of class Ia and Ib: IleRS, ArgRS, GlnRS and GluRS.Study of the GlnRS-tRNA Gln com plex lead to the con clu sion that it in ter acts with the ac cep tor, dihydrouridine and anticodon stems of tRNA [7].The KMSKS forms a loop, the con for ma tion of which is con served in free form of the men tioned en zymes, but changes dra mat i cally upon sub strate bind ing.This change prob a bly af fects the struc ture of HIGH re gion [6].The flex i bil ity of the KMSKS loop could be a con se quence of aminoacylation com plex ity, re quir ing MetRS sub strates to be bound to the ac tive site at subse quent steps of the re ac tion.The con nec tive polypeptide do main of MetRS from T. thermophilus is built mainly of antiparallelly ar ranged eight b strands (Fig. 1) [6].The core of CP do main is a four-stranded b sheet, con served in all class Ia and Ib AARSs.An other four stranded b sheet is in serted between b strands of the core.This in ser tion coor di nates one Zn 2+ ion.The zinc fin ger structure is cru cial for en zyme ac tiv ity.It plays a key role in methionine ac ti va tion as well as in cor rect po si tion ing of the 3¢ end of tRNA [8].
Re sults of stud ies car ried out on MetRSs from dif fer ent bac te rial sources sug gest that the zinc ion con trib utes to the cor rect ori en ta tion of the en zyme ac tive site.
There is a dif fer ence in to pol ogy of CP domain ob served be tween T. thermophilus and E. coli MetRSs.Namely, the zinc-binding domain of E. coli MetRS seems to be a re sult of du pli ca tion of the struc ture found in T. thermophilus pro tein [5].More over, a compar i son of se quences of known MetRSs enabled to dis tin guish four groups (fam i lies) differ ing in the num ber of knuck les and of zinc fin gers in the CP re gion [5].Con nec tive peptides of Eukaryota, Archaea and Spirochaetales prob a bly bind two zinc ions.Two other fam ilies pos sess ing two knuck les and one zinc ion or one knuckle co or di nat ing zinc are rep resented by E. coli and T. thermophilus MetRSs, re spec tively.The fourth group con tains enzymes struc tur ally sim i lar to T. thermophilus MetRS, al though they do not bind any zinc ion.MetRS from My co bac te rium tu ber cu lo sis be longs to this group [9].
Su per im po si tion of the Rossmann folds of E. coli and T. thermophilus MetRSs has re vealed dis tinct ori en ta tions of the CP do mains with re spect to the cat a lytic cen ter.This can ar gue for the con nec tive pep tide be ing mo bile with re spect to the en zyme ac tive site [5].The tryptophan flu o res cence of E. coli MetRS pointed to changes in the en zyme con for mation ac com pa ny ing methionylation step and MetRS-tRNA Met com plex for ma tion [10][11][12].
The SC fold of T. thermophilus MetRS is followed by an a-he lix rich cy lin dri cal do main, unique for class Ia syn the tas es (Fig. 1).It has been shown that amino-acid res i dues in volved in tRNA Met anticodon rec og ni tion are clustered on one face of this a-he lix bun dle structure.The rec og ni tion sur face of T. thermophilus MetRS con tains in vari ant Asn355, Arg359 and Trp424 [6].Mu ta gen e sis performed for MetRS of E. coli has shown that cor re spond ing Asn391, Arg395 and Trp461 also in ter act with the anticodon [13][14][15][16].The C-terminal he lix of E. coli MetRS folds back towards the KMSKS con tain ing loop, prob a bly play ing a role in pro duc tive bind ing of the tRNA ac cep tor stem in side the KMSKS and CP re gions.Two amino-acid re gions sur round ing res idues 253 and 300 of E. coli MetRS are well con served in all known se quences of its orthologues (Fig. 2).These res i dues are pos sibly in volved in methionine bind ing [17][18][19].This pre dic tion is re in forced by re sults of stud ies on mu ta tions of His301 and Trp305 of E. coli en zyme [17,20].It has been also pointed out that the two re gions men tioned above face one an other form ing a hy dro phobic cleft, sim i lar to that of TyrRS, where L-tyro sine bind ing oc curs [21].Two main do mains pres ent in the ma jor ity of AARSs struc tures -the cat a lytic cen ter and the anticodon bind ing do main -re flect the L-shaped struc ture of the tRNA mol e cule.It has been shown in the case of E. coli MetRS that mu ta tions of par tic u lar res i dues lo cated in the anticodon bind ing do main and in a helix join ing it to the cat a lytic core of the en zyme break the do main-domain func tional com muni ca tion in a synthetase complexed with tRNA Met [22].These res i dues have no con tact with tRNA Met but they con trib ute to the stabil ity of the MetRS-tRNA Met com plex and to the cat a lytic ef fi ciency of aminoacylation.It has been pro posed that they can be im por tant for in duced-fit-directed bind ing of methionine spe cific tRNA [22].

