-
Unique protein architecture of alanyl-tRNA synthetasefor
aminoacylation, editing, and dimerizationMasahiro Naganumaa,
Shun-ichi Sekinea,b, Ryuya Fukunagaa,1, and Shigeyuki
Yokoyamaa,b,2
aDepartment of Biophysics and Biochemistry, Graduate School of
Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo
113-0033, Japan; and bRIKENSystems and Structural Biology Center,
Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama
230-0045, Japan
Edited by Paul R. Schimmel, The Scripps Research Institute, La
Jolla, CA, and approved April 6, 2009 (received for review February
17, 2009)
Alanyl-tRNA synthetase (AlaRS) specifically recognizes the
majoridentity determinant, the G3:U70 base pair, in the acceptor
stem oftRNAAla by both the tRNA-recognition and editing domains. In
thisstudy, we solved the crystal structures of 2 halves of
Archaeoglo-bus fulgidus AlaRS: AlaRS-�C, comprising the
aminoacylation,tRNA-recognition, and editing domains, and AlaRS-C,
comprisingthe dimerization domain. The
aminoacylation/tRNA-recognitiondomains contain an insertion
incompatible with the class-specifictRNA-binding mode. The editing
domain is fixed tightly via hydro-phobic interactions to the
aminoacylation/tRNA-recognition do-mains, on the side opposite from
that in threonyl-tRNA synthetase.A groove formed between the
aminoacylation/tRNA-recognitiondomains and the editing domain
appears to be an alternativetRNA-binding site, which might be used
for the aminoacylationand/or editing reactions. Actually, the amino
acid residues requiredfor the G3:U70 recognition are mapped in this
groove. The dimer-ization domain consists of helical and globular
subdomains. Thehelical subdomain mediates dimerization by forming a
helix–loop–helix zipper. The globular subdomain, which is important
forthe aminoacylation and editing activities, has a
positively-chargedface suitable for tRNA binding.
crystal structure � dimerization domain � aminoacyl-tRNA
synthetase �proofreading � wobble base pair
Aminoacyl-tRNA synthetases (aaRSs) catalyze the ligation
ofcognate amino acids and tRNAs, and thus establish thegenetic code
in protein biosynthesis. They are modular proteinscomposed of an
aminoacylation domain and a few additionaldomains for discrete
functions, such as tRNA binding, oligomer-ization, and amino acid
proofreading (1, 2). The 20 aaRSs aredivided into 2 classes, I and
II, based on the 2 unrelated types ofaminoacylation domains (3, 4).
The aminoacylation reactionoccurs at the catalytic site on the
aminoacylation domain, andthe reaction generally consists of 2
steps: the initial activation ofthe amino acid with ATP to generate
the aminoacyl-adenylate,followed by the transfer of the aminoacyl
moiety to the 3� endof the tRNA. Although the aminoacylation is
generally accurate,several aaRSs cannot completely avoid the
misactivation of anoncognate amino acid, when it is similar to the
cognate one. Tosolve this problem, these aaRSs use a proofreading
mechanism,in which the incorrect products are hydrolyzed at the
active sitein the editing domain.
Alanyl-tRNA synthetase (AlaRS) is one of the class II aaRSsand
consists of 4 domains: the N-terminal class II
aminoacylationdomain, the tRNA-recognition domain, the editing
domain, andthe C-terminal oligomerization (dimerization or
tetrameriza-tion) domain (Fig. 1A) (1, 2). AlaRS occupies a special
positionin the history of aaRS research. Escherichia coli AlaRS
wasamong the first aaRSs that were cloned, sequenced, and
char-acterized genetically and biochemically (1, 5, 6). tRNAAla
con-serves a unique G3:U70 wobble base pair in the acceptor
stem,and this base pair dictates the tRNA identity toward AlaRS(7,
8). This remarkable finding, that a small number of nucleo-tide
residues serve as the predominant determinant for thetRNA identity,
accelerated the search for the identity determi-
nants of other aaRS–tRNA pairs. It was also striking that
thepredominant identity determinant of a tRNA exists in
theacceptor–stem duplex, rather than the anticodon and the
dis-criminator base (9, 10). Actually, AlaRS can aminoacylate
small,isolated portions of tRNA, such as a ‘‘minihelix’’ and a
‘‘micro-helix,’’ as long as they have the G3:U70 base pair (11).
TheG3:U70 base pair is considered to be recognized from the
minorgroove side (12, 13).
