Structural basis for the substrate recognition and catalysis of peptidyl-tRNA hydrolase.
ABSTRACT Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are produced by aborted translation, to recycle tRNA for further rounds of protein synthesis. Pth is ubiquitous in nature, and its enzymatic activity is essential for bacterial viability. We have determined the crystal structure of Escherichia coli Pth in complex with the tRNA CCA-acceptor-TΨC domain, the enzyme-binding region of the tRNA moiety of the substrate, at 2.4 Å resolution. In combination with site-directed mutagenesis studies, the structure identified the amino acid residues involved in tRNA recognition. The structure also revealed that Pth interacts with the tRNA moiety through the backbone phosphates and riboses, and no base-specific interactions were observed, except for the interaction with the highly conserved base G53. This feature enables Pth to accept the diverse sequences of the elongator-tRNAs as substrate components. Furthermore, we propose an authentic Pth:peptidyl-tRNA complex model and a detailed mechanism for the hydrolysis reaction, based on the present crystal structure and the previous studies' results.
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ABSTRACT: In all organisms, the large ribosomal subunit contains multiple copies of a flexible protein, the so-called 'stalk'. The C-terminal domain (CTD) of the stalk interacts directly with the translational GTPase factors, and this interaction is required for factor-dependent activity on the ribosome. Here we have determined the structure of a complex of the CTD of the archaeal stalk protein aP1 and the GDP-bound archaeal elongation factor aEF1α at 2.3 Å resolution. The structure showed that the CTD of aP1 formed a long extended α-helix, which bound to a cleft between domains 1 and 3 of aEF1α, and bridged these domains. This binding between the CTD of aP1 and the aEF1α•GDP complex was formed mainly by hydrophobic interactions. The docking analysis showed that the CTD of aP1 can bind to aEF1α•GDP located on the ribosome. An additional biochemical assay demonstrated that the CTD of aP1 also bound to the aEF1α•GTP•aminoacyl-tRNA complex. These results suggest that the CTD of aP1 interacts with aEF1α at various stages in translation. Furthermore, phylogenetic perspectives and functional analyses suggested that the eukaryotic stalk protein also interacts directly with domains 1 and 3 of eEF1α, in a manner similar to the interaction of archaeal aP1 with aEF1α. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.Nucleic Acids Research 11/2014; · 8.81 Impact Factor
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ABSTRACT: The genomic RNA of encephalomyocarditisvirus (EMCV) encodes a single polyprotein, and the primary scission of the polyprotein occurs between nonstructural proteins, 2A and 2B, by an unknown mechanism. To gain insight into the mechanism of 2A-2B processing, we first translated the 2A-2B region in vitro with eukaryotic and prokaryotic translation systems. The 2A-2B processing occurred only in the eukaryotic systems, not in the prokaryotic systems, and the unprocessed 2A-2B protein synthesized by a prokaryotic system remained uncleaved when incubated with a eukaryotic cell extract. These results suggest that 2A-2B processing is a eukaryote-specific, co-translational event. To define the factors required for 2A-2B processing, we constituted a protein synthesis system with eukaryotic elongation factor (eEF)1, eEF2, eukaryotic release factor (eRF)1, eRF3, aminoacyl-tRNA synthetases, tRNAs, ribosome subunits and a plasmid template that included the hepatitis C virus internal ribosome entry site. We successfully reproduced 2A-2B processing in the reconstituted system even without eRFs. Our results indicate that this unusual event occurs without eIFs, eRFs, and any specific factor.Journal of Biological Chemistry 09/2014; · 4.60 Impact Factor
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ABSTRACT: Peptidyl-tRNA hydrolase (Pth) catalyses the release of tRNA and peptide components from peptidyl-tRNA molecules. Pth from a Gram-positive bacterium Streptococcus pyogenes (SpPth) was cloned, expressed, purified and crystallised. Three-dimensional structure of SpPth was determined by X-ray crystallography at 2.19 Å resolution. Structure determination showed that the asymmetric unit of the unit cell contained two crystallographically independent molecules, designated A and B. The superimposition of C(α) traces of molecules A and B showed an r.m.s. shift of 0.4 Å, indicating that the structures of two crystallographically independent molecules were identical. The polypeptide chain of SpPth adopted an overall α/β conformation. The substrate-binding cleft in SpPth is formed with three loops: the gate loop, Ile91-Leu102; the base loop, Gly108-Gly115; and the lid loop, Gly136-Gly150. Unlike in the structures of Pth from Gram-negative bacteria, the entry to the cleft in the structure of SpPth appeared to be virtually closed. However, the conformations of the active site residues were found to be similar.FEBS open bio. 01/2014; 4:915-22.
Structural basis for the substrate recognition and
catalysis of peptidyl-tRNA hydrolase
Kosuke Ito1,*, Ryo Murakami1, Masahiro Mochizuki1, Hao Qi2, Yoshihiro Shimizu2,
Kin-ichiro Miura2, Takuya Ueda2and Toshio Uchiumi1
1Department of Biology, Faculty of Science, Niigata University, 8050 Ikarashi 2-no-cho, Nishi-ku, Niigata
950-2181 and2Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University
of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
Received February 9, 2012; Revised July 11, 2012; Accepted July 28, 2012
Peptidyl-tRNA hydrolase (Pth) cleaves the ester
peptidyl-tRNA molecules, which are produced by
aborted translation, to recycle tRNA for further
rounds of protein synthesis. Pth is ubiquitous in
nature, and its enzymatic activity is essential for
bacterial viability. We have determined the crystal
structure of Escherichia coli Pth in complex with
the tRNA CCA-acceptor-T)C domain, the enzyme-
binding region of the tRNA moiety of the substrate,
at 2.4A˚resolution. In combination with site-directed
mutagenesis studies, the structure identified the
amino acid residues involved in tRNA recognition.
