Title: the cell wall of Mycobacterium tuberculosis: Structure
and Mechanism of L,D-transpeptidase 2
Authors: Sabri B. Erdemli, Radhika Gupta, William R. Bishai,
Gyanu Lamichhane, L. Mario Amzel, Mario A. Bianchet
17 April 2012
26 September 2012
26 September 2012
Cite this article as: Erdemli SB, Gupta R, Bishai WR, Lamichhane G, Amzel L M,
Bianchet MA, the cell wall of Mycobacterium tuberculosis: Structure and Mechanism
of L,D-transpeptidase 2, Structure, doi:10.1016/j.str.2012.09.016
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• The structure of Mtb LdtMt2 shows the binding of a peptidoglycan fragment.
• The structure of the complex maps the interaction between acyl-donor and enzyme.
• Ldts show a conserved C-terminal ykuD domain with a divergent N-terminal domain.
• Based on the structure of this complex a reaction mechanism is proposed.
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Targeting the cell wall of Mycobacterium tuberculosis:
Structure and Mechanism of L,D-transpeptidase 2
Sabri B. Erdemli1,&, Radhika Gupta1§, William R. Bishai§,
Gyanu Lamichhane§, L. Mario Amzel&,* and Mario A. Bianchet#,&,*
#Department of Neurology, &Department of Biophysics and Biophysical Chemistry, and
§Center for Tuberculosis Research, Division of Infectious Diseases, Johns Hopkins
University School of Medicine, Baltimore, MD, USA
1 Authors contributed equally to this work
*Corresponding authors: email@example.com, firstname.lastname@example.org
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With multi-drug resistant cases of tuberculosis increasing globally, better antibiotic drugs and
novel drug-targets are becoming an urgent need. Traditional β-lactam antibiotics that disrupt
the D,D-transpeptidases are not effective against mycobacteria, in part because mycobacteria
rely mostly on β–lactam insensitive L,D-transpeptidases for biosynthesis and maintenance of
their peptidoglycan layer. This reliance plays a major role in drug-resistance and persistence of
Mycobacterium tuberculosis (Mtb) infections. The crystal structure at 1.7 Å resolution of the
Mtb L,D-transpeptidase LdtMt2 containing a bound peptidoglycan fragment, reported here,
provides information about catalytic site organization as well as substrate recognition by the
enzyme. Based on our structural, kinetic, and calorimetric data, we propose a catalytic
mechanism for LdtMt2 in which both acyl-acceptor and acyl-donor substrates reach the catalytic
site from the same, rather than different, entrances. Together, this information provides vital
insights for the development of novel drugs targeting this validated yet unexploited enzyme.
by L,D-transpeptidases such as the one studied here. These enzymes transfer the peptide bond
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Multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium
tuberculosis (Mtb) are emerging at alarming rates (Fauci, 2008). A possible reason for this is
poor patient compliance with existing treatments, which require multiple drugs to be taken
daily for at least 6 months (ATS, 1997) to eliminate the resilient ~1% of bacilli, commonly known
as persisters (Jindani et al., 2003; Lewis, 2007). Any strategy aimed at shortening duration of
tuberculosis therapy must be effective in eliminating the persisters. Mtb persists in the lungs of
infected individuals in a slow growing, non-replicating dormant state in which the bacillus is
resistant to the host immune response and is transiently tolerant of antibiotics (Betts et al.,
2002; Bishai, 2000). Mtb persisters extracted from lung lesions show morphological changes
similar to those observed in bacilli that are subject to nutrient starvation or anaerobic stress
(Betts et al., 2002). These changes involve, among other physiologic alterations, the remodeling
of the Mtb peptidoglycan layer (Lavollay et al., 2008), a vital layer consisting of an elaborate
network of peptidoglycan chains with cross-linked peptide stems.
