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.
1 | P a g e
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: firstname.lastname@example.org, email@example.com
2 | P a g e
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
3 | P a g e
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
6 | P a g e
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
7 | P a g e
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).
8 | P a g e
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
9 | P a g e
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
10 | P a g e
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),
11 | P a g e
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
12 | P a g e
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).
pH profile of the reaction shows a maximum catalytic rate at pH 7.8 (Fig. 7D, Table S3). The
13 | P a g e
Imipenem has approximately 17 times higher affinity for the enzyme than meropenem (Fig.
5 C, D). While binding of imipenem to LdtMt2 is exothermic, the same reaction with meropenem
is endothermic (Fig. 5C, D). The meropenem positive ΔS of binding, a consequence of its large
hydrophobic surface area, partially compensates for the unfavorable enthalpic contribution.
In addition to meropenem and imipenem, a potent β-lactamase inhibitor, clavulanic-acid,
was tested as ligand of ex-LdtMt2. ITC experiments did not detect binding by this compound to
wild-type ex-LdtMt2 even at concentrations in the mM range (data not shown). The β-Me
disulfide-adduct (Fig. 4B) observed when protein samples are treated with β-Me interferes with
imipenem and meropenem binding: the KD of these compounds increases an order of magnitude
(data not shown).
β – Lactamase activity of ex- LdtMt2
The kinetics of opening the lactam-ring of imipenem by ex-LdtMt2 displays the expected
irreversible inhibitor characteristics (Fig. 7A, B). The enzymatic reaction stops after one
turnover. The ratio of the total amount of imipenem rings opened to the amount of enzyme
added is close to one (0.96 ± 0.07, Fig. 7B).
To further characterize the enzyme, the β-lactamase activity of ex-LdtMt2 was measured using
the chromogenic substrate nitrocefin as a reporter (O'Callaghan et al., 1972). A low β-lactamase
activity was observed, with a Km of 16 µM and a kcat of 0.01 sec-1 (Fig. 7C; see also Table S2). The
enzymatic reaction shows the biphasic kinetics observed before in β-lactamases (Fig. 7E and F).
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An initial burst of activity (Fig. 7E) is followed by a linear slower rate (kde-acylation = 33.4 nM/min;
Fig. 7F) characteristic of a bifurcated kinetic path consistent with the formation of an
alternative, more labile, acyl-enzyme intermediate species (Monks and Waley, 1988).
Two pieces of evidence reduce the possibility that contamination with β-lactamases from
the bacterial expression-host is the source of the weak activity observed. First, the C354A
mutant of ex-LdtMt2 expressed and purified the same way as the wild-type shows no detectable
β-lactamase activity (data not shown). Second, no inhibition of ex-LdtMt2 β-lactamase activity
was detected with clavulanic acid (data not shown), that at the concentrations tested (ten of
mMs) should have inhibited most knwon bacterial β-lactamases.
LdtMt1 (Gupta et al., 2010). These data underscore the importance of L,D-transpeptidase activity
15 | P a g e
The degree of cross-linking and proportion of each type of linkage in the peptidoglycan layer
vary among different bacterial species as well as during different growth phases. For example,
the degree of cross-linking of the E. coli peptidoglycan is approximately 50%, with 4?3 linkages
accounting for 95% (90% in stationary phase) of the linkages (Vollmer and Holtje, 2004). In
contrast, the degree of cross-linking of the Mycobacterium spp peptidoglycan is as high as 80 %,
with 3?3 linkages comprising 70% (80% in stationary phase) of total linkages (Matsuhashi,
1966). This high frequency of 3?3 linkages in mycobacteria contrasts sharply with the low level
commonly observed in most other bacteria. For example, in E. coli and E. faecium, 3?3 linkages
represent only a relatively small fraction of the total --5 % during growth phases and 10 % in
stationary phase (Tuomanen and Cozens, 1987).
During the stationary phase of growth, formation of new 4?3 linkages is impaired because
the mature peptidoglycan layer has few pentapeptide stems, the required substrates of D,D-
transpeptidases, and the de novo synthesis of precursors is significantly reduced (Goffin and
Ghuysen, 2002). In contrast, L,D-transpeptidases could still cross-link the available tetrapeptide
Expression of the Mtb L,D transpeptidase LdtMt1 is upregulated in the transcriptome of
laboratory models of persisters (Betts et al., 2002; Keren et al., 2011) and shows a seventeen-
fold increase during the activation of the dormancy regulon of Mtb (Lavollay et al., 2008).