The structure also revealed that Pth interacts with
the tRNA moiety through the backbone phosphates
and riboses, and no base-specific interactions were
observed, except for the interaction with the highly
conserved base G53. This feature enables Pth to
accept the diverse sequences of the elongator-
tRNAs as substrate components. Furthermore, we
propose an authentic Pth:peptidyl-tRNA complex
model and a detailed mechanism for the hydrolysis
reaction, based on the present crystal structure and
the previous studies’ results.
During the course of protein synthesis, peptidyl-tRNA is
prematurely produced from the ribosome as a result of
aborted translation (1). For example, amino acid starva-
tion, tRNA starvation and translation of a truncated
mRNA will cause ribosome stalling and the release of
peptidyl-tRNAs (2–4). Ribosomal recycling factor, elong-
ation factor G and initiation factor 3, as well as other
factors, contribute to this release (5–8). The accumulation
of the peptidyl-tRNAs thus released is toxic for the cell,
because they can either impair the initiation of translation
or slow protein synthesis, due to the limitation of the cor-
responding tRNAs (9–11). However, this situation is pre-
vented by the activity of peptidyl-tRNA hydrolase (Pth)
peptidyl-tRNA, by cleaving the ester bond between the
C-terminal end of the peptide and the 20- or 30-hydroxyl
of the ribose at the 30-end of the tRNA, thereby freeing the
tRNA for reuse in protein synthesis (13,14). The activity
of Pth is essential for the viability of bacteria (10,15).
The biochemical features of Pth have mainly been
studied using the enzyme from Escherichia coli. Pth recog-
nizes both the peptide and tRNA moieties of the substrate
(16). Both of the moieties consist of diverse sequences
depending on the substrate; nevertheless, Pth can accept
various types of peptidyl-elongator tRNAs as substrates
(14,17). In addition, Pth accepts peptidyl-tRNAHis, which
has the peculiar feature of possessing one extra G nucleo-
tide at position ?1, as a substrate (18,19). The mechanism
for this broad substrate specificity remains unknown. On
the other hand, it is known that N-blocked aminoacyl-
tRNAs are the shortest substrates of the enzyme (13,14);
by Pth. (14,20). Indeed, this formylated initiator tRNA
must be kept intact, to be recruited by initiation factor 2
and to participate in the formation of the ribosomal initi-
ation complex. The resistance of formylated initiator
tRNA to Pth is considered to be a consequence of the
unpaired 1:72 nucleotides, which are a unique character-
the detailed mechanism remains unknown. Moreover, it
is not yet clear how the enzyme discriminates peptidyl-
tRNAs from aminoacyl-tRNAs as the substrates, which
is an essential mechanism for the normal peptide
The Pth enzymes are ubiquitous in nature and can be
classified into two types—Pth, sometimes referred to as
Pth1 and Pth2. Pth exists in bacteria (21), whereas Pth2
is present in archaea (22,23). There is no sequence or
structural similarity between Pth and Pth2. On the other
is hardly cleaved
*To whom correspondence should be addressed. Tel/Fax: +81 25 262 7029; Email: email@example.com
Published online 25 August 2012Nucleic Acids Research, 2012, Vol. 40, No. 2010521–10531
? The Author(s) 2012. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
hand, eukaryotes possess multiple types of Pths, including
the Pth and Pth2 types and others (12). Considering the
rising incidence of bacterial resistance to known antibi-
otics, Pth is an attractive target and is now in the spotlight,
because, as mentioned above, bacteria possess a single
type of Pth and its activity is essential for their viability,
whereas eukaryotes possess multiple types of Pths (12,24).
To develop novel antibiotics, the characterization of the
interaction between Pth and its substrate is needed at an
The structures of the Pth proteins from several species
have been determined, to elucidate the essential transla-
tion maintenance and tRNA-recycling functions of this en-
zyme and to develop novel antibiotics (25–28). However,
the molecular mechanism of the reaction remains unclear,
because all of these structures have been determined in the
substrate-free form. Thus, as the first step, we have
determined the crystal structure of E. coli Pth in
complex with the CCA-acceptor-T?C domain of tRNA
(Figure 1), the enzyme-binding region of the tRNA moiety
(as described later), at 2.4A˚ resolution. In addition, we
According to these results, we identified the amino acid
residues involved in the tRNA moiety recognition and
also revealed the mechanism by which Pth accepts the
diverse sequences of the elongator-tRNAs as substrate
components. Furthermore, we present an authentic
Pth:peptidyl-tRNA complex model and a detailed mech-
anism for the hydrolysis reaction, which clearly explain
the previous studies’ results.
MATERIALS AND METHODS
Sample preparation and crystallization
Sample production, purification and crystallization of E.
coli Pth in complex with the CCA-acceptor-T?C domain
of E. coli tRNAAla(G1 to A7 and A49 to A76 of E. coli
GGC) were performed as previously described (29).
Briefly, E. coli Pth was overexpressed in E. coli strain
BL21(DE3) with an N-terminal His-tag. The recombinant
protein was purified by a Ni-chelating column and then
treated with thrombin to remove the N-terminal His-tag.
