doi:10.1016/j.jmb.2007.06.053
J. Mol. Biol. (2007) 372, 186–193
Structural Plasticity and Enzyme Action: Crystal
Structures of Mycobacterium tuberculosis
Peptidyl-tRNA Hydrolase
M. Selvaraj 1 , Siddhartha Roy 1 , N.S. Singh 2 R. Sangeetha 2
Umesh Varshney 2 and M. Vijayan 1 ⁎
1
Molecular Biophysics Unit,
Indian Institute of Science,
Bangalore 560012, India
2
Microbiology and Cell Biology
Laboratory, Indian Institute of
Science, Bangalore 560012,
India
Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the
attached peptide in peptidyl-tRNA in order to avoid the toxicity resulting
from its accumulation and to free the tRNA available for further rounds in
protein synthesis. The structure of the enzyme from Mycobacterium
tuberculosis has been determined in three crystal forms. This structure and
the structure of the enzyme from Escherichia coli in its crystal differ
substantially on account of the binding of the C terminus of the E. coli
enzyme to the peptide-binding site of a neighboring molecule in the crystal.
A detailed examination of this difference led to an elucidation of the
plasticity of the binding site of the enzyme. The peptide-binding site of the
enzyme is a cleft between the body of the molecule and a polypeptide
stretch involving a loop and a helix. This stretch is in the open conformation
when the enzyme is in the free state as in the crystals of M. tuberculosis
peptidyl-tRNA hydrolase. Furthermore, there is no physical continuity
between the tRNA and the peptide-binding sites. The molecule in the E. coli
crystal mimics the peptide-bound enzyme molecule. The peptide stretch
referred to earlier now closes on the bound peptide. Concurrently, a channel
connecting the tRNA and the peptide-binding site opens primarily through
the concerted movement of two residues. Thus, the crystal structure of
M. tuberculosis peptidyl-tRNA hydrolase when compared with the crystal
structure of the E. coli enzyme, leads to a model of structural changes
associated with enzyme action on the basis of the plasticity of the molecule.
© 2007 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: protein synthesis; peptide-binding site; tRNA-binding site; open
and closed conformation; mobility of active site
Introduction
The translating ribosome often prematurely releases peptide as peptidyl-tRNA, accumulation of
which is toxic to the cell.1,2 Such accumulation could
also result in non-availability of free tRNA for
protein synthesis. Such a situation is prevented by
the action of peptidyl-tRNA hydrolase (Pth), which
hydrolyzes the ester link between the peptide and
the 2′ or 3′-OH group of the sugar at the 3′ end of
tRNA.3 Pth was first identified in E. coli.4–6 HomoAbbreviations used: Pth, peptidyl-tRNA hydrolase;
EcPth, Pth from E. coli; CRS2, chloroplast RNA splicing 2.
E-mail address of the corresponding author:
[email protected]
logues of this protein, often referred to as Pth1, exist
in bacteria. A different protein, referred to as Pth2,
exists in archaea. Eukaryotes possess multiple Pth
activities. The structure of the Pth from E. coli (EcPth)
has been determined by X-ray crystallography.7 The
structural work has been supplemented by detailed
mutational and other biochemical investigations.7–10
These studies have provided a reasonable understanding of the regions of the molecule and the
residues involved in peptide and tRNA binding. It
has been shown that peptidyl-tRNA containing a
tetrapeptide or a longer peptide is the best substrate
of the enzyme.11,12 The structures of a couple of
archaeal Pth molecules13,14 and that of a similar
human enzyme15 have been determined. Their structures are substantially different from that of the enzyme from E. coli.
0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.
187
Plasticity and Action of M. tuberculosis Pth
We have been pursuing a program on the structural biology of proteins from Mycobacterium tuberculosis and the related Mycobacterium smegmatis,16–22
for contributing to the understanding of the biology
of the pathogen and with a view to elucidating the
structures of possible drug targets for the design of
inhibitors. As part of this program, the structure of
Pth from M. tuberculosis (MtuPth) has been determined in three crystal forms. Although the overall
structure of MtuPth is similar to that of EcPth,
substantial differences exist in the structures, as
observed in the EcPth crystals and the MtuPth
crystals, primarily on account of the binding of the C
terminus of the protein to the peptide-binding site of
a neighboring molecule in EcPth crystals. These
differences have been shown to represent the
structural plasticity of the binding regions of the
enzyme. On the basis of this plasticity, we propose a
model for structural changes during enzyme action
involving the opening and closing of a channel
connecting the peptide and tRNA-binding sites, and
the movement of a structural element involving a
loop and a helix.