An E. coli AlaRS fragment comprising the aminoacylation
andtRNA-recognition domains (the N-terminal 461 residues)
canspecifically aminoacylate tRNAAla (1). The crystal structure
ofthe corresponding fragment (AlaRS-N) from the bacteriumAquifex
aeolicus was reported (14, 15). It revealed that AlaRSdoes not
dimerize through the aminoacylation domain, in con-trast to the
other class II aaRSs. The structures of amino acid-and ATP-bound
AlaRS-N revealed how the cognate alanine andthe noncognate glycine
and serine interact with the aminoacy-lation site. AlaRS is one of
the aaRSs that use the proofreadingmechanism, in that mischarged
products, such as Gly-tRNAAlaand Ser-tRNAAla, are transferred to
the editing domain, wherethe ester bond is hydrolyzed (2). A defect
in the AlaRS editingactivity causes cell death in the mouse nervous
system (16). It wasrecently reported that the E. coli AlaRS editing
domain pos-sesses a region, distinct from the N-terminal domains,
thatrecognizes the G3:U70 base pair (17). Therefore, AlaRS
maytransfer the acceptor stem of tRNAAla from the first binding
sitein the aminoacylation domain to the second site in the
editingdomain, in contrast to the other editing aaRSs (classes I
and II),which have been proposed to shuttle the flexible
single-strandedCCA terminus of the tRNA between the aminoacylation
andediting catalytic sites (18–22). The C-terminal domain of
AlaRSis not only essential for the oligomerization, but also
importantfor the aminoacylation and editing reactions (17, 23).
Smallproteins homologous to the AlaRS editing domain,
designatedAlaX, are found in many organisms (24, 25). They are
active inthe trans hydrolysis of misacylated tRNAAla in vitro (24).
Thecrystal structures of AlaX-S (specific to Ser-tRNAAla) andAlaX-M
(specific to Ser-tRNAAla and Gly-tRNAAla) from thearchaeon
Pyrococcus horikoshii have been reported (26, 27).
The structures of the editing and oligomerization domains,
thebasis of oligomerization, and the domain arrangement in
thefull-length AlaRS have remained elusive. We previously
suc-ceeded in the crystallization of 2 fragments of AlaRS from
the
Author contributions: S.Y. designed research; M.N., S.S., and
R.F. performed research; M.N.,S.S., and S.Y. analyzed data; and
M.N., S.S., and S.Y. wrote the paper;.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The coordinates and structure factors have been
deposited in the ProteinData Bank, www.pdb.org (PDB ID codes 2ZTG
and 2ZVF).
1Present address: Department of Biochemistry and Molecular
Pharmacology, University ofMassachusetts Medical School, Worcester,
MA 01605.
2To whom correspondence should be addressed. E-mail:
[email protected].
This article contains supporting information online at
www.pnas.org/cgi/content/full/0901572106/DCSupplemental.
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archaeon Archaeoglobus fulgidus, AlaRS-�C, comprising
theaminoacylation, tRNA-recognition, and editing domains,
andAlaRS-C, comprising the dimerization domain (23). In thepresent
study, we determined their crystal structures at 2.2- and3.2-Å
resolutions, respectively. The AlaRS-�C structure re-vealed a
unique arrangement of the editing domain, relative tothe
aminoacylation/tRNA-recognition domains, and the ar-chaea-specific
insertions/deletions. The AlaRS-C structure pro-vided the basis of
dimerization, via the formation of a helix–loop–helix zipper
(HLHZ). The structures suggested the domainorganization in the
full-length AlaRS dimer, and thus couldserve as a platform for
future analyses of how the aminoacyla-tion/tRNA-recognition domains
and the editing domain of
AlaRS independently recognize the G3:U70 base pair
oftRNAAla.
ResultsStructure Determination. A. fulgidus AlaRS is a homodimer
of 906amino acid residue polypeptides (23). It was genetically
dividedinto 2 parts, AlaRS-�C (residues 1–739) and AlaRS-C
(residues736–906) (23), and both structures were solved (Table
S1).AlaRS-�C is composed of the class-II specific
aminoacylationdomain, the tRNA-recognition domain, and the editing
domain.The structure of AlaRS-�C complexed with an
alanyl-adenylateanalog, 5�-O-[N-(L-alanyl)sulfamoyl] adenosine
(Ala-SA), wasdetermined at 2.2 Å (Fig. 1B). The crystallographic
asymmetricunit contained 1 AlaRS-�C molecule. The refined model has
Rand Rfree factors of 21.5% and 26.4%, respectively.
AlaRS-Ccomprises the dimerization domain, and its structure was
deter-mined at 3.2 Å (Fig. 1C). Two AlaRS-C molecules form
ahomodimer, and there are 4 dimers in the asymmetric unit.
Therefinement converged to R and Rfree factors of 20.5% and
27.6%,respectively (Table S1).
The Aminoacylation Domain. The A. fulgidus AlaRS-�C
structurerevealed the aminoacylation domain (residues 1–257),
composedof a central antiparallel �-sheet (�1–�8 and �10) and 5
�-helices(�1–�5), which is typical of the class-II aaRSs. It is
superposableon that of A. aeolicus AlaRS-N, with an rmsd of 1.9 Å
for 179C� atoms. A. fulgidus AlaRS possesses an
archaea-specificN-terminal extension (AddA1, residues 1–58),
including �1, �2,�1, and �2 (Fig. 2 and Fig. S1 A). �1 and �2 are
integrated in thecentral �-sheet, and thus AddA1 is part of the
aminoacylationdomain. Although the bacterial and eukaryal AlaRSs
lackAddA1, they instead possess 2 insertions (depicted as
InsB/E1and InsB/E2 in A. aeolicus AlaRS), which occupy the
corre-sponding space. A. fulgidus AlaRS also contains an
insertion(InsA1, residues 226–232) including �9 (Fig. 2 and Fig. S1
A).Lys-229, at the tip of InsA1, seems to occupy the position
ofLys-73 in the E. coli enzyme, which cross-links to tRNAAla
(28).