The protein was further purified using Ni-chelating,
benzamidine-conjugated and anion-exchange resins. The
CCA-acceptor-T?C domain of E. coli tRNAAlawas
synthesized by in vitro transcription using T7 RNA poly-
merase and purified with an anion-exchange column. For
crystallization, E. coli Pth and the CCA-acceptor-T?C
domain were mixed in a molar ratio of 1:2.5 at a final
protein concentration of 14mg/ml, in 10mM HEPES–
KOH (pH 7.6) containing 10mM MgCl2 and 7mM
?-mercaptoethanol. Crystals were obtained with the
sitting-drop vapor-diffusion method using a reservoir
solution containing 100mM sodium acetate buffer (pH
5.2), 20% (w/v) 1,4-butanediol and 30mM glycyl-glycyl-
glycine at 20?C.
Data collection and structure determination
Prior to data collection, crystals were soaked in reservoir
solution containing 10% (v/v) ethylene glycol as a cryo-
protectant. Diffraction data were collected at BL41XU of
SPring-8 (Harima, Japan) using a Mar 225 CCD detector.
The data were processed and scaled using the program
XDS (30) and analyzed by the CCP4 program suite (31).
Initial phases were obtained by the molecular replacement
method with the program MOLREP (32), using the struc-
tures of E. coli Pth (PDB accession code 2PTH) as a
acceptor-T?C domain was then obtained by phase im-
provement with the program ARP/wARP (33) and could
be traced using the program PHENIX (34). The model
was subsequently improved by iterative cycles of manual
model building with the program COOT (35) and
Figure 1. Full-length tRNA and the CCA-acceptor-T?C domain. (A) The base sequence of E. coli tRNAAla
acceptor stem is colored pink, the D loop is lime, the anticodon arm is violet, the variable loop is blue, the T?C arm is cyan and the CCA terminus
is yellow. The CCA-acceptor-T?C domain is surrounded by a red dashed line. (B) Ribbon diagram of yeast free tRNAPhe(PDB accession code
6TNA) (40), with the same color-coding as in (A). The CCA-acceptor-T?C domain is surrounded by a red dashed line.
GGC, drawn in the cloverleaf form. The
10522Nucleic Acids Research, 2012,Vol.40, No. 20
The quality of the model was checked with the program
PROCHECK (37). Computer graphic representations
were prepared using the program PyMOL (http://www
.pymol.org). The surface electrostatic potentials were
calculated using the program APBS (38) and visualized
with the program PyMOL.
Preparation of N-acetyl-alanyl-tRNAAla
Escherichia coli tRNAAlawas synthesized by in vitro
transcription as described previously (29). After transcrip-
tion, the RNA sample was purified by 12% polyacryl-
amide gel electrophoresis under denaturing conditions
with 8M urea. The tRNAAlawas retrieved from gel by
soaking crushed gel in 0.5M ammonium acetate buffer
(pH 6.0) containing 1mM EDTA, 10mM magnesium
with 2-propanol. Purified tRNAAlawas aminoacylated
at 37?C in 22ml of 50mM HEPES–KOH (pH 7.6) con-
taining 20mM KCl, 10mM MgCl2, 4mM ATP, 20mM ?-
mercaptoethanol, 0.01% BSA, 10mM tRNAAla, 20mM
[14C]L-alanine (150Ci/mol) and 150nM purified E. coli
Alanyl-tRNA synthetase (39). After a 1-h incubation, an
aminoacylated sample was extracted with phenol and
chloroform, followed by precipitation with 2-propanol in
the presence of 0.3M sodium acetate. The tRNA pellet
was dissolved in 1.1ml of 5mM sodium acetate buffer
(pH 5.5) and acetylated at 0?C for 15min by adding
1.1ml of dimethylsulfoxide, 0.22ml of glacial acetic
acid and 0.22ml acetic anhydride, followed by precipita-
tion with 2-propanol. The pellet was dissolved in 0.5ml
of 0.36M sodium acetate buffer (pH 5.5) containing
10mM CuSO4and incubated at 37?C for 30min. After
incubation, to remove copper ions, the sample solution
was applied to a PD-10 column (GE Healthcare)
equilibrated with 20mM sodium acetate buffer (pH 5.5)
samples were precipitated with 2-propanol and washed
with 80% ethanol. Finally, the pellet was dissolved in
5mM sodium acetate buffer (pH 5.5) and stored at
Peptidyl-tRNA hydrolase assay
Peptidyl-tRNA hydrolase activity was measured at 28?C
in 100ml assays containing 20mM HEPES–KOH (pH
7.6), 10mM MgCl2, 3.5 to 25mM acetyl-[14C]Ala-
tRNAAlaand 2–6nM Pth variants. The reaction was
quenched by addition of 100ml of 10% TCA, and 20ml
of 4mg/ml carrier RNA from yeast. After quenching, the
sample was precipitated with 220ml of 2-propanol in the
presence of 0.3M sodium acetate. 200ml of the super-
natant was added to 1ml insta-gel plus (PerkinElmer),
counting. Km and kcat values were generated by using
the program GraphPad Prism version 2.0 (GraphPad
14C radioactivity was measured by scintillation
RESULTS AND DISCUSSION
First, we attempted to crystallize E. coli Pth with a
full-length tRNA. However, we were unable to obtain
such crystals, probably because the flexible structure of
tRNA caused problems in the crystallization. Thus, we
next tried to crystallize E. coli Pth with the CCA-
acceptor-T?C domain of tRNA (Figure 1), because this
region of tRNA is considered to be structurally stable, and
the enzyme binds to the tRNA moiety via this region
(described in the next section). As a result, we obtained
the crystals of Pth in complex with this tRNA construct.