Formation of most common type of cross-link, the (D,D) 4?3 linkage (Fig. 1A: top), is
catalyzed by the D,D-transpeptidase activity of enzymes commonly referred to as penicillin
binding proteins. These enzymes catalyze the transfer of the peptide bond between the fourth
residue (D chiral-center) and the fifth residue of a pentapeptide donor stem to a side chain
amide group (also a D chiral-center) at the third residue of an adjacent acceptor stem (usually a
di-amino acid). A second type of cross-link, the (L,D) 3?3 linkage (Fig. 1A: bottom), is catalyzed
between the third residue (L chiral-center) of a tetrapeptide donor stem to the side chain amide
overexpression of the enzyme better represents the peptidoglycan layer of Mtb persisters.
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group of the third residue (D chiral-center) of an adjacent acceptor-stem. In both types of
transpeptidases, the catalysis proceeds by a two-step mechanism: acylation of the enzyme by
the penultimate peptide of the donor-stem with the release of the stem C-terminal residue,
followed by de-acylation of this acyl-enzyme intermediate by an acceptor stem.
The 3?3 linkages were first identified In 1974 by Wietzerbin et al. in the peptidoglycan of
mycobacteria (Wietzerbin et al., 1974), but their significance and biosynthetic pathway
remained unknown until recently, when their predominance in Mtb was demonstrated and
LdtMt1 (or MT0125, the product of gene Rv0116c) was identified as an L,D-transpeptidase that
generates 3?3 linkages (Lavollay et al., 2008). Frequently associated with virulence and β-
lactam resistance, predominance of 3?3 linkage has been observed in spontaneous mutation of
E. faecium (Mainardi et al., 2005), in other mycobacteria M. abscessus (Lavollay et al., 2011),
and in the spore-forming bacteria Clostridium difficile (Peltier et al., 2011).
Recently, another 3?3 L,D-transpeptidase, LdtMt2 (MT2594, the product of gene Rv2518c),
was characterized in Mtb (Gupta et al., 2010). An Mtb strain lacking LdtMt2 loses virulence and
has attenuated growth during the chronic phase of the disease (Gupta et al., 2010). In addition,
this strain is more susceptible to the therapeutic combination of amoxicillin and clavulanic acid,
suggesting that the (3?3) L,D-transpeptidase activity is a major contributor to β-lactam
resistance. It was also shown that a vaccine utilizing an M. bovis BCG strain that overexpresses
L,D-transpeptidases has enhanced protective efficacy against Mtb persisters (Nolan and
Lamichhane, 2011), suggesting that the higher degree of 3?3 linkages resulting from the
Kinetic measurements show that LdtMt2 has residual hydrolase-activity towards certain β-
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Despite the obvious significance of L,D-transpeptidases in Mtb growth and virulence, no
structural or binding information exists for the five members of this class of Mtb enzymes
identified to date (Gupta et al., 2010). Here, we present the characterization of the
extramembrane portion of LdtMt2 (ex-LdtMt2), including the determination of its structure at 1.7-
Å resolution. Ex-LdtMt2 is composed of two domains: an N-terminal immunoglobulin–like domain
and a C-terminal catalytic ErfK/YbiS/YhnG domain (Pfam accession number PF03734). Based on
this structure, comparative modeling of the identified Mtb homologues indicates that the N-
terminal domain fold and the enzyme’s overall conformation distinguish this group from other
structurally characterized ErfK/YbiS/YhnG-domain-containing proteins such as Bacillus subtilis
ykuD (Bielnicki et al., 2006) and Enterococcus faecium L,D-transpeptidase Ldtfm (Biarrotte-Sorin
et al., 2006).
The catalytic site of LdtMt2, located at the entrance of a narrow tunnel connecting two
cavities that open to the solvent, is similar to that observed in Ldtfm (Biarrotte-Sorin et al.,
2006). Unexpectedly, the crystal structure shows the presence of a fragment of the
peptidoglycan of the expression host (E. coli) in the catalytic site of the enzyme. Until now, no
related L,D-transpeptidase has been structurally characterized with a bound peptidoglycan
fragment. LdtMt2 recognizes the peptidoglycan (acyl-donor-like) using residues located in both
cavities, including the catalytic residues Cys354 and His336. The observed mode of binding of
this short peptidoglycan fragment led us to propose a catalytic mechanism for these
transpeptidases that differs from that previously posited (Biarrotte-Sorin et al., 2006).
lactams. Isothermal titration calorimetry experiments show that the carbapenems (Bonfiglio et
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al., 2002), imipenem and meropenen, bind only the catalytically active enzyme and they do so
with apparent submicromolar affinities. The structural, biochemical, and thermodynamic data
presented here provide vital information about the interactions of the enzyme with its
substrates and inhibitors that will help advancing this unexploited enzyme into a target for the
design of novel compounds for the treatment of Mtb infections.