A clear electron density corresponding to Ala-SA is visible
inthe active-site cleft (Fig. 1B and Fig. S2). The manner
ofinteraction with Ala-SA in the aminoacylation active site
isdescribed in SI Text.
The tRNA-Recognition Domain. The �-helix-rich middle domain
ofAlaRS-�C (residues 258–484), which is supposed to interactwith
the tRNA acceptor arm, is composed of 11 �-helices and a2-stranded
short parallel � sheet (Fig. 1B and Fig. S1 A). Thisdomain can be
divided into 2 subdomains, designated here as
A
B Aminoacylation
tRNA recognitionMid2
Mid1
N
C
C
N
Editing
Linker
Globular subdomains
Helical subdomains
N
C
Editing core
β barrel
InsA2
AddA1
Helix-loop
InsA1
motif1
C
Aminoacylation tRNA recognition Editing Oligomerization
AlaRS
AlaRS-ΔC
AlaRS-C
1 906
1 739
736 906
Fig. 1. The structures of AlaRS-�C and AlaRS-C. (A) Domain
organizations ofA. fulgidus AlaRS, AlaRS-�C, and AlaRS-C. Shown are
the aminoacylationdomain (green), Mid1 (blue) and Mid2 (cyan) in
the tRNA-recognition domain,the �-barrel (yellow) and editing-core
(orange) subdomains in the editingdomain, and the helical (midnight
blue) and globular (light blue) subdomainsin the dimerization
domain. (B) A ribbon representation of AlaRS-�C. Themodel is
colored as in A, and the N-terminal addition (AddA1) and
insertions(InsA1 and InsA2) are highlighted in purple and brown,
respectively. Motif 1in the aminoacylation domain (violet), the
helix–loop in the editing domain(salmon), and the linker connecting
the tRNA-recognition and editing do-mains (red) are shown. Ala-SA
in the aminoacylation site and the editing-sitezinc ion are
depicted as cpk models. (C) The AlaRS-C dimer is shown as a
ribbonmodel. One molecule of the dimer is colored as in A, and the
other moleculeis colored gray.
InsA1
InsB/E1
InsB/E2
InsA2
AddA1
A.fulgidus A.aeolicusMid2
Mid2
A B
Fig. 2. The aminoacylation and tRNA-recognition domains. (A) The
amino-acylation and tRNA-recognition domains of A. fulgidus AlaRS,
colored as inFig. 1B, are shown. (B) The A. aeolicus AlaRS-N
structure, shown in the sameorientation. The 2 regions missing in
A. fulgidus (InsB/E1 and InsB/E2) arecolored brown, and Mid2 is
shown in gold.
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Mid1 (residues 258–419) and Mid2 (residues 420–484).
Thesesubdomain structures in A. fulgidus AlaRS are similar to
theircounterparts in A. aeolicus AlaRS (14), as revealed by the
rmsdsof 2.4 and 2.3 Å, respectively. Mid1 tightly contacts the
amino-acylation domain by hydrophobic interactions, whereas
Mid2protrudes from the rest of the protein body. The �-helix
(�13)connecting Mid1 and Mid2 is continuous, whereas the
corre-sponding helix is distorted in the middle in A. aeolicus
AlaRS-N.Thus, Mid2 in A. fulgidus AlaRS-�C is oriented outward
by�20°, compared with that of A. aeolicus AlaRS-N (Fig. S3). It
isfurther tilted by �50°, because the last �-helix of Mid2
isconnected to the editing domain by the linker. In the beginningof
the subdomain, the Mid1, archaeal AlaRSs possess an inser-tion of
�50 amino acid residues, which is missing in the bacterialAlaRSs.
In the A. fulgidus AlaRS-�C structure, this insertion(InsA2,
residues 277–330) assumes a helix–loop–helix structure(�8–�9) and
is bent back to form part of the active-site cleft. Thepresence of
this insertion seems to be incompatible with thetRNA interaction
manner proposed previously for the bacterialAlaRS (14), as
discussed below.
The Editing Domain. The editing domain of A. fulgidus
AlaRS(residues 501–737) consists of 2 subdomains, the
N-terminal�-barrel subdomain (residues 501–588) and the C-terminal
�/�subdomain (the editing core, residues 589–737), composed of
2central �-helices (�17 and �18, residues 590–614 and
642–657,respectively) sandwiched by 3- and 4-stranded
antiparallel�-sheets (Fig. 1B and Fig. S1B). The editing domain is
super-posable on P. horikoshii AlaX-M [Protein Data Bank (PDB)