Result and Discussion
Molecular structure
The structure of MtuPth has been determined in
three crystal forms at resolutions of 1.98 Å, 2.35 Å
and 2.49 Å using the molecular replacement method
(Table 1). Form I and form III are orthorhombic,
P212121, with one molecule in the asymmetric unit.
The two have similar cell parameters, and they differ
mainly in the lower solvent content of form III. Form
II also has similar cell parameters, but is monoclinic,
P21, with two molecules in the asymmetric unit. The
C-terminal stretch varying in length from 9–12 residues in the 191 residue polypeptide chain could
not be located in electron density maps. Thus, the
number of residues defined in the four crystallographically independent molecules varied between
179 and 182. The structures of the four molecules
are remarkably similar. The r.m.s.d. in Cα positions
when pairs of them are superimposed varies from
0.2–0.5 Å.
As in the case of the homologous EcPth, MtuPth
adopts an α/β fold with a twisted β-sheet flanked by
helices (Figure 1). The mixed β-sheet has four
parallel strands in the middle. This feature is flanked
by anti-parallel strands. Another strand, parallel
with the central four, completes the sheet, which is
sandwiched between two longish α-helices on one
side (α1 and α5) and two on the other (α2 and α3).
The remaining short helix (α4) forms part of a mobile
element.
EcPth and MtuPth are homologous with a sequence identity of 38% and, as shown by rescue of E.
coli AA7852 (pthts), growth at the non-permissive
temperature of 42 °C, in the presence of a plasmidborne copy of MtuPth, the mycobacterial enzyme is
Table 1. Data collection statistics and refinement
parameters
A. Data collection
Space group
Unit-cell parameters
a (Å)
b (Å)
c (Å)
β (deg.)
VM (Å Da−3)
Solvent content (%, v/v)
No. subunits/
asymmetric unit
Resolution limits (Å)
Highest shell (Å)
No. measured
reflections
No. unique reflections
Completeness (%)
Rsyma
Average I/∑(I)
Multiplicity
Form I
Form II
Form III
P212121
P21
P212121
36.30
61.85
73.97
–
2.1
41
1
35.83
73.76
59.79
92.35
2
38
2
35.84
57.06
72.59
–
1.9
34
1
30.0–1.98
2.04–1.98
81,818
30.0–2.35
2.43–2.35
37,029
30.0–2.49
2.58–2.49
22,020
11,461 (1174) 12,613 (1236)
95.2 (99.8)
96.8 (95.4)
7.7 (48.7)
11.8 (41.1)
22.7 (3.7)
9.05 (2.7)
7.1
2.9
B. Refinement and model statistics
R-factor (%)
22.5
25.1
Rfree (%)
No. atoms/asymmetric unit
Protein
1337
Water
180
Chloride
–
r.m.s.d. from ideal
Bond lengths (Å)
0.006
Bond angles (deg.)
1.3
Dihedral angles (deg.)
23.5
Improper angles (deg.)
0.93
Ramachandran plot
statisticsb
Most favoured
93.8
regions (%)
Additionally allowed
6.2
regions (%)
Generously allowed
0.0
regions (%)
Disallowed regions (%)
0.0
5428 (522)
98.2 (95.0)
8.2 (38.4)
15.14 (3.6)
4.1
19.6
24.8
19.8
24.1
2701
328
–
1342
78
1
0.008
1.4
24.0
0.94
0.007
1.4
23.9
0.85
93.2
90.5
6.4
9.5
0.3
0.0
0.0
0.0
a
Rsym = ∑h∑l∣Ihl–bIhN∣/∑h∑l bIhN, where Il is the lth observation of reflection h and bIhN is the weighted average intensity for
all observations l of reflection h.
b
Calculated for non-glycine and non-proline residues using
PROCHECK.
functional in E. coli. In fact, the rescue by MtuPth is
indistinguishable from that by a plasmid-borne
copy of wild-type EcPth. As controls, the empty
vector (pACDH) does not support growth of E. coli
AA7852 at non-permissive temperature and the
strain grows at the permissive temperature of 30 °C,
irrespective of the nature of the plasmid. The
homology between the M. tuberculosis and E. coli
proteins is reflected in the three-dimensional structure as well (Figure 1). When the EcPth molecule is
superimposed on the four independent MtuPth
molecules, 169–173 pairs of Cα positions superimpose with r.m.s.d. ranging from 1.3–1.5 Å with a
maximum deviation in the range of 3.1–4.0 Å. The
remaining pairs deviate by greater extents. The two
N-terminal residues invariably belong to this set.