The details of the sample preparation and crystallization
are described in our previous report (29). The crystals
belonged to the hexagonal space group P61, with
unit-cell parameters a=b=55.1, c=413.1A˚. The struc-
ture was solved by molecular replacement at 2.4A˚ reso-
lution, with an Rworkof 19.3% and an Rfreeof 23.7%. The
data collection and structure refinement statistics are
provided in Table 1.
Pth:CCA-acceptor-T?C domain in the asymmetric unit,
Table 1. Data collection and refinement statistics
Unit-cell parameters (A˚)
No. of the Pth:CCA-acceptor–T?C domain
Solvent content (%)
Resolution range (A˚)
No. of measured reflections
No. of unique reflections
No. of atoms/asymmetric unit
r.m.s.d. from ideal values
Bond length (A˚)
Bond angle (?)
Ramachandran plot statistics
Most favored regions (%)
Allowed regions (%)
Generously allowed regions (%)
Disallowed regions (%)
aValues in parentheses are for the highest resolution shell.
symmetry-related reflections and <I(hkl)> is its average.
cRfreewas calculated by using 5% of randomly selected reflections that
were excluded from the refinement.
i Ii(hkl), where Ii(hkl) is
Nucleic Acids Research, 2012,Vol.40, No. 2010523
and the two complexes are quite similar. The r.m.s.d.’s
over equivalent Ca atoms and backbone phosphorus
atoms of the two complexes are 0.08 and 0.09A˚, respect-
ively. Thus, one complex structure of the Pth:CCA-
acceptor-T?C domain in the asymmetric unit is shown
in Figure 2. The overall structure of Pth consists of a
single a/b globular domain. Seven b-strands form a
twisted mixed central b-sheet, surrounded by a total of
six helices. The structure of Pth in the complex is similar
to that of the substrate-free enzyme (25), except for some
loop regions (r.m.s.d. of 1.32A˚ over 193 equivalent Ca
atoms) (Supplementary Figure S1). On the other hand,
the superposition of the CCA-acceptor-T?C domain
and free yeast tRNAPhe(40) showed that, although these
structures are essentially similar, there are structural dif-
ferences at the flexible 30-CCA terminus and at the
acceptor stem, (r.m.s.d. of 3.21A˚
backbone phosphorus atoms) (Supplementary Figure
S1). The structural difference within the acceptor stem
over 35 equivalent
part probably arose from the deletion of the D arm, anti-
codon arm and variable loop. However, a previous report
demonstrated that the catalytic efficiency of Pth for the N-
acetyl-histidyl-CCA-acceptor-T?C domain of tRNAHisis
comparable to that for the N-acetyl-histidyl-full-length
tRNAHis(41), indicating that the deletion does not
affect the interaction with Pth.
Pth interacts with the tRNA acceptor-T?C domain on
the T?C stem side via two sites (Figure 2). At the first site,
the positively charged patch around the active site cavity
of Pth interacts with the backbone of the acceptor stem in
an electrostatic manner (referred to as the ‘acceptor site’).
At the second site, the C-terminal a-helix of Pth interacts
with the minor groove of the T?C stem in a shape-
complementary manner (referred to as the ‘T?C site’).
In addition to these two interaction sites, the loop–helix
a4 segment over the active site cavity of Pth is proximal
to the 30-CCA terminus, although a direct interaction was
not observed (referred to as the ‘CCA site’). Peptidylation
Figure 2. Overall structure of Pth in complex with the CCA-acceptor-T?C domain of tRNA. (A) Two views of the overall structure of the
Pth:CCA-acceptor-T?C domain complex, represented by a ribbon diagram. As in Figure 1, the acceptor stem is colored pink, the T?C arm is
cyan and the CCA terminus is yellow. Pth is colored green. To clarify the configuration for Pth:tRNA binding, the left panel is shown with the same
viewing angle of the CCA-acceptor-T?C domain as that of the full-length tRNA in Figure 1B. The right panel shows the view rotated by ?90?along
the vertical axis from the left panel. (B) Two views of the overall structure of the Pth:CCA-acceptor-T?C domain complex, showing the surface of
Pth colored according to its calculated electrostatic potential (blue, positively charged range +4kT/e; red, negatively charged ?4kT/e). The view-
points are the same as those in (A).
10524Nucleic Acids Research, 2012,Vol.40, No. 20
or N-acetyl-aminoacylation is probably a prerequisite for
the interaction. The details of these three sites are
described in a following section. The present structure
also indicates that the D arm, anticodon arm and
variable loop cannot exist in locations where Pth can
interact with them. Therefore, although the CCA-
acceptor-T?C domain used here is a partial construct of
tRNA, it is sufficient for the description of all of the
aspects of the interaction between Pth and the tRNA
moiety of the substrate.
We compared the structure of the Pth:CCA-acceptor-
T?C domain complexwith
protein:tRNA complexes determined so far, in which the
proteins recognize the acceptor and T?C stem regions on
the T?C stem side. The structure comparison revealed
that, whereas Pth recognizes the acceptor stem and the
T?C stem of the substrate by its single domain, other
proteins, such as EF-Tu (42), CCA-adding enzyme
(43,44), GatB (45,46) and RNase Z (47), recognize them
by multi-domain or multi-subunit systems. These facts
indicate that Pth has a unique tRNA recognition
feature. For more details, see the Supplementary Results
and Discussion and Supplementary Figure S2.
those of theother
Interaction at the acceptor site
At the acceptor site, the positively charged patch of Pth,
which is composed of the highly conserved basic amino
acid residues Lys103, Lys105 and Arg133 (Supplementary
Figure S3), interacts with the tRNA acceptor stem in an
electrostatic manner (Figure 3A and 3D). Specifically, the
Nz atoms of Lys103 and Lys105 form hydrogen bonds
with the phosphate groups of G4 and G2, respectively.