1979), and a C-terminal catalytic domain (CD, residues 254 to 407), consisting of a β-sandwich
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Ex-LdtMt2 crystal structure
The extramembrane portion of the L,D-transpeptidase from M. tuberculosis, LdtMt2 (ex-LdtMt2,
residues 120 to 408), was cloned and expressed in E. coli. A crystal of a seleno-methionine
(SeMet) derivative of ex-LdtMt2 was used to determine the structure using Multiple Anomalous
Dispersion (MAD) methods. The structure was refined to a final Rwork of 0.19 and an Rfree of 0.23
using diffraction data collected from a di-μ-Iodo-bis[ethylenediamine]-di-Pt[II] (PIP) derivative
crystal (Fig. 1B and Table 1; see also Fig S1), which had the best diffraction among all the
crystals tested (1.7 Å resolution). Crystals of ex-LdtMt2 belong to the orthorhombic space group
I212121 and contain two monomers (A and B) in the asymmetric unit. These two molecules do
not correspond to a physiological dimer, since ex-LdtMt2 behaves as a monomer in solution as
determined by size exclusion chromatography (data not shown). In addition, areas buried by the
two possible dimers in the crystal asymmetric unit, 810 Å2 and 1360 Å2, are not consistent with a
dimer stable in solution (Krissinel, 2011).
A portion of the N-terminal region of ex-LdtMt2, spanning residues 122 to 132, is observed
only in monomer B with low occupancy (50%) and disconnected from the bulk of the fold.
Aminoacids from 150 to 407 are observed in both monomers of the crystal asymmetric unit with
an rms deviation of 0.31 Å between the aligned 258 Cα-atoms of monomers A and B. Each
monomer consists of two globular domains: an N-terminal domain (residues 150 to 250) folded
as an anti-parallel β-barrel resembling an immunoglobulin domain (IgD) (Amzel and Poljak,
with two mixed β-sheets characteristic of the ErfK/YbiS/YhnG-fold (Bielnicki et al., 2006). A
et al., 2006; Dodson and Wlodawer, 1998).
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short linker (residues 251 to 253) joins the two domains (Fig. 1B). A small C-terminal sub-
domain (CTSD; residues 379 to 407) extends the ErfK/YbiS/YhnG-fold. In this sub-domain,
Trp394 and two Trp residues of the C-terminal helix α3 (398 and 401) make a zipper-like
interaction with the IgD N-terminal domain that fixes the relative orientation of the two
The N-terminal domain has characteristics not commonly found in Ig-domains (Amzel and
Poljak, 1979): the interstrands loops are unusually long, including a three-turn helix (α1) in the
loop connecting strands β3 and β4.
In the refined structure, PIP was found to have reacted with solvent exposed Nδ atoms of
histidine residues and with sulfur atoms of methionine residues (His214 in monomer A, and
Met153, Met157 Met237 and His347 in both monomers), to form five (four in monomer B) iodo-
Pt(II) ethylenediamine adducts (Fig. S1) (O'Halloran et al., 1987).
A two-step enzymatic mechanism resembling that of D,D-transpeptidases was proposed for the
ErfK/YbiS/YhnG-fold family of transpeptidases, except that, the catalytic serine of D,D-
transpeptidases is replaced by an absolutely conserved cysteine residue (Mainardi et al., 2007).