Perhaps more interesting is the substantial deviation
in the C-terminal residues. In EcPth, three residues
188
Plasticity and Action of M. tuberculosis Pth
with EcPth. Also, amino acid residues essential for
Pth activity are conserved in it, yet it is incapable of
rescuing a pthts E. coli strain for reasons that are not
clear. Indeed, as might be expected from the extent
of similarity among the three sequences, the threedimensional structure of CRS2 is closer to that of
MtuPth than to that of EcPth. The C terminus of
CRS2 does not interact with the peptide-binding
region of a neighboring molecule and is disordered
as in MtuPth.
Plasticity and enzyme action
Figure 1. Superposition of Cα positions in MtuPth in
form 1 (blue) and EcPth (brown). For clarity, the C-terminal
helix of EcPth, which is not defined in MtuPth crystals, has
been removed. Residue numbering is according to the
MtuPth sequence. The secondary structural elements are
labeled from the N terminus to the C terminus.
at the terminus are locked into the binding site of a
neighboring molecule in the crystal. Consequently,
the C-terminal stretch, which contains a helix, is
fully ordered. This does not happen in any of the
crystals of MtuPth. Consequently, the C-terminal
residues are not defined in the molecule. The Cterminal region of even well-ordered residues
exhibit considerable deviations from the position
of the corresponding residues in EcPth. Apart from
the residues at the N and C termini, the central
region of the 136–150 stretch in MtuPth deviates
substantially from its position in EcPth, with a
maximum deviation of close to 9 Å in Cα positions.
As discussed later, this deviation is of considerable
functional significance. Deviations of lesser amount
are exhibited by other loops as well. Some of them
are again functionally important. Also noteworthy is
the shift in α5, presumably as a result of the
movement in the contiguous C-terminal stretch.
Thus, deviations between EcPth and MtuPth are
most pronounced in the C-terminal region beyond
residue 136 and in the two N-terminal residues. The
remainder of the molecule, 134 residues, in EcPth
and MtuPth (form I), superimpose with an r.m.s.d.
in Cα positions of 0.93 Å.
Another protein that is structurally homologous
to MtuPth, and indeed to EcPth, is the functionally
unrelated chloroplast RNA splicing 2 (CRS2).23 It
has a sequence identity of 40% with MtuPth and 32%
The major difference between MtuPth and EcPth is
in the region believed to be involved in peptide and
tRNA binding. In the crystal structure of the E. coli
enzyme, the binding cleft, bound by the body of the
molecule and the 136–150 residue stretch containing a
loop and a short helix, is occupied by the C-terminal
stretch of a neighboring molecule (Figure 2). Three
residues of this stretch interact with Asn12, Tyr17,
Asn70 and Asn116 (residue numbering as in
MtuPth). On the basis of detailed structural and
biochemical considerations, this interaction has been
assumed to represent the formation of a complex
between Pth and the peptide segment adjacent to the
CCA end of the peptidyl-tRNA substrate.7 Interestingly, this interaction involves only the main-chain
atoms of the three residue segment in the stretch and
is therefore sequence-independent.
The crystal structure of MtuPth is entirely different
and does not involve the intermolecular interaction
referred to above. The most pronounced difference
between the molecular structures of MtuPth and
EcPth is in the orientation of the loop composed of
residues 136–150 (Figures 1 and 2). It is closed over
the bound peptide segment in EcPth. In the absence
of the bound peptide, the loop has an open
conformation in MtuPth. This movement also has
direct relevance to tRNA binding. Lys142, which
occurs at the middle of the loop and has been shown
to be involved in interaction with tRNA, points
towards the binding region in E. coli (Figure 2).7 The
corresponding residue, Arg143, points to the solution in nearly the opposite direction in MtuPth.