The NZ1 atom of Arg133 also forms a hydrogen bond
with the phosphate group of G3. The NZ2 atom of
Arg133 makes a bifurcated hydrogen bond with the O20
and O30atoms of the G2 ribose. The substitution of these
basic amino acid residues with neutral ones reduced the
substrate-binding efficiency (19,48–50). In addition to
these interactions, the main chain amide nitrogen of
Gly109 forms hydrogen bonds with the phosphate group
of G2 and the O30atom of the G1 ribose. The Od2 atom of
Asp96 also forms a hydrogen bond with the O20atom of
the G2 ribose. The replacement of Asp96 with alanine,
however, has little effect on the catalytic efficiency (41).
This replacement may be compensated by Arg133.
The mutation of Gly100 with aspartic acid is respon-
sible for the Pth (Ts) phenotype, which can grow normally
at 30?C, but cannot sustain growth at the non-permissive
temperature of 42?C (48). The present structure shows
that, although a direct interaction is not observed,
Gly100 exists in a loop connected to a b-strand that
includes Lys103 and Lys105. Thus, the slight conform-
ational change of the loop, caused by the mutation of
Gly100, might perturb the structure around Lys103 and
Lys105 at the non-permissive temperature and conse-
quently destabilize the Pth:substrate complex formation,
as suggested in a previous report (50).
In the present structure, the phosphate group of G1 is
covalently linked to an unexpectedly extended guanosine
nucleotide (G-1) (Supplementary Figure S4). This extra
G-1 may have been caused by the untemplated incorpor-
ation of GMP during in vitro transcription by T7 RNA
polymerase, arising from the 50-terminal consecutive
guanosine nucleotides of the RNA sequence (29,51). We
identified the transcription products as a mixture of the
CCA-acceptor-T?C domains with and without the extra
G-1 and other molecules, by MALDI–TOF MS analyses
(Supplementary Figure S5). However, the structure shows
that the extra G-1 sustains the flexible 30-CCA terminus
(Supplementary Figure S4), and this situation would result
in the preferential crystal formation of the complex
including the CCA-acceptor-T?C domain with the extra
G-1. Indeed, we tried to obtain crystals of the complex by
using the acceptor-T?C domain that does not include the
extra G-1 by rigorous purification; nevertheless, we could
not obtain such a crystal.
Fromant et al. investigated the combined effects of the
removal of the 50-terminal phosphate of the substrate and
of various amino acid substitutions in Pth on the kcat/Km
values and demonstrated that the removal of the 50-
terminal phosphate exerted no marked combined effect
for the K105A and R133A mutants, in contrast to the
other mutants (49). In addition, Lys105 and Arg133 are
very close together on Pth. Therefore, the 50-terminal
phosphate of the substrate was expected to be clamped
by Lys105 and Arg133 (49). On the other hand, in the
present crystal structure, as mentioned above, the phos-
phate group of G1, which corresponds to the intrinsic 50-
terminal phosphate, is covalently linked to the extra G-1
nucleotide, and this extra G-1 is involved in the crystal
packing. For this reason, the conformation around the
phosphate group of G1 is slightly distorted and lies
removal of the extra G-1 and a slight conformational
change around G1 allowed the original 50-terminal phos-
phate to be modeled to bind to the positively charged
patch around the active site cavity, which includes
Lys105 and Arg133, without disturbing the interaction
between Pth and the tRNA moiety, and without disrupt-
ing the base pairs (Figure 4A). More specifically, the 50-
terminal phosphate is expected to hydrogen bond with the
main chain amide nitrogen of His110. This area is posi-
tively charged, and Lys105 and Arg133 would contribute
to its net positive charge. Thus, our current model is con-
sistent with the previous mutagenesis study that reported
the decreased catalytic efficiency by the alanine substitu-
tions of Lys105 and/or Arg133 (49). In addition, a
previous NMR chemical shift perturbation study demon-
strated that the spectral properties of the main chain
amide nitrogen of His110 are strongly affected by the
addition of a duplex RNA that mimics the CCA-
acceptor-T?C stem part of tRNA (41). Further experi-
ments are necessary to determine the precise binding site
of the 50-terminal phosphate group.
away from the surface of Pth. However, the
Interaction at the T)C site
At the T?C site, the C-terminal a-helix of Pth penetrates
the substrate (Figure 3B and D). Specifically, the Nd1 atom
Nucleic Acids Research, 2012,Vol.40, No. 2010525
the phosphate group of G63. The involvement of His188 in
the interaction was unexpected and thus was not analyzed
previously. Therefore, wereplaced His188 withalanine and
acetyl-alanyl-tRNAAlaas a substrate (Table 2). The
results revealed that the H188A mutant resulted in a
5.4-fold decrease in the kcat/Km value, as compared to
that of the wild-type enzyme. This reduction was mainly
caused by an increase in the Km value. In addition to
His188, the crystal structure showed that the Od1 and
Nd2 atoms of Asn185 hydrogen bond with the N2 atom
of the G53 base and the O20atom of the U(T)54 ribose,
respectively. These interactions are consistent with a
previous NMR chemical shift perturbation study that
implied the involvement of Asn185 in the substrate
binding (41). As in the case of His188, we substituted
using N-acetyl-alanyl-tRNAAlaas a substrate. The N185A
mutant resulted in a 5.7-fold decrease in the kcat/Kmvalue,
as compared to that of the wild-type enzyme, whereas the
kcatvalue was essentially unchanged. These results are con-
sistent with thosefrom aprevious site-directed mutagenesis
study, in which N-acetyl-lysyl-tRNALyswas used as a sub-
strate (41). In addition, we measured the catalytic param-
eters of the double mutant of N185A and H188A. The
mutant resulted in a further decrease in the kcat/Kmvalue
and a further increase in the Kmvalue, whereas the kcat
value was barely influenced. These results indicate that
Asn185 and His188 play important roles in facilitating pro-
ductive substrate binding.