A highly conserved sequence motif of this group of L,D-transpeptidases, HXX14-17[S/T]HGChN
(where 'h' stands for a hydrophobic residue, Fig. 2) contains three residues (in bold letters)
analogous to the catalytic triad of cysteine-proteases: a cysteine, a histidine, and a third residue
that accept an H-bond from the histidine stabilizing the competent imidazole tautomer (Bielnicki
transpeptidase activity has been reported (Gupta et al., 2010). Of these proteins, only
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In ex-LdtMt2, those catalytic residues, His336, Ser337, and Cys354, reside under a flap formed
by a long insert (residues 299 to 323) that is not part of the canonical ErfK/YbiS/YhnG-fold
(Bielnicki et al., 2006). This insert, strand β15, loop LF and strand β16, folds over the β-sheet
formed by strands β17, β18 and β19 (residues 324 to 357). The closed flap creates two large
cavities (vestibules) connected by a narrow tunnel. These cavities are open to solvent, one at
the end of the molecule, and the other at the interface between the CD and IgD (Fig. 1C, outer
and inner cavities, respectively). Strand β16 (residues 318 to 323), the C-terminal end of β18
(residues 336 and 337), the loop Lc (residues 338 to 352) and the strand β19 (residues 353 to
357) line the outer cavity (Fig. 1D). The residues of the conserved motif (Fig. 2) are readily
accessible through the outer cavity, with the catalytic residues located deep within it. With the
exceptions of His336 and Ser337 (both in β18), the conserved motifs extend through the end of
loop Lc and strand β19 . Ser351, His352, and Gly353 are located in loop Lc while Cys354 and
Asn356 in strand β19 (Fig. 1D). Other highly conserved residues in similar sequences of gram-
positive bacteria, Tyr318 and Gly332 in LdtMt2 (Fig. 2), are located at the inner cavity. Tyr318 and
Met303 line the internal side of the flap and, together with Cys354, form the walls of the narrow
tunnel. His336 and His352 flank the tunnel at the outer cavity (Fig. 1D). The carbonyl group of
Ser337 accepts a H-bond from the Nδ of His336 (2.8 Å). This H-bond stabilizes the tautomer of
His336 protonated at Nδ.
Comparison of LdtMt2 with Mtb paralogs
The presence of five genes in Mtb that code for proteins with potential L,D-
LdtMt1 (Lavollay et al., 2008) and LdtMt2 (Gupta et al., 2010) have been shown to be functional
bound from the lysis supernatant and carried through the purification process. Ex-LdtMt2
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L,D-transpeptidases. The multiple alignment of LdtMt2 with the other Mtb proteins shows
pairwise sequence identities range from 33 % to 45 % (Fig. 2), suggesting a high structural
similarity of these proteins to ex-LdtMt2. Comparative models of these proteins were built using
LdtMt2 as a template (Fig. 3). The overall fold of the IgD is conserved among these proteins. Three
homologues, MT0202 (also know as Rv0192, 45% identity with LdtMt2), LdtMt1 (36 %) and
MT1477 (Rv1433, 33 %), have a short loop connecting strands β7 and β10 and lack strands β8 and
β9 of the ex-LdtMt2 IgD (Fig. 2 and Fig. 3A). The bulk of the CD has little variation among
homologues; MT0501 (Rv0483, 34 %) has the shortest loop LF among the five proteins and
MT1477 shows an eighteen-residue insertion after β12. The C-terminal sub-domains of these
homologues, however, show significant differences with LdtMt2. The only exception is MT0501,
which contains a CTSD of the same size and features as LdtMt2. MT1477 and LdtMt1 completely
lack this sub-domain and MT0202 has a significantly shortened version. Most of the motif
residues (Fig. 2) are conserved in these proteins, with the exception of the replacement of His
352 (LdtMt2 numbering; Fig. 2) by an asparagine in MT0501. In the neighborhood of the active
site a methionine in MT0501 replaces the highly conserved Trp340 of LdtMt2 (Fig. 2 and Fig. 3B).