Another important difference between the structures of EcPth and MtuPth is in the location of Asp98
and Gly113 (Figure 3). The minimum interatomic
distance between the two residues is 6.7 Å in EcPth,
while it varies between 3.1 Å and 3.4 Å in the
different crystal forms of MtuPth. In fact, Asp98 and
Gly113 are in contact with each other in the MtuPth
structures. In EcPth, a channel spans the peptide and
the tRNA-binding regions; the channel is closed in
MtuPth (Figure 4). Gly113 and Asp98 are roughly at
the boundary between the peptide and the tRNAbinding regions. Residues Asn12, Tyr17, Asn70 and
Asn116, which are important in peptide binding, are
on one side of the boundary; residues Lys107 and
Arg134 are important in tRNA binding and are on
the other side of the boundary (Figure 3).7,9 The
catalytic residue His22 is close to this boundary. It
has been suggested that Lys107 and Arg134 could
Plasticity and Action of M. tuberculosis Pth
189
Figure 2. A stereo view of the binding region of MtuPth in form I (blue), superimposed on the same region of EcPth
(brown) using Cα positions. A few critical residues are shown in stick representation. The location of the C-terminal
tetrapeptide of the neighboring molecule in the crystal structure of EcPth is represented by main-chain atoms shown as
spheres.
function as an ”ion clamp”.9 These residues are surrounded by basic residues, such as His94, Arg103,
Arg105 and Arg132, thus providing a positively
charged patch for interaction with tRNA. Mutational studies of the E. coli enzyme have shown that
most of these residues need to have a basic sidechain for efficient tRNA binding.9
The above observations provide a model for the
mobility of the protein associated with enzyme
action. The binding of the substrate peptide leads to
the closure of the cleft through the inward movement of the 136–150 stretch as in EcPth. Concurrently, Asp98 and Gly113 move apart, generating a
channel spanning the peptide and tRNA-binding
regions. Peptidyl-tRNA can now bind to the
enzyme. Thus, the concerted movement deduced
from the structure is necessary to enable peptidyltRNA to bind to Pth. Some of the critical residues
involved in the concerted motion are illustrated in
Figure 5, as they appear in the structures of MtuPth
and EcPth. In MtuPth, which represent the free
enzyme, the guanidyl group of Arg139 (the corresponding residue is histidine in EcPth) interacts with
the carboxylate group of Asp98, which keeps the
aspartyl residue in the closed position. As can be
deduced from the location of the histidyl residue in
EcPth, which represents the holoenzyme, this saltbridge breaks when the 136–150 stretch closes on the
bound peptide. The aspartyl residue moves to a new
position at which it can form, as simple modeling
indicates, a salt-bridge with the new position of
Arg139. Concurrently, another movement is triggered by a conformational change in Asn116. In
MtuPth, the side-chain carbonyl oxygen atom of this
residue is hydrogen bonded to the side-chain of the
catalytic His22. Upon peptide binding, the residue
undergoes a conformational change to enable it to
interact with the bound peptide. This triggers a
movement in the 113–116 stretch. In MtuPth, Nδ of
His115 can form a hydrogen bond with the carbonyl
oxygen atom of Asp95. In EcPth, the imidazole ring
flips by about 180° and can no longer form this
hydrogen bond. Along with His115 and Asn116,
Gly113 also moves away from the 95–98 stretch. The
net result of these movements is to widen the
channel connecting the peptide and the tRNAbinding regions and remove the block caused by
the close proximity of Asp98 and Gly113.
A comparison of available bacterial Pth sequences
suggests that the above model is likely to be
applicable to all of them. Among the residues
involved in peptide binding, Asn12, Asn70 and
Asn116 are totally conserved, while Tyr17 is
replaced only by phenylalanine. Residues 107 and
134 involved in tRNA binding are lysine or arginine
in almost all sequences. The catalytic residue His22
190
Plasticity and Action of M. tuberculosis Pth
Figure 3. Location of important residues in the binding region of (a) MtuPth and (b). EcPth. The difference in the
mutual disposition of Asp98 and Gly113 (represented as van der Waals spheres) is highlighted.
Figure 4. The binding region (magenta) in a surface representation of (a) MtuPth and (b) EcPth. The channel
connecting the peptide and the tRNA binding regions is clearly discernible.
191
Plasticity and Action of M. tuberculosis Pth
Figure 5. Residues and interactions involved in the concerted motion in MtuPth (blue) and EcPth (brown). Broken
lines indicate hydrogen bonds. Residue numbering is according to the MtuPth sequence.
is totally conserved. Also conserved are Asp95 and
His115, which have been shown to be involved in
catalysis.10 Residues 98 and 113 have essentially a
structural role in closing or opening the channel
connecting the peptide and tRNA-binding regions.