Interestingly, in all growth phases of Mtb, expression of LdtMt2 is ten fold higher than that of
as a chemotherapeutic target.
16 | P a g e
Structural comparison with other L,D-transpeptidases
The structure determined here shows that the bulk of the ex-LdtMt2 CD (251-379 aa) has a
fold similar to those of the C-terminal domain of B. subtilis ykuD (Bielnicki et al., 2006) and the
catalytic domain of the L,D-transpeptidase of E. faecium Ldtfm (Biarrotte-Sorin et al., 2006).
LdtMt2 shows 23 % sequence identity with Ldtfm, mostly concentrated in the CD. Despite having
similar two-domain architectures and catalytic domains, the three enzymes differ significantly in
their N-terminal domains (Fig. 8). YkuD shows a small N-terminal domain with a fold, LysM, that
is associated with cell wall degradation (Bateman and Bycroft, 2000). The Ldtfm stalk domain is a
60 Å long cylinder formed by a four α-helix bundle, with each helix of the bundle connected by a
small two-strand antiparallel β-sheet (Fig. 8A). The relative orientation of the two domains of
Ldtfm is maintained by a long loop that follows the third α-helix of the stalk domain resulting in
an 82 Å long cylindrical molecule (Fig. 8A). In contrast, the orientation of the two domains of ex-
LdtMt2 is maintained by direct interaction of residues of the CTSD with residues of the IgD,
resulting in a maximum dimension of only 60 Å (Fig. 8A).
The solvent accessibility of the catalytic site is very different in these three bacterial
enzymes: ykuD shows a completely open site, whereas LdtMt2 and Ldtfm show a site cover by the
β-hairpin flap (Fig. 8B). The β-hairpin flap of LdtMt2 is similar to that of Ldtfm, although it has a
seven residues longer loop LF that narrows the inner cavity of LdtMt2. The catalytic site of ykuD
shows the greatest divergence of the three sites (Fig. 8C). Although, these three enzymes
preserve a similar arrangement of the catalytic triad residues (Fig. 8C). Most of LdtMt2 and Ldtfm
catalytic site residues are conserved with only a few changes that (nevertheless) maintain the
overall shape of the site (Fig. 8C).
17 | P a g e
Mtb LdtMt2 homologues
Comparative modeling of LdtMt2 homologues in Mtb predicts localized variations in their IgD
and CD that may perturb the IgD-CD interaction. Loop modifications at the interface between
the IgD and CD, and the loss of CTSD interaction with the IgD (Fig. 3A), could change each
enzyme’s overall shape and flexibility. It is worth noting that these homologues are membrane-
associated proteins and have different levels of expression during different phases of growth
(Gupta et al., 2010). Their different shape and flexibility could reflect the fact that these
enzymes are specialized to target different peptidoglycan layer constituents, act at different
levels in the three-dimensional peptidoglycan layer (Vollmer and Holtje, 2004), or form a
particular cross-linking pattern by acting only on stems that present a spatial arrangement that
is optimum for a given enzyme. These multiple enzymes may provide a mechanism for
modulating the three-dimensional structure of the peptidoglycan layer during different phases
Binding of Imipenem to LdtMt2
The shape of the binding isotherm indicates the presence of equilibrium between bound
and unbound species (Fig. 5C). Kinetics experiments using fluorescence and absorption
spectrophotometric-techniques to follow the inactivation by imipenem of two closely related
enzymes, Ldtfm (Triboulet et al., 2011) and LdtMt1 (Dubee et al., 2012), showed the presence of
an enzyme-inhibitor complex before the irreversible acylation. Such complex is consistent with
the apparent tight binding of imipenem to LdtMt2 we observed using ITC (KD = 54.8 ± 8.8 nM).