IDcode 2E1B], with an rmsd of 1.5 Å for 202 C� atoms. The
�/�subdomain also superposes well on P. horikoshii AlaX-S (PDBID
code 1WXO) lacking the �-barrel subdomain, with an rmsdof 1.4 Å for
134 C� atoms.
A cavity is formed at the subdomain interface. At thebottom,
His-600, His-604, His-707, and Cys-703 coordinate ametal ion, which
is supposed to be the editing active center(Fig. S4A). The
tetrahedral coordination and the intenseanomalous peak observed at
the metal site suggested that themetal is a zinc, as in the case of
the AlaX proteins. Thr-603,Gln-620, Gln-682, and Gln-701 constitute
the cavity wall.His-600, Thr-603, His-604, Gln-620, His-707, and
Cys-703 areconserved among the AlaRSs. Glu/Gln occupies the
positioncorresponding to Gln-701. Gln-620, Gln-682, and
Gln-701correspond to Thr-30, Asp-92, and Asp-114, respectively, in
P.horikoshii AlaX-S, which are involved in interactions withserine
(Fig. S4B) (27). In AlaX-S, Thr-33 is also involved in theserine
interaction, but AlaRS lacks the corresponding residue.The
conserved Thr-603 in AlaRS could structurally compen-sate for the
absence of this residue. The glycine-rich loop of the�-barrel
subdomain resides at the entrance of the cavity andmight interact
with 3� end of the tRNA.
Position of the Editing Domain. The editing domain of A.
fulgidusAlaRS is connected to the last �-helix of the
tRNA-recognitiondomain by a 38-Å-long loop, consisting of 16 amino
acidresidues (residues 485–500). The editing domain contacts
theaminoacylation domain to form a hydrophobic core (Fig.
3B).Ile-670, Tyr-681, Phe-678, and Val-685, from a
helix–loopstructure (residues 669–689) in the editing domain,
interactwith Tyr-84 in motif 1 (residues 61–88), Trp-90, Phe-107,
andVal-112 of the aminoacylation domain. Arg-89, Trp-619,
andArg-679 are stacked. The editing domain also contacts Mid1of the
tRNA-recognition domain (Fig. 3C). His-617, Thr-635,and Phe-637 in
the editing domain and Val-356, Val-403,Thr-407, Ile-411, and
Leu-414 in Mid1 form a hydrophobiccore. Asn-616, Arg-638, and
Asp-727 in the editing domaininteract with Asp-402, Glu-410, and
Arg-367, respectively, inMid1.
It is remarkable that the position of the editing domainrelative
to the aminoacylation domain differs from those inother class II
aaRSs with reported structures. For example,compared with E. coli
threonyl-tRNA synthetase (ThrRS)(20), the editing domain resides on
the opposite side of theaminoacylation domain in AlaRS-�C (Fig.
3A). The amino-acylation active site is �37 Å away from the editing
active sitein A. fulgidus AlaRS, which is comparable with the �39
Ådistance in ThrRS (20, 29). We previously obtained a 3.7-Ådataset
from an AlaRS-�C crystal belonging to a differentspace group (23).
The structure was solved by molecularreplacement, using the present
AlaRS-�C structure. Theposition of the editing domain relative to
the aminoacylationdomain and their interface are the same.
In most cases, the dimerization of class II aaRSs is
mediatedthrough motif 1 (30, 31). Nevertheless, A. aeolicus
AlaRS-Nreportedly does not form a dimer, because the
tRNA-recognition domain hinders dimerization through motif 1
(14).Consistent with this finding, A. fulgidus AlaRS-�C motif 1
doesnot mediate dimerization, but interacts with the
helix–loopstructure (residues 668–688) in the editing domain to
form an
ThrRS editing domain AlaRS-∆C editing domain
A
Ile670
Val112
Tyr84
Trp90
Phe678Val685
Tyr681
Arg89
Arg679
Phe107
Trp619
B
Arg367
Asp727
Glu410
Arg638
Phe637
Ile411Leu414
Val356
Thr635
His617Asn616
Asp402
Val403
Thr407
C
Fig. 3. The editing domain. (A) The position of the AlaRS
editing domain.The structure of AlaRS-�C, depicted as a tube model,
is superposed on that ofThrRS by the aminoacylation domain. The
AlaRS-�C model is colored as in Fig.1B, and ThrRS is colored gray.
(B) The interface of the editing and aminoacy-lation domains. The
helix–loop in the editing domain and motif 1 in theaminoacylation
domain are colored salmon and brown, respectively. Theresidues
involved in the interactions are shown as white stick models.
(C)The interface of the editing and tRNA-recognition domains. The
editing coresubdomain and Mid1 are colored orange and blue,
respectively.
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interdomain interface. It is interesting to note that
AlaX-M,which lacks the helix–loop structure, exists as a monomer
insolution (26). In contrast, the helix–loop mediates the
ho-modimerization of AlaX-S (27).
The C-Terminal Dimerization Domain. A. fulgidus AlaRS forms
adimer through an interaction between the C-terminal dimeriza-tion
domains of the 2 molecules (23). AlaRS-�C, lacking thedimerization
domain, exists as a monomer in solution (23). Wefirst determined
the structure of the isolated dimerizationdomain of A. fulgidus
AlaRS, AlaRS-C (Fig. 1C). The structurerevealed that the
dimerization domain consists of a long helicalsubdomain and a
globular subdomain. The helical subdomaincontains 2 �-helices of 32
and 53 Å and a linker in between. Thissubdomain exclusively
mediates the dimer interaction to form acharacteristic HLHZ (Fig.