The Nz atom of Lys182 exists in the proximity of the
O30atom of U(T)54 and the backbone phosphate group of
Figure 3. Interaction between Pth and the tRNA moiety of the substrate. (A–C) Close-up view of the acceptor site (A), the T?C site (B) and the
CCA site (C). Pth is represented by a ribbon model, while the tRNA moiety is represented by a phosphate trace model, with the same color-coding as
in Figure 2A. The amino acid residues and nucleotides involved in the interaction are depicted by stick models. The catalytic center His20 is also
shown by a stick model. Interactions are indicated with dashed lines. Possible hydrogen bonds from Lys142 and Lys182 are indicated with solid lines.
(D) Schematic diagram of the interaction between Pth and the tRNA moiety of the substrate. Interactions are indicated by dashed lines. Possible
hydrogen bonds from Lys142 and Lys182 are indicated with solid lines. The color-coding of the bases is the same as in (A–C): the bases from the
acceptor stem are colored pink, the T?C arm is cyan and the CCA terminus is yellow. Riboses and phosphate groups are colored light blue and
10526 Nucleic Acids Research, 2012,Vol.40, No. 20
U(?)55, although a direct interaction is not observed.
However, the plasticity of the equivalent segment of Pth
from Mycobacterium tuberculosis has been demonstrated
by an NMR experiment (27), suggesting that Lys182 inter-
acts with either or both U(T)54 and U(?)55 during the
reaction process. Indeed, the Km value of the K182A
mutant is >8-fold higher than that of the wild-type
enzyme, whereas the kcatvalue only displayed a minor
Interaction at the CCA site
Probably due to either the absence of peptidylation or
N-acetyl-aminoacylation, or perhaps the influence of the
crystal packing, no direct interaction between Pth and the
CCA terminus was observed in the present structure.
However, the Nz atom of Lys142, which resides in the
loop–helix a4 segment over the active site cavity, exists
in the proximity of the backbone phosphates of C74 and
the discriminator A73 (Figure 3C). On the other hand, the
plasticity of the loop–helix a4 segment is suggested from
the high average temperature factor of this region
(Supplementary Figure S6). Moreover, the plasticity of
the equivalent segment of Pth from M. tuberculosis has
These facts suggest that Lys142 interacts with the
backbone phosphates of either or both C74 and the dis-
criminator A73 during the reaction process. Indeed, the
Kmvalue of the K142A mutant is >5-fold larger than that
of the wild-type enzyme, whereas the kcatvalue was found
to be comparable between the two (25). It seems likely
that the loop-helix a4 segment captures the flexible
CCA terminus of the substrate, by an electrostatic inter-
action via Lys142, to recruit or stabilize the binding of the
peptidyl-adenosine moiety within the active site cavity.
Mechanism of the broad specificity towards tRNA
It has been demonstrated that Pth recognizes both the
peptide and tRNA moieties of the substrate (16). Both
Figure 4. Structure of the Pth:peptidyl-tRNA complex model. (A) Structure around G1, in the case without the extra G-1. (B) Structure around the
active site cavity. Left panels in (A) and (B): the amino acid residues involved in the interaction between Pth and the peptidyl-tRNA are depicted by
stick models on a transparent ribbon model of Pth. As in Figure 2A, the acceptor stem is colored pink, the CCA terminus is yellow, and Pth is green.
In addition, the peptide moiety is represented by an orange stick model. Right panels in (A) and (B): the viewpoint is the same as that of each left
panel. Pth is shown in a surface model, colored as in Figure 2B. In (A), to make the structure around G1 more visible, the peptidyl-A76 moiety is
omitted. In (B), the ester bond between the peptide and tRNA moiety is indicated by a circle. In all panels, hydrogen bonds are indicated with
dashed lines. The removal simulation of the extra G-1 and the peptidyl-A76 docking simulation were performed manually as described in the text,
and then the geometry was idealized using the program COOT (35).
Table 2. Catalytic parameters of Pth variants
aRelative kcat/Kmvalues are shown, given an arbitrary value of 100 with
the WT enzyme.
Nucleic Acids Research, 2012,Vol.40, No. 20 10527
of these moieties consist of diverse sequences depending
on the substrate; nevertheless, Pth can accept many kinds
of peptidyl-elongator tRNAs as substrates (14,17). The
present structure has revealed the structural basis for the
sequence-independent recognition of the tRNA moiety.
Specifically, in the acceptor site, the amino acid residues
on the positively charged patch of Pth interact with the
backbone phosphates and ribose of the acceptor stem, and
a base-specific interaction was not observed (Figure 3A
and D). Likewise, in the T?C site, the amino acid
residues on the C-terminal a-helix of Pth interact with
the backbone phosphate and ribose of the T?C stem,
and a base-specific interaction was also not observed,
except for the highly conserved G53 (Figure 3B and D).