MetRSs STRUC TURAL DI VER SITY
Methionyl-tRNA syn the tas es be long to those of AARSs which show struc tural di ver sity depend ing on the source the en zyme was isolated from.On that base MetRSs of dif fer ent or i gin have been di vided into five struc tural groups (Fig. 3) [23].A min i mal core en zyme is found in or gan isms be long ing to all king doms of life and in eukaryotic organella.A large group of eubacterial and some archaeal MetRSs pos sess ad di tional C-terminal domains.An early study car ried out on E. coli MetRS proved that this do main was dis pensable for en zyme ac tiv ity but was re spon si ble for its dimerization [24,25].In ter est ingly, it is highly ho mol o gous to eubacterial pep tide Trbp 111, found in E. coli and A. aeolicus genomes [26,27].Trbp 111 binds non specifically tRNAs [27].A sim i lar C-terminal domain was iden ti fied in MetRSs from worm C. elegans, plants O. sativa and A. thaliana and some eubacteria ( Borrelia burgdorferi, Treponema pallidum) [23].A func tional study of that do main in monomeric O. sativa MetRS has led to the con clu sion that it pro vides the en zyme with better cat a lytic ef fi ciency in bind ing the amino acid ac cep tor arm of tRNA Met and fa cil i tates its aminoacylation [23].This kind of C-terminal MetRS pep tide is sim i lar to a monomeric cytokine EMAP II (endo the lial monocyte-activating polypeptide II) .EMAP II was orig i nally iden ti fied as a prod uct of murine methylcholanthrene A (Met A)-in duced fibrosarcoma cells [28] but it is ex pressed also in nor mal cells upon apoptosis -a phys i o log i cal pro cess by which multicellular or gan isms get rid of in jured, infected or de vel op men tally un nec es sary cells.EMAP II is gen er ated af ter proteolytic cleavage of p43, one of the com po nents of multisynthetase com plex, where it prob a bly fa cil itates tRNA-substrate bind ing [26].In ter estingly, an EMAP II-like cytokine was also gener ated af ter proteolytic cleav age of hu man TyrRS [32].Its ef fect en com passes in duc tion  Only crit i cal re gions of the ac tive site (sig na ture sequences) and pos si ble methionine bind ing sites are shown.Among the pre sented pri mary struc tures are MetRSs of eukaryotic or i gin (Hsapc -Homo sa pi ens cyto plas mic, Celec -Caenorhabditis elegans cy to plas mic, Scerc -Saccharomyces cerevisiae cy to plas mic, Spomc -Schizosaccharomyces pombe cy to plas mic, Osatc -Oryza sativa cy to plas mic, Athac -Arabidopsis thaliana cytoplas mic), archaebacterial en zymes (Mther -Methano-of mi gra tion of mononuclear phagocytes (MPs) and polymorphonuclear leu ko cytes (PMNs), stim u la tion of tu mor ne cro sis factor-a (TNFa) and tis sue fac tor pro duc tion, as well as ac ti va tion of cell sur face ex pres sion of P-and E-selectins.It is sur pris ing, that this same kind of struc ture ap pended to MetRS or p43 di rects tRNA to the ac tive site of synthetase.