The observation of a tight complex enzyme-inhibitor previous to enzyme irreversible acylation,
the one-to-one relation between inactivated enzyme and consumed inhibitor (Fig. 7A, B), and
hole” formed by the backbone NH-groups of residues 352 to 354 at the C-terminus of loop Lc. In
18 | P a g e
the lack of binding of the inhibitor to the catalytically inactive C354A mutant suggest that the
formation of a reversible tetrahedral intermediate (see below) is required for a strong binding of
imipenem to LdtMt2.
LdtMt2 catalytic mechanism
Biarrotte-Sorin et al. (2006) proposed that in this kind of L,D-transpeptidases the two paths to
the catalytic cysteine (via the inner or the outer cavity; Fig. 1C), are used one for the acyl-donor
and the other for the acyl-acceptor substrates. However in this mechanism, the enzyme would
need to go through multiple conformational changes of the flap to accommodate entrance of
substrates and release of products using the catalytic site tunnel. The mode of binding of the
peptidoglycan fragment found in the structure of LdtMt2 suggests a much simpler mechanism, in
which the acyl-donor peptidoglycan stem binds to both cavities through the tunnel (Fig. 1C and
Fig. 4) and remains bound during most of the catalytic cycle, reducing the need for
conformational changes during the catalytic cycle of the enzyme. We built models of the
peptidoglycan-stem substrates bound to the active site of the enzyme based on the structure of
LdtMt2 and use it to propose atomic details of the catalytic mechanism of LdtMt2 (Fig. 9). In this
model, the catalytic site tunnel and features at either side of the tunnel tightly bind the stem di-
aminoacid (A2pm3). At one end of the tunnel, within the inner cavity, Tyr318 and the carbonyl
group of Gly332 recognize the D chiral-center of the di-aminoacid side chain, making hydrogen
bonds to its carboxylate and amide group. The tunnel recognizes the aliphatic portion of the side
chain. At the other end, within the outer cavity, the L chiral-center is surrounded by an “anion
terminus that may explain the preference of the enzyme for tetrapeptide stems (Lavollay et al.,
19 | P a g e
this position, the acyl-group of the m-A2pm3 L chiral-center is within reach of Cys354. These
features are consistent with a nucleophilic attack by this cysteine on the acyl-carbon.
In the crystal structure of ex-LdtMt2, the Sγ atom of Cys354 points toward the inside
entrance of the catalytic site, away from the putative acyl-group. Identical cysteine
conformations (χ ≅ -73o) have been consistently observed in all the other crystal structures of
ErfK/YbiS/YhnG-domain proteins (Biarrotte-Sorin et al., 2006; Bielnicki et al., 2006). Therefore,
one can expect that earlier in the catalytic cycle the cysteine may adopt another conformation
(χ ≅ 58o) that points toward the carbonyl carbon of the scissile bond. In this conformation the
SH group would be briefly at 2.9 Å from the Nε atom of His336, allowing deprotonation of the
Cys354 SH-group for the nucleophilic attack at the peptide carbonyl group. The imidazolium
cation of the protonated His336 may then be the donor for the protonation of the departing D-
Ala amide. His336 and Asn356 make hydrogen bonds to this CO-group, providing further
recognition of the donor-stem L chiral-center (Fig. 4A). The pH dependence of the reaction rate
shows an increase of β-lactamase activity compatible with the ionization of a group with a pKa
of around 7.8 (Fig. 6D, see also Table S3). Participation of the Cys354-thiolate/His336-
Imidazolium ion pair in the concerted acid-base catalysis of the acyl-transfer is compatible with
this pH optimum.
The rest of the interactions between the LdtMt2 and the bound peptidoglycan fragment
involve the side chain of Asn356, which coordinates the main-chain NH-group and the terminal
carboxylate of D-Ala4 (D-Ala4 binding site). Trp340 provides steric constraints to the stem
2008). Interestingly, in one of the Mtb homologues of LdtMt2, MT0501, a less bulky methionine
from participating in new cross-links. Linked stems can dock in the vestibule with their
20 | P a g e
residue replaces Trp340 (Fig. 3B), suggesting that this enzyme can accommodate pentapeptides,
perhaps broadening its specificity from D-Ala to non-canonical D-aminoacids observed by Cava
et al. (2011). Towards the N-terminus of the peptidoglycan stem, the conserved His352,
provides a hydrogen bond to groups of the iso-glutamyl side-chain.