They are Asp and Gly, respectively, in a vast
majority of the sequences. Gly136 and Val150,
which hinge the mobile 136–150 stretch to the rest
of the molecule, are totally conserved.
ligand, reminiscent of loop closure in other proteins
such as, for example, GTP-binding proteins,24 and
β-lactoglobulin.25,26 This closure leads to concerted
movement of several residues, resulting in the
opening of a channel connecting the peptide and
the tRNA-binding sites. The residues involved in
these movements, like those that take part in peptide and tRNA binding, are highly conserved in
bacterial Pth, lending further support to the proposed model.
Concluding remarks
The difference in the molecular packing in the
crystal structures of MtuPth and EcPth leads to a
proposal on the structural changes associated with
ligand binding to the enzyme. The molecules in the
EcPth crystal represent the peptide-bound state on
account of the interaction of the C-terminal residues
with the binding site of a neighboring molecule,
while those in the MtuPth crystals, which exhibit an
entirely different mode of packing, represent the free
enzyme. Peptide binding is accompanied by the
closure of a peptide stretch of the enzyme on the
Materials and Methods
Structure determination and analysis
The recombinant protein was expressed, purified,
crystallized and data collected from the crystals as
described.27 The structure of MtuPth was solved using
molecular replacement employing PHASER.v.1.2.28 The
structure of EcPth (2PTH) was used as the search model.
After initial phasing, the model was subjected to rigid
body, positional, torsional angle dynamics and individual
B-factor refinement using CNS 1.1,29 after omitting 5% of
192
the reflections for Rfree calculations. COOT was used for
modeling.30 Density was seen for the first 179 amino acid
residues out of the 191 during the early stages of model
building. In the course of model building and refinement,
electron density appeared for one to three more residues.
Thus in form I, in molecules A and B in form II and in form
III, the model consists of 179, 180, 182 and 180 residues,
respectively. Water oxygen atoms were identified in the
final stages of refinement based on peaks greater than 2.5σ
in Fo–Fc maps and 0.8σ in 2Fo–Fc maps. Refinement
parameters, along with data collection statistics, are given
in Table 1.
The models were validated using PROCHECK.31 Secondary structure elements were determined from program
STRIDE.32 Structural superpositions were made using
ALIGN.33 Interatomic distances were calculated using
CONTACT from the CCP4 program suite.34 All Figures
for molecular representation were prepared using
PyMol†. CastP was used for calculating the cavities and
pockets on the protein surface.35 CLUSTALW was used for
alignment of the sequences of bacterial Pth.36 The
sequences were taken from the NCBI database.
Plasmids
The pACDH (TcR), an ACYC origin of replication based
plasmid has been described.37 The pACDMtuPth was
generated by subcloning of an NcoI-HindIII fragment
from pET11dMtuPth, into similarly digested pACDH.27
pACDEcoPth was generated by subcloning of the EcPth
open reading frame (NdeI-HindIII) from a pre-existing
ColE1 origin of replication-based (pTrc99C-derivative)
plasmid into a similarly digested derivative of pACDHNdeI (pACDH wherein the NcoI site has been changed to
an NdeI site).
Bacterial strains and growth
E.coli TG1 was used for recombinant DNA techniques.
E. coli AA7852 (a gift from Dr J. Menninger), harboring a
temperature-sensitive allele of the Pth gene, was used to
check for functional complementation by MtuPth. Bacterial cultures were grown in LB medium.38 For growth on
solid medium, agar (final concentration 1.5%, w/v) was
added to LB medium. When needed, tetracycline was
used at a concentration of 7.5 μg/ml.
Protein Data Bank accession codes
The atomic coordinates and the structure factors of all
the three crystal forms of Pth have been deposited in the
RCSB Protein Data Bank with accession codes 2Z2I, 2Z2J,
and 2Z2K).
Acknowledgements
Intensity data were collected at the X-ray Facility
for Structural Biology, supported by the Department
of Science and Technology. Computations were
performed at the Supercomputer Education and
† http://www.pymol.org
Plasticity and Action of M. tuberculosis Pth
Research Centre, and the Bioinformatics Centre and
the Graphics Facility, both supported by the Department of Biotechnology (DBT). The work forms
part of a DBT-sponsored research program. S.R. and
N.S.S. are CSIR research fellows. M.V. is a Distinguished Biotechnologist awardee of the DBT.
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Edited by G. Schulz
(Received 13 April 2007; received in revised form 2 June 2007; accepted 18 June 2007)
Available online 27 June 2007