Trbp 111 and EMAP II have highly sim i lar se quences and ter tiary struc ture, called the OB-fold (oligonucleotide-oligo sacchari de-binding fold) [33,34].This kind of b-bar rel has been also found at N-termini of AspRS [35], LysRS [36] and AsnRS [37], where it is respon si ble for spe cific bind ing of tRNA anticodon.The func tion of OB-fold lo cal ized in an other AARS -T.thermophilus PheRSis still un known [38].Two last struc tural vari ants of MetRSs are found only in eukaryotic cells.The en zyme being a com po nent of multisynthetase com plex found in higher eukaryotes cy to plasm, possesses a large N-terminal ex ten sion, me di ating as so ci a tion with other pro teins form ing that par ti cle [39,40], and a shorter C-terminal ap pen dix.Its se quence is re lated to that of linker do main of GluProRS, which forms another kind of RNA-binding mod ule [41].MetRSs iso lated from yeast cy to plasm possess only the N-terminal ex ten sion.This ad ditional do main en ables as so ci a tion with Arc1p pep tide (aminoacyl-tRNA synthetase cofactor 1 (ARC1) gene prod uct), which is also a homologue of EMAP II/Trbp 111 [42,43].MetRS to gether with GluRS and Arc1p form a func tional com plex, where the lat ter pep tide plays an im por tant role in tRNA bind ing and de liv ery to the ac tive sites of the two syn thetas es [43].
The crys tal struc tures of bac te rial MetRSs were de ter mined us ing pro teins bear ing cat alytic and anticodon bind ing do mains, with the C-terminal ex ten sion re moved by ge netic en gineer ing [5,6].Se quence sim i lar ity sug gests that the miss ing MetRS do main is of ter tiary struc ture sim i lar to that re cently de ter mined for hu man EMAP II [34].This set of informations en abled us to pro pose a pos si ble struc ture for both cat a lytic and ad di tional C-terminal do mains of plant MetRS (Fig. 4).Ma trixes for mod el ing the O. sativa en zyme were E. coli MetRS and hu man EMAP II, respec tively.The last a-he lix of the bac te rial en - zyme anticodon-binding do main folds back towards the ac tive site.On the other hand, a study of EMAP II-like do main of plant en zyme showed that it could in ter act with tRNA Met accep tor stem [23].Both facts lead to the con clusion that EMAPII-like C-terminal ex ten sion of plant en zyme is lo cated near its ac tive site, so as to be able to take a part in tRNA de liv ery and cor rect po si tion ing for aminoacylation catal y sis [23].
The OB-fold pres ent in EMAP II and Trbp 111 seems to be char ac ter is tic for other peptides of sim i lar se quence, e.g.C-termini of H. sa pi ens TyrRS and E. coli MetRS.They are rep re sented by model struc tures shown in Fig. 5, all gen er ated us ing EMAP II as a ref erence.Our Trbp 111 model is con sis tent with the pub lished struc ture (not shown) [33].