In the CCA site, as discussed above, Lys142 probably
interacts with the discriminator-CCA moiety through the
(Figure 3C and D). These features allow Pth to accom-
plish sequence-independent recognition of the tRNA
Pth accepts peptidyl-tRNAHis, which has the peculiar
feature of possessing one extra G nucleotide at position
?1 (G-1), as a substrate (18,19). A previous kinetic
study demonstrated that the substrate efficiencies of
histidyl-tRNAHismolecules were comparable to that of
upon the loss of the 50-phosphate group of the G-1-
obliterated N-acetyl-histidyl-tRNAHis, the kcat/Kmvalue
decreased 7-fold (19). These results indicated that the
phosphate group of G1 plays a crucial role in the
reaction of Pth, in contrast to the dispensability of G-1.
On the other hand, in the case of diacetyl-lysyl-tRNALys
as a substrate, the removal of the 50-phosphate group
decreased the kcat/Km value by 17-fold (49), as in the
case of the removal of the 50-phosphate group from the
these facts, Fromant et al. proposed that the locations of
the G1 phosphate group of tRNAHisand the 50-phosphate
group of standard elongator tRNAs on Pth are identical,
whereas the G-1 of tRNAHisis in a position that does not
hinder the Pth reaction (19). Actually, there is sufficient
space on top of G1, on the surface of the enzyme, to
accommodate G-1 of tRNAHis(Figure 4A). Therefore,
we consider the previous proposition to be structurally
In the Pth:CCA-acceptor-T?C domain structure obtained
here, the 30-terminal adenosine A76 did not bind to the
active site of Pth. Peptidylation or N-acetyl-aminoacy-
lation may be required to place the 30-terminal adenosine
A76 in the binding site. However, the region on Pth that
interacts with A76 can be predicted, using the present
structure. In addition, enzymatic analyses and NMR
chemical shift perturbation studies have indicated the
amino acid residues involved in the substrate binding
substrate-free Pth from E. coli, three residues (K191–
A192–Q193), at the C-terminus of a neighboring enzyme
molecule in the crystal, were bound to the active site, and
this interaction was assumed to represent the formation of
a complex between Pth and the peptide segment of the
substrate (25). Taking this information into account, we
modeled the peptidyl-A76 moiety in the active site cavity,
as discussed below.
There is a pocket suitable for accommodating the
adenine ring in the active site cavity (Figure 4B). Due to
backbone on the Pth:CCA-acceptor-T?C domain struc-
ture obtained here, the adenine ring of A76 could be pos-
itioned in the pocket, so that the N6 atom of the adenine
ring form hydrogen bonds with the main chain carbonyl
oxygen of Gly111, and the N3 atom of the adenine ring
makes van der Waals contacts with the side chains of
Leu95 and Val149 (Figures 4B and 5A). This docking is
reasonable, because NMR chemical shift perturbation
studies revealed the involvement of Leu95, Gly111 and
Val149 in substrate analog binding (41,52). Leu95 has
also been demonstrated to be involved in the catalysis
by a site-directed mutagenesis study (41). We next
placed the K191–A192–Q193 tripeptide of the substrate-
free Pth structure into the equivalent position of the
bond between the K191–A192–Q193 tripeptide and the
30-OH of A76 could be formed, by the slight conform-
ational change of Gln193 (Supplementary Figure S7).
Therefore, we rearranged the conformations of the side
chains of Asn68 and Asn114 to resemble those in the
substrate-free Pth structure, in which the Nd2 atoms of
these amino acid residues form hydrogen bonds with the
C-terminal carboxyl groups of the K191–A192–Q193 tri-
peptide. As a result, in the model, the Nd2 atoms of Asn68
and Asn114 form hydrogen bonds with the ester carbonyl
oxygen of the substrate. In addition, as in the case of the
substrate-free Pth, the Nd2 atom of Asn10 and the main
chain amide nitrogen of Asn68 form hydrogen bonds
with the main chain carbonyl oxygens of Ala192 and
Lys191 of the peptide moiety of the substrate, respectively.
Indeed, the involvement of Asn10, Asn68 and Asn114 in
substrate binding has been demonstrated by enzymatic
analyses and an NMR chemical shift perturbation study
complex model, Pth recognizes only the main chain
atoms of the substrate and the side chains of the substrate
point toward away from the enzyme (Figures 4B and 5A).
Thus, this model explains the sequence independence of
the peptide moiety recognition by the enzyme. In addition,
this model clearly explains why the enzyme does not digest
aminoacyl-tRNAs. Because, Asn10, which is a determin-
ant for discrimination between peptidyl- and aminoacyl-
tRNAs (52), forms a hydrogen bond with the first amide
oxygen of the peptide moiety, and this interaction cannot
be accomplished by aminoacyl-tRNAs.
In the previous docking study, the adenine ring of A76
was expected to stack on Phe66 (41). However, this pre-
diction is not fully consistent with other studies, for the
following reasons. First, the degree of the kcat/Kmvalue
reduction by the replacement of Phe66 with alanine is very
small, as compared to the cases of other putative
10528Nucleic Acids Research, 2012,Vol.40, No. 20
substrate-binding amino acid residues in the active site
cavity (25,41,49,50). Second, the chemical shift variation
of Phe66 upon the addition of a substrate analog was not
very high, as compared to the cases of other putative
substrate-binding amino acid residues in the active site
cavity (52). Third, in light of the present complex struc-
ture, it seems unlikely that the CCA terminus extends so
that the adenine ring of A76 stacks on Phe66, thus pos-
itioning the ester bond between the peptide and A76 near
His20, in the catalytic center (Figure 4B). If the adenine
ring of A76 stacks on Phe66, then this situation would
either locate the ester bond farther away from Phe66, or
cause steric hindrance between the peptide and tRNA
moieties of the substrate. In contrast, in our model, the
ester bond exists near His20, as described in the next
section. Furthermore, we performed a docking simulation
using AutoDock Vina (53). The best result fit well to the
tripeptidyl-A76 moiety of our model (see ‘Supplementary
Materials and Methods’ section and Supplementary
Figure S7). No interaction between the adenine ring and
Phe66 was observed in all of the candidate docking models
suggested by the program.