4A and Fig. S5). Val-744, Met-747,Leu-750, and Leu-751 in �20, and
Leu-765, Val-769, Phe-772,Phe-773, Trp-776, Gln-779, Ile-783,
Leu-786, Val-789, Ile-790,Leu-793, and Ile-797 in �21, in 1
protomer of the dimer,respectively, form leucine-zipper-like
interactions with theircounterparts in the other monomer. The
N-terminal portion of
�21 (Pro-762, Leu-765, Pro-766, and Val-769) in 1 protomer
alsointeracts with the C-terminal portion of �20 (Ala-754,
Ile-757,and Leu-758). The linker mediates the formation of the
hydro-phobic core at the �20–�21 junction, where the coiled-coil
istwisted (Fig. 4A). Similar HLHZ structures are also present
inseveral transcriptional regulators, such as the Myc
protoonco-gene product and its relatives (32).
The C-terminal globular subdomain is composed of a6-stranded
antiparallel �-sheet and 3 �-helices (Fig. 4B and Fig.S1B). This
subdomain tightly packs against �21 of the HLHZ toform a
hydrophobic core. Trp-794, Leu-798, and Met-799, in�21, and
Val-811, Val-815, Leu-829, Leu-838, and Phe-851, inthe globular
subdomain, are involved in the hydrophobic inter-actions. One
surface of the globular subdomain is positivelycharged, by the
contributions of Lys-855, Arg-859, Arg-863,Arg-867, Lys-870,
Arg-876, and Lys-877. It is interesting that theconserved
glycine-rich segment (870KGSGGGR876) forms a�-strand that is part
of the �-sheet (Fig. 4B). A structuralsimilarity search using the
DALI server (33) revealed that theglobular subdomain is similar to
that of the ssDNA 5�-3�exonuclease RecJ (34) and exopolyphosphatase
(35), with highZ scores of 11.5 and 9.1, respectively.
DiscussiontRNA Interactions. In the crystal structures of
tRNA-boundclass-II aaRSs, including ThrRS, seryl-tRNA synthetase,
andaspartyl-tRNA synthetase, the amino acid acceptor arm of thetRNA
binds to a common site on the class II aminoacylationdomain (20,
36, 37). The binding site corresponds to the grooveformed between
Mid1 and Mid2 in the tRNA-recognitiondomain of A. fulgidus AlaRS.
However, if the tRNA acceptorarm binds to the groove of A. fulgidus
AlaRS via the commonmode (mode 1), then the nucleotides at
positions 1–5 and 68–73have a serious steric clash with the
archaea-specific insertion ofa helix–loop–helix (InsA2) within the
groove (Fig. 5). To avoidthe putative clash, a drastic
conformational change should occur.Because InsA2 interacts with �7
and �10 to form a hydrophobiccore, the reorientation of InsA2 seems
to be unlikely. When thetRNA relocates to the other tRNA-binding
site for editing, it
N N
C C
A
α
α α
B
α
Fig. 4. The C-terminal dimerization domain. (A) The dimer
interactions viathe helical subdomains. An HLHZ formed in the dimer
is shown in a stereoview.One molecule is colored midnight blue, and
the side chains are shown as redstick models. The other molecule is
colored gray. (B) The globular subdomainis shown in a ribbon
representation. Basic amino acid residues forming thebasic patch
are shown as stick models. The conserved Gly-rich
segment(870KGSGGGR876) is highlighted in red.
Arg371
Arg731
Val460
Ala-SA
Mid2
InsA2
1 2
Fig. 5. Models of tRNA binding. A tRNA model was created by
superpositionof the aminoacylation domains of AlaRS-�C and the E.
coli ThrRS�tRNAThr
complex, and the acceptor-arm portion of the tRNA (residues 1–7
and 66–76)is shown as a dark-yellow transparent surface model (mode
1). The secondtRNA model, bound to an alternative tRNA-binding
site, is also depicted as ablue surface model (mode 2). The A76
residues in the first and second modelsare highlighted in yellow
and blue, respectively. Amino acid residues impor-tant for the
aminoacylation or editing activity (17, 28, 38, 39, 45, 46) are
shownas stick models. Ala-SA in the aminoacylation site and the
editing-site zinc ionare depicted as cpk models. A stereo version
of this figure is presented asFig. S6.
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should dissociate to move over the protuberant Mid2 subdomainand
then rebind. However, an alternative mode (mode 2) is thatthe tRNA
acceptor stem binds to a groove formed between theMid2 subdomain
and the editing domain (‘‘alternative groove’’)of A. fulgidus AlaRS
(Fig. 5). In the present structure, the linkerconnecting Mid2 and
the editing domain is located in thealternative groove, but the
linker appears to be quite flexible andto change its conformation
upon tRNA binding. The alternativegroove has entrances to both the
aminoacylation and editingactive sites, which would facilitate tRNA
relocation betweenthem. Consequently, the alternative mode 2 is
more likely thanthe common mode 1 for A. fulgidus AlaRS.