MetRS AND PRO TEIN-PROTEIN IN TER AC TIONS
As it was men tioned above, in ter ac tions between MetRSs and other pro teins are me diated by ad di tional do mains ap pended to the cat a lytic core of en zyme.They take a part in the as sem bly of multisynthetase com plex [44][45][46] or in ter ac tions with tRNA bind ing pro teins, en hanc ing MetRS cat a lytic ef ficiency [43].Com plexes iso lated from cy toplasm of Metazoa con tain eight syn the tas es -GluProRS, IleRS, LeuRS, MetRS, GlnRS, ArgRS.LysRS, AspRS, and three non enzymatic pep tides of mo lec u lar mass of 43 kDa, 38 kDa and 18 kDa.In ter ac tions be tween all the com po nents of the multienzymatic complex were stud ied ex ten sively us ing ge netic 3D mod els were gen er ated us ing SWISS-MODEL pro gram avail able on EXPASY server (http://www.expasy.ch/swissmod/SWISS-MODEL.html).An E. coli MetRS (PDB ID 1QQT) [5] and hu man EMAP II cytokine (1EUJ) [34] were taken as ma trixes for struc tural align ment.Se quence re gions cor re spond ing to ob tained mod els are marked in gray on the top scheme.[40,47] and chem i cal crosslinking meth ods [48].The ge netic ap proach has re sulted in deter mi na tion of sev eral con tacts oc cur ring between AARSs N-or C-terminal ap pen di ces [40], namely, the N-terminal noncatalytic domain of MetRS could make ho mol o gous (MetRS-MetRS) and heterologous (MetRS-ArgRS, MetRS-GlnRS) in ter ac tions.On the other hand, stud ies con ducted us ing chem i cal crosslinking have shown that some syn the tases are in close prox im ity in the com plex (e.g.MetRS and AspRS) but they do not in ter act through N or C ter mini [48].This ob ser va tion sug gests that ad di tional con tacts in volv ing core do mains of en zymes are also im por tant.Looking for pos si ble el e ments in volved in such in ter ac tions we have an a lyzed MetRSs amino-acid se quences de pos ited in EMBL/ GenBank.This search re sulted in iden ti fi cation of a tri pep tide -Arg-Gly-Asp (RGD)pres ent in 17 of 40 in spected pri mary structures (Ta ble 1).This mo tif is a char ac ter is tic fea ture of the pro teins in volved in cell ad hesion events, e.g.fibronectin [49], vitro nectin [50], and fibrinogen [51].In the struc ture of those pro teins RGD forms a loop which is directly en gaged in in ter ac tions with spe cific integrins [52].We won der whether the presence of RGD pep tide in MetRSs mostly of eukaryotic and archaebacterial or i gin (Fig. 6) can be re lated to pos si ble pro tein-protein inter ac tions, which, for ex am ple, could lo cate the en zyme in a par tic u lar cell com part ment (through bind ing to mem brane pro teins) or link ing it with an other pep tide as sist ing MetRS func tion or be ing a mem ber of some multienzyme com plex.Such ques tions need, of course, fur ther ex per i men tal ex plo ra tions, start ing from look ing for pos si ble MetRSs ligands.

AD DI TIONAL AC TIV I TIES OF MetRSs
Aminoacyl-tRNA syn the tas es are multifunctional pro teins.Apart from tRNAs aminoacylation they play an im por tant role in main tain ing the translational fi del ity, RNA pro cess ing and cel lu lar trans port, apoptosis, syn the sis of sig nal mol e cules and trans - lational or transcriptional reg u la tion [53].MetRS par tic i pates in the main te nance of translational fi del ity through ed it ing of homocysteine (Hcy), a non-protein amino acid which is an oblig a tory pre cur sor of methionine [54,55].Due to its struc tural sim i lar ity to methionine Hcy can en ter the first step of pro tein biosynthesis and be ac ti vated form ing MetRS-bound homocysteinyl-adenylate, which is not trans ferred to tRNA but re jected in an intramolecular con ver sion re ac tion, lead ing to pro duc tion of homocysteine thiolactone.This same Hcy ed it ing mech a nism was also con firmed for IleRS, LeuRS, ValRS and LysRS [56,57].How ever, it has been shown that in cul tured hu man cells Hcy can be in cor po rated into pro teins in two ways: u posttranslationally, through acetylation of side chain amino groups of lysine res i dues by thiolactone, u translationally, through in cor po ra tion of S-nitroso-Hcy into pro tein [58].