Hydrolysis reaction of Pth
We found that the configuration around the active site
cavity of the Pth:peptidyl-tRNA complex model described
above is comparable to that of the acyl-enzyme intermedi-
ate of an elastase, in which the C-terminal carboxyl group
of a cleaved peptide is linked via an ester bond to the
catalytic Ser residue (54). That is, in the active site of
Pth (Figure 5A), His20 and Asp93 are present, and a
hydrogen bond is formed between the Nd1 atom of
His20 and the Od1 atom of Asp93, in a similar manner
as the His57 and Asp102 residues of the catalytic triad of
the acyl-enzyme intermediate (Figure 5B). Relative to
these histidine and aspartic acid residues, the ester
groups of the peptidyl-tRNA and the acyl-enzyme inter-
mediate exist in equivalent positions, although these histi-
dine and aspartic acid residues are on opposite sides,
relative to their ester bond planes. Hence, we could
model a water molecule that can nucleophilically attack
the ester carbon of the peptidyl-tRNA, in a similar
manner as the acyl-enzyme intermediate. Thereby, this
water molecule formed a hydrogen bond with the Ne2
atom of His20 in the Pth:peptidyl-tRNA complex model.
In the acyl-enzyme intermediate, the main chain amide
nitrogens of the Gly193 and Ser195 residues form
hydrogen bonds with the ester carbonyl oxygen in the
oxyanion hole. Similarly, in the complex model, the Nd2
atoms of Asn68 and Asn114 form hydrogen bonds with
the ester carbonyl oxygen of the peptidyl-tRNA.
In addition to the structural similarity with the
acyl-enzyme intermediate, a previous enzymatic analysis
demonstrated that His20 acts as the catalytic base in the
hydrolysis reaction of Pth, and Asp93 contributes to the
lowering of the pKavalue of this His20 (50). Furthermore,
a site-directed mutagenesis study revealed that single
residue substitutions of Asn68 and Asn114 with alanine
caused considerable changes in the kcat values by two
orders of magnitude, whereas the Kmvalues were compar-
able to that of the wild-type enzyme (49). Thus, as also
described by Goodall et al. (50), these data suggest that
Pth catalyzes peptidyl-tRNA hydrolysis by following a
reaction mechanism similar to that of the acyl-enzyme
intermediate of an elastase: Asp93 stabilizes the basic
form of His20, such that it will accept a proton from a
proximal water molecule, which then nucleophilically
attacks the ester carbon of the peptidyl-tRNA and gener-
ates the tetrahedral oxyanion intermediate. This inter-
mediate is stabilized by the hydrogen bonds from Asn68
and Asn114. The decomposition of the tetrahedral
general acid catalysis by the Asp93-polarized His20,
producing the peptide and the tRNA.
In conclusion, we have determined the crystal structure of
E. coli Pth in complex with the tRNA CCA-acceptor-T?C
domain, the enzyme-binding region of the tRNA moiety
of the substrate. The structure revealed the amino acid
Figure 5. Peptidyl-tRNA binding and catalysis. (A) Active site of the Pth:peptidyl-tRNA complex model. Stick models of Pth (green), A76 (yellow)
and the peptide moiety (orange) are represented on a transparent ribbon model of Pth. (B) Active site of the acyl-enzyme intermediate of an elastase
(PDB accession code 1HAX) (54). Stick models of elastase (green) and the peptide (orange) are represented on a transparent ribbon model of
elastase. In (A) and (B), hydrogen bonds are indicated with dashed lines. The water molecules, which are poised to nucleophilically attack the ester
carbon, are represented by red spheres.
Nucleic Acids Research, 2012,Vol.40, No. 2010529
residues involved in the tRNA recognition and the mech-
anism by which Pth accepts the diverse sequences of the
elongator-tRNAs as substrate components. Furthermore,
we presented the authentic Pth:peptidyl-tRNA complex
model and the precise mechanism for the hydrolysis
reaction, based on the present crystal structure and the
previous studies’ results. This study represents a signifi-
cant advancement toward the complete understanding of
the reaction mechanism of Pth.
The structure factors and coordinates of the model have
been deposited in the Protein Data Bank (PDB accession
Supplementary Data are available at NAR Online:
Supplementary Materials and Methods, Supplementary
Results and Discussion, Supplementary Figures 1–7, and
Supplementary References [55–60].
The authors thank Dr Shoji Odani and Dr Tomohiro
Miyoshi of Niigata University for their helpful advice
about this study. The synchrotron-radiation experiments
were performed at BL41XU of SPring-8 with the approval
of JASRI (Harima, Japan) (Proposal No. 2008B2182).
The synchrotron-radiation experiments were also per-
formed at BL17A of PF and at NW12A of PF-AR with
the approval of KEK (Tsukuba, Japan) (Proposal No.
Grant-in-Aid for Young Scientists (Start-up) [20870018 to
K.I.] and Grant-in-Aid for Young Scientists (B) [21770108
to K.I.] from the Japan Society for the Promotion of
Science (JSPS); the Uchida Energy Science Promotion
Foundation grant [22-1-10 to K.I.]; UNION TOOL CO
grant (to K.I.); Grant for the Promotion of Niigata
Grants-in-Aidfor Education and Research at the
Institute of Science and Technology from Niigata
University (to K.I.). Funding for open access charge:
Conflict of interest statement. None declared.
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