For the bacterial AlaRS from A. aeolicus, the tRNA was dockedin
mode 1 (14). Because A.aeolicus AlaRS lacks the
archaea-specificinsertion (InsA2), tRNA binding is not hindered. In
addition, theacceptor stem could be proximal to Asp-398,
corresponding toAla-409 in E. coli AlaRS, which is thought to be
indirectly involvedin the G3�U70 interaction (38). The
aminoacylation and tRNA-recognition domains of A.aeolicus AlaRS
were successfully sepa-rated from the editing domain for
crystallography (14), whereas thecorresponding
aminoacylation/tRNA-recognition fragment andthe editing domain
fragment of E. coli AlaRS were both preparedfor functional studies
(17, 38). In contrast, in the case of the archaealAlaRS from A.
fulgidus, it was difficult to prepare the correspondingfragments,
probably because of the hydrophobic interaction be-tween the
aminoacylation/tRNA-recognition domains and the ed-iting domain
(Fig. 3). Therefore, the bacterial AlaRSs might havethe editing
domain in a different location from that in the archaealAlaRS,
relative to the aminoacylation/tRNA-recognition domains,thus
allowing the tRNA to shift easily between the 2 active sites. InE.
coli AlaRS, the aminoacylation/tRNA-recognition domains andthe
editing domain are both able to recognize the G3�U70 base pairin
the tRNA acceptor stem (17), involving Arg-314 on the
tRNA-recognition domain and Arg-693 on the editing domain of E.
coliAlaRS (17, 39). Intriguingly, in the present A. fulgidus
AlaRSstructure, Arg-371 (Mid1) and Arg-731, which correspond to
theG3�U70 recognition residues Arg-314 and Arg-693, respectively,
areclose to the putative tRNA binding site in the alternative
groove(Fig. 5, mode 2). The tRNA-recognition domain including
Arg-371resides on the minor groove side of the acceptor stem, and
thisbinding mode (mode 2) is more preferable for the minor
grooverecognition of the G3�U70 base pair than mode 1 (12, 13).
There-fore, we cannot exclude the possibility that the bacterial
AlaRSshave a similar domain arrangement to that of A. fulgidus
AlaRS, andbind tRNA via mode 2. However, the editing domain could
interactwith the G3�U70 base pair in the major groove (Fig. 5).
Arg-371 andArg-731 are too far from each other to simultaneously
interact withthe G3�U70 base pair. Our model in mode 2 is
compatible with thefact that E. coli AlaRS aminoacylates the 3�-OH
of A76 (40).
The C-terminal dimerization domain of A. fulgidus AlaRS isalso
crucial for the tRNA interaction, because the deletion of thedomain
dramatically reduces the aminoacylation activity (23).The A.
fulgidus AlaRS-C structure reveals that the globularsubdomain of
the dimerization domain possesses a positively-charged face,
including the conserved Gly-rich segment (Fig.4B). The globular
subdomain, therefore, is a candidate for thetRNA-binding site, to
support aminoacylation reactions. For E.coli AlaRS, the region of
residues 808–875 is a nonspecifictRNA-binding site (17), which
includes the Gly-rich segment.
The Full-Length AlaRS Structure. The present structures
ofAlaRS-�C and AlaRS-C provide clues to infer the full-lengthAlaRS
structure. The distance between the N termini in theAlaRS-C dimer
is only 14 Å, which could restrict the positionsof the other
domains. Because the editing domain C terminus isconnected to the
dimerization domain N terminus, the 2 editingdomains in the dimer
should be close to each other, whereas theaminoacylation and
tRNA-recognition domains would be dis-
tant from the 2-fold axis. In the AlaRS-�C crystal structure,
theediting domain interacts back-to-back with that of the
symmetry-related molecule correlated by the crystallographic 2-fold
axis.Met-650, Ile-656, Leu-657, and Met-716 mediate the
interaction,and the buried surface area is �400 Å2. In the crystal
lattice, thedistance between the editing-domain C-termini is �19 Å,
whichallows their connection to the dimerization-domain N
terminiwithout a large conformation change. Overall, the
full-lengthAlaRS dimer is likely to assume a butterfly-like
structure (Fig.6). Although this model still requires validation,
it could serve asa platform for future analyses.
Materials and MethodsProtein Preparation. See SI Text.
Crystallization and Data Collection. See SI Text.
Structure Determination and Refinement. The structure of
AlaRS-�C was solvedby the single-wavelength anomalous dispersion
method. The Se-site andinitial phase determinations and solvent
flattening were performed with theAutoSHARP program (41). All 15 of
the Se sites were identified. Densitymodification and initial model
building using the RESOLVE program placed51% of the amino acid
residues, and the remaining residues were builtmanually with the
COOT program (42, 43). Structure refinement was carriedout with the
CNS program (44). A randomly-chosen 5% of the data were setaside
for cross-validation. The refinement included several rounds of
simulat-ed-annealing, positional, and individual B factor
refinements. The refinementconverged to final R and Rfree factors
of 21.5% and 26.4%, respectively (TableS1). In the Ramachandran
plot, 87.4%, 11.9%, and 0.6% of the residues fell inthe most
favored, additional allowed, and generously allowed regions,
re-spectively. No residues were in the disallowed region.
The structure of AlaRS-C was solved by the SAD method with the
AutoSHARPprogram (41). Of the 56 Se sites, 48 were identified.