Electron density not belonging to protein residues was present in the catalytic site of the
SeMet and the PIP derivative crystals. This density, longer and more branched than either a
succinic acid or a PEG molecule from the crystallization media, was interpreted as belonging to a
di-peptide fragment of a muropeptide stem, probably a fragment of the E. coli peptidoglycan
probably recognizes common features between its physiological substrate and a muropeptide of
purifying the protein under denaturing conditions; see Supplemental Experimental Procedures),
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E. coli. The peptidoglycan of Mtb contains muropeptides composed of an N-acetylglucosamine
joined by a β (1-4) glycosidic bond to an N-acetylmuramic acid decorated with 4 or 5 aminoacid-
long peptide stems (NAcGlc-β[1,4]-MurNAc-L-Ala1-γ-D-GLu2-m-A2pm-NH2
(Barreteau et al., 2008). The electron density, 14 Å in length, broadens at three places that
correspond to carboxylates of the peptidoglycan (Fig. 4 A, see also Fig. S2). The broadest
section, at the center of the catalytic site, is connected by narrow zig-zag densities to two broad
densities located at the tunnel entrances across from the active site tunnel. Based on the
separation and size of these features, a γ-D-Glu-m-A2pm dipeptide was built into this electron
density (Fig. 4A), with the terminal peptide L chiral-center placed near the catalytic His336 and
the D chiral-center in the inner cavity of the tunnel, hydrogen-bonded by Tyr318 and main-chain
carboxyl of Gly332. The SH group of Cys354 is 4.6 Å from the carboxylate group of the dipeptide
A2pm moiety (Fig. 4A). This carboxylate makes H-bonds with the Nε of His336 and the Nδ1 of
Asn356, both residues of the conserved motif (Fig. 2). The γ-D-Glu-m-A2pm dipeptide may
correspond to the second and third peptide moieties of a peptidoglycan stem.
Chemical modification of Cys354 in the absence of ligand
During the structural analysis, several of the structures of different native crystal forms showed
modifications of Cys354 by buffer components or molecular oxygen (Fig. 4B and C; Table S1).
When β-mercaptoethanol (β-Me) is present, a disulfide bridge is formed between the Sγ of
Cys354 and β-Me (Fig. 4B). The rest of the molecule remains unperturbed (rms 0.17 Å for 524
Cα’s of the crystal asymmetric unit). In protein crystals without endogenous ligand (obtained by
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Cys354 is in its sulfenic acid form (Fig. 4C). The rest of the structure, including the active site
does not show significant changes (rms 0.23 Å for 524 Cα’s of the crystal asymmetric unit).
Binding of carbapenems to wild type and C354A LdtMt2
Two classes of antibiotics inhibit polymerization of the cell wall peptidoglycan: β-lactams and
glycopeptides. Carbapenems are a subfamily of β-lactam antibiotics that irreversibly inhibit
classical D,D-transpeptidases (Bonfiglio et al., 2002). Three carbapenems -- imipenem (Fig. 5A),
meropenem (Fig. 5B), and ertapenem -- have been shown to also be active against L,D-
transpeptidases. Adducts of these carbapenems to Ldtfm (Mainardi et al., 2007), LdtMt1 (Lavollay
et al., 2008) and LdtMt2 (Gupta et al., 2010) were identified using mass spectrometry.
To characterize direct binding of imipenem and meropenem to LdtMt2 in the absence of
enzymatic activity, we performed isothermal titration calorimetry using a catalytically inactive
mutant C354A. Surprisingly, no heat of complex formation was observed with the mutant
protein with either imipenem (Fig. 6A) or meropenem (data not shown) at ligand concentrations
up to the mM range. In contrast, ITC experiments with the wild-type ex-LdtMt2 show strong
binding of both carbapenems to the enzyme (Fig. 5C, D).
Lack of binding by the C354A mutant is not due to a large structural change: the crystal
structure of the mutant (Fig. 6B, Table S1) shows no significant changes with respect to the wild-
type (rms deviation of 0.26 Å for 516 Cα-atoms of the asymmetric unit). The absence of a thiol
group at the residue 354 slightly widens the narrowest section of the tunnel, producing only
minor disturbances in other binding site residues not enough change to explain the lack of
binding (Fig 6B).