Both sit u a tions can oc cur in cells from en dothe lial cell cul tures main tained on Hcy in fo -Vol.48 Methionyl-tRNA synthetase 345 late-limited me dia.Translational in cor po ration of Hcy into pro teins abol ishes their bi olog i cal ac tiv ity and has sig nif i cant med i cal con se quences, con trib ut ing to in duc tion of pathogenesis in atherosclerosis [54,58].Multienzyme com plexes sim i lar to those local ized in the cy to plasm of higher eukaryotes have been re cently found also in the nu cleus [59].More over, an in ves ti ga tion of cel lu lar dis tri bu tion of dif fer ent hu man AARSs has led to the dis cov ery, that MetRS was uniquely lo cal ized in nu cleolus of proliferative cells [60].The MetRS was translocated to nu cle olus upon a mitogenic sig nal in duced by dif ferent growth fac tors (in su lin, platelet-derived growth fac tor, epi der mal growth factor).The nu cle o lus is that part of nu cleus where rRNA biogenesis takes place.It has been ob served that the pres ence of MetRS in nu cle o lus was de pend ent on the in teg rity of rRNA and the ac tiv ity of RNA poly mer ase I. Im mu no log i cal tests have con firmed that MetRS is re quired for nu cle o lar syn the sis of rRNA [60].This enzyme can be a part of the cel lu lar sys tem co ordi nat ing biogenesis of ri bo some and pro tein syn the sis.

CON CLU SIONS
From the sys tem at i cally grow ing body of data con cern ing methionyl-tRNA synthetase an in ter est ing pic ture emerges, show ing that the en zyme might be of di verse struc ture, related not only to its func tion but also to cel lular or ga ni za tion.This lat ter point still needs fur ther ex plo ra tion, es pe cially in the case of plant en zymes.Genomics and proteomics, devel op ing dy nam i cally dur ing the last few years, try to an swer gen eral ques tions concern ing the or ga ni za tion of cel lu lar ap pa ra tus as a whole.Taking into ac count the in volvement of many AARSs in dif fer ent met a bolic path ways, go ing be yond pro tein biosynthesis it self, one can ex pect new find ings show ing a com pli cated net work of re la tions of syn the tases to other com po nents of the cell.

Fig ure 3 .
Fig ure 3. Struc tural di ver sity of MetRSs.A min i mal core en zyme has been found in or gan isms be long ing to all king doms as well as in organella.Most of Eubacteria pos sess MetRSs bear ing an ad di tional C-terminal do main re spon si ble for dimerization, homol o gous to A. aeolicus Trbp 111 pep tide, which is also a dimer and is able to bind tRNAs.A sim i lar C-terminal do main, more re lated to a cytokine EMAP II, is adjoined to eukaryotic and some bac te rial MetRSs (plant MetRS be long ing to this group is a mono mer).EMAP II and Trbp 111 are close se quence and struc ture homologues.Two last struc tural vari ants of en zyme have been found only in eukaryotic cells.MetRS pos sess ing an N-terminal ex ten sion and short C-terminal ap pen dix form ing an other kind of tRNA-binding do main is a mem ber of multienzymatic com plex found in higher eukaryotes (N-terminal do main me di ates as so ci a tion in the com plex).En zymes pos sess ing only an N-terminal ex ten sion ex ist in yeast cy to plasm, where they form a com plex with GluRS and Arc1p pro tein, which is an EMAP II homologue, re spon si ble for tRNA rec og ni tion and de liv ery.

Fig ure 5 .
Fig ure 5. Ter tiary struc ture of hu man EMAP II [34] and mod els made on its base: C-terminal do main of hu man TyrRS, Trbp 111 from A. aeolicus and C ter mi nus of E. coli MetRS.Modelling was done by SWISS-MODEL.

Ta ble 1. The pres ence of RGD se quence in known MetRSs.
En zymes con tain ing RGD tri pep tide are marked in red.The tree was ob tained using NEIGH BOR and CONSENSE programs from PHYLIP pack age.