Model building was per-formed manually by using the COOT program
(42). Refinement was done withthe CNS program, and the R and Rfree
factors for the final model are 20.5% and27.6%, respectively (Table
S1). In the Ramachandran plot, 88.5%, 11.2% and0.2% of the residues
fell in the most favored, additional allowed, and generouslyallowed
regions, respectively. No residues were in the disallowed
region.
ACKNOWLEDGMENTS. We thank the staffs of the Photon Factory
(Tsukuba,Japan) and SPring-8 BL41XU (Hyogo, Japan) beam lines for
assistance with ourdata collection. This work was supported in part
by a Ministry of Education,Culture, Sports, Science, and Technology
Global Centers of Excellence Program(Integrative Life Science Based
on the Study of Biosignaling Mechanisms), aMinistry of Education,
Culture, Sports, Science, and Technology Grant-in-Aid forScientific
Research, and the Ministry of Education, Culture, Sports, Science
andTechnology Targeted Proteins Research Program. R.F. was
supported by ResearchFellowships from the Japan Society for the
Promotion of Science.
Fig. 6. A model of the full-length AlaRS dimer. Two copies of
AlaRS-�C,which are correlated by the crystallographic 2-fold axis,
and an AlaRS-C dimer,are shown. The N termini of AlaRS-C were
placed near the C termini ofAlaRS-�C. The model was colored as in
Fig. 1.
Naganuma et al. PNAS � May 26, 2009 � vol. 106 � no. 21 �
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-
1. Jasin M, Regan L, Schimmel P (1983) Modular arrangement of
functional domainsalong the sequence of an aminoacyl tRNA
synthetase. Nature 306:441–447.
2. Beebe K, Ribas De Pouplana L, Schimmel P (2003) Elucidation
of tRNA-dependentediting by a class II tRNA synthetase and
significance for cell viability. EMBO J 22:668–675.
3. Cusack S (1995) Eleven down and nine to go. Nat Struct Biol
2:824–831.4. Eriani G, Delarue M, Poch O, Gangloff J, Moras D
(1990) Partition of tRNA synthetases
into two classes based on mutually exclusive sets of sequence
motifs. Nature 347:203–206.
5. Jasin M, Regan L, Schimmel P (1984) Dispensable pieces of an
aminoacyl tRNA syn-thetase which activate the catalytic site. Cell
36:1089–1095.
6. Putney SD, et al. (1981) Primary structure of a large
aminoacyl-tRNA synthetase. Science213:1497–1501.
7. Hou YM, Schimmel P (1988) A simple structural feature is a
major determinant of theidentity of a transfer RNA. Nature
333:140–145.
8. McClain WH, Foss K (1988) Changing the identity of a tRNA by
introducing a G-Uwobble pair near the 3� acceptor end. Science
240:793–796.
9. Vasil’eva IA, Moor NA (2007) Interaction of aminoacyl-tRNA
synthetases with tRNA:General principles and distinguishing
characteristics of the high-molecular-weightsubstrate recognition.
Biochemistry (Moscow) 72:247–263.
10. Beuning PJ, Musier-Forsyth K (1999) Transfer RNA recognition
by aminoacyl-tRNAsynthetases. Biopolymers 52:1–28.
11. Francklyn C, Schimmel P (1989) Aminoacylation of RNA
minihelices with alanine.Nature 337:478–481.
12. Musier-Forsyth K, Schimmel P (1992) Functional contacts of a
transfer RNA synthetasewith 2�-hydroxyl groups in the RNA minor
groove. Nature 357:513–515.
13. Musier-Forsyth K, et al. (1991) Specificity for
aminoacylation of an RNA helix: Anunpaired, exocyclic amino group
in the minor groove. Science 253:784–786.
14. Swairjo MA, et al. (2004) Alanyl-tRNA synthetase crystal
structure and design foracceptor-stem recognition. Mol Cell
13:829–841.
15. Swairjo MA, Schimmel PR (2005) Breaking sieve for steric
exclusion of a noncognateamino acid from active site of a tRNA
synthetase. Proc Natl Acad Sci USA 102:988–993.
16. Lee JW, et al. (2006) Editing-defective tRNA synthetase
causes protein misfolding andneurodegeneration. Nature
443:50–55.
17. Beebe K, Mock M, Merriman E, Schimmel P (2008) Distinct
domains of tRNA synthetaserecognize the same base pair. Nature
451:90–93.
18. Fukai S, et al. (2000) Structural basis for double-sieve
discrimination of L-valine fromL-isoleucine and L-threonine by the
complex of tRNAVal and valyl-tRNA synthetase. Cell103:793–803.
19. Fukunaga R, Yokoyama S (2005) Aminoacylation complex
structures of leucyl-tRNAsynthetase and tRNALeu reveal two modes of
discriminator-base recognition. NatStruct Mol Biol 12:915–922.
20. Sankaranarayanan R, et al. (1999) The structure of
threonyl-tRNA synthetase-tRNAThr
complex enlightens its repressor activity and reveals an
essential zinc ion in the activesite. Cell 97:371–381.
21. Silvian LF, Wang J, Steitz TA (1999) Insights into editing
from an Ile-tRNA synthetasestructure with tRNAIle and mupirocin.
Science 285:1074–1077.
22. Tukalo M, Yaremchuk A, Fukunaga R, Yokoyama S, Cusack S
(2005) The crystal structureof leucyl-tRNA synthetase complexed
with tRNALeu in the post-transfer-editing con-formation. Nat Struct
Mol Biol 12:923–930.
23. Fukunaga R, Yokoyama S (2007) Crystallization and
preliminary X-ray crystallographicstudy of alanyl-tRNA synthetase
from the archaeon Archaeoglobus fulgidus. ActaCrystallogr F
63:224–228.
24. Ahel I, Korencic D, Ibba M, Söll D (2003) Trans-editing of
mischarged tRNAs. Proc NatlAcad Sci USA 100:15422–15427.
25. Schimmel P, Ribas De Pouplana L (2000) Footprints of
aminoacyl-tRNA synthetases areeverywhere. Trends Biochem Sci
25:207–209.
26. Fukunaga R, Yokoyama S (2007) Structure of the AlaX-M
trans-editing enzyme fromPyrococcus horikoshii. Acta Crystallogr D
63:390–400.
27. Sokabe M, Okada A, Yao M, Nakashima T, Tanaka I (2005)
Molecular basis of alaninediscrimination in editing site. Proc Natl
Acad Sci USA 102:11669–11674.
28. Hill K, Schimmel P (1989) Evidence that the 3� end of a tRNA
binds to a site in theadenylate synthesis domain of an
aminoacyl-tRNA synthetase. Biochemistry 28:2577–2586.
29. Dock-Bregeon AC, et al. (2004) Achieving error-free
translation; the mechanism ofproofreading of threonyl-tRNA
synthetase at atomic resolution. Mol Cell 16:375–386.
30. Logan DT, Mazauric MH, Kern D, Moras D (1995) Crystal
structure of glycyl-tRNAsynthetase from Thermus thermophilus. EMBO
J 14:4156–4167.
31. Mosyak L, Reshetnikova L, Goldgur Y, Delarue M, Safro MG
(1995) Structure ofphenylalanyl-tRNA synthetase from Thermus
thermophilus. Nat Struct Biol 2:537–547.
32. Nair SK, Burley SK (2003) X-ray structures of Myc-Max and
Mad-Max recognizing DNA.Molecular bases of regulation by
protooncogenic transcription factors. Cell 112:193–205.
33. Holm L, Sander C (1998) Touring protein fold space with
Dali/FSSP. Nucleic Acids Res26:316–319.
34. Yamagata A, Kakuta Y, Masui R, Fukuyama K (2002) The crystal
structure of exonu-clease RecJ bound to Mn2� ion suggests how its
characteristic motifs are involved inexonuclease activity. Proc
Natl Acad Sci USA 99:5908–5912.
35. Ugochukwu E, Lovering AL, Mather OC, Young TW, White SA
(2007) The crystalstructure of the cytosolic exopolyphosphatase
from Saccharomyces cerevisiae revealsthe basis for substrate
specificity. J Mol Biol 371:1007–1021.
36. Cavarelli J, et al. (1994) The active site of yeast
aspartyl-tRNA synthetase: Structural andfunctional aspects of the
aminoacylation reaction. EMBO J 13:327–337.
37. Biou V, Yaremchuk A, Tukalo M, Cusack S (1994) The 2.9-Å
crystal structure of T.thermophilus seryl-tRNA synthetase complexed
with tRNASer. Science 263:1404–1410.
38. Ho C, Jasin M, Schimmel P (1985) Amino acid replacements
that compensate for a largepolypeptide deletion in an enzyme.
Science 229:389–393.
39. Ribas de Pouplana L, Buechter D, Sardesai NY, Schimmel P
(1998) Functional analysis ofpeptide motif for RNA microhelix
binding suggests new family of RNA-binding do-mains. EMBO J
17:5449–5457.
40. Hecht SM, Chinualt AC (1976) Position of aminoacylation of
individual Escherichia coliand yeast tRNAs. Proc Natl Acad Sci USA
73:405–409.
41. Emsley P, Cowtan K (2004) Coot: Model-building tools for
molecular graphics. ActaCrystallogr D 60:2126–2132.
42. Vonrhein C, Blanc E, Roversi P, Bricogne G (2006) Automated
structure solution withautoSHARP. Methods Mol Biol 364:215–230.
43. Terwilliger TC (2000) Maximum-likelihood density
modification. Acta Crystallogr D56:965–972.
44. Brunger AT, et al. (1998) Crystallography and NMR system: A
new software suite formacromolecular structure determination. Acta
Crystallogr D 54:905–921.
45. Davis MW, Buechter DD, Schimmel P (1994) Functional
dissection of a predictedclass-defining motif in a class II tRNA
synthetase of unknown structure. Biochemistry33:9904–9911.
46. Shi JP, Musier-Forsyth K, Schimmel P (1994) Region of a
conserved sequence motif in aclass II tRNA synthetase needed for
transfer of an activated amino acid to an RNAsubstrate.
Biochemistry 33:5312–5318.
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nloa
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15,
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