In the observed tautomer, forced by the hydrogen bond to the carbonyl of Ser337, His336
has its Nε ready to accept a H+ from Cys354 (Fig. 9A step 1). The cysteine thiolate formed by this
H+ abstraction attacks the acyl-carbon and forms a tetrahedral intermediate stabilized by the
“anion hole” and by the just protonated His336 (Fig. 9A step 2). After the intermediate thioester
formation and protonation by His336, D-Ala4 is released (Fig. 9A step 3). Then another peptide
stem can enter the catalytic site, also through the external vestibule, and bind to active site
residues with the side-chain amide of the A2pm3’ residue (also a D chiral-center and isomorphic
to D-Ala4), (Fig. 9B). His336 is in position to activate the amine group of the acceptor stem D
chiral-center (Fig 9A step 4). Nucleophilic attack by this amine group, forms the new peptide
bond that cross-links the two stems with release and protonation of the cysteine thiolate by
His336 (Fig. 9 step 5-6). It is clear that the last step in the transpeptidation reaction requires
that donor and acceptor peptidoglycan stems both be at the catalytic site for the reaction to
occur (Fig. 9B). The wide and shallow entrance of the outer cavity has the required
characteristics to accommodate both peptidoglycans stems (Fig. 9B).
Higher order cross-linked stems are also observed in Mtb, though with low frequency (3 %)
(Lavollay et al., 2008), suggesting that already linked peptidoglycan stems are not precluded
unreacted D chiral-center reaching the catalytic residues without any conformational change in
21 | P a g e
the enzyme. The use of the unreacted L chiral-center of an already crosslinked stem as acyl-
donor imposes more steric constrains and most likely involves the opening of the flap. After
formation of the new cross-link, release of the higher-order linked peptide stems will require
opening the flap again.
The crystal structure of the complex of a Mtb L,D-transpeptidase with a peptidoglycan reported
here provides a three-dimensional map of the extensive interactions between the peptidoglycan
fragment and LdtMt2, involving residues that are conserved in all members of this Mtb family of
L,D-transpeptidases. Knowing this map is an important step toward a rational design of high
affinity drugs engineered to inhibit L,D-transpeptidase activity.
22 | P a g e
Protein preparation, purification and crystallization
A truncated construct of MT2594 (Rv2518c) spanning residues 120-408 (ex-LdtMt2) was
expressed in E. Coli BL21(DE3) strains and in a methionine auxotroph strain supplemented with
SeMet. Proteins were separated from the lysates using (Ni)-affinity chromatography (Ni-Histrap
GE HealthCare Inc.). Size exclusion chromatography was performed using a sepharose column
(Sepharose 12 GE HealthCare Inc.). Native and SeMet-derivatized protein samples were used in
the crystallization trials. Diffraction quality crystals of all forms used in this study were obtained
in 48 hrs at 20 oC with hanging drops vapor diffusion methods. Drops of 1µl of protein (20
mg/ml) and 1µl of reservoir solution were equilibrated against a reservoir containing 0.1 M
HEPES (pH 7.5), 1 M succinic acid and 1% (w/v) PEG MME 2000, except for SeMet crystals in
where HEPES at pH 7.0 was used. For heavy atom derivates, crystals were and soaked in 10 µl of
reservoir solution containing the heavy atom compounds at the desire concentration. In the
case of PIP (di-µ-iodo-bis(ethylenediamine)-di-Pt(II)), ex-LdtMt2 crystals were soaked with 100
µM for 48 hrs. (See Supplemental Experimental procedures for additional details)
Data Collection, Structure Determination and Refinement
Diffraction data of the PIP derivative, mutant, and β-Me disulfide-adduct and oxidized forms of
the native were collected using an in house CuKα X-ray source (FRE+ super bright ™ generator
with a Saturn 944+ CCD Detector; Rigaku USA, Inc.). The SeMet-derivative crystal used in the
phase determination was collected on beamline X4A of the National Synchrotron Light Source of
the Brookhaven National Laboratory (Table 1). X-ray diffraction experiments were carried out
ceph-3-em-4-carboxylic Acid, EMD Inc., (Fig. 7D)— a chromogenic β-lactam. The β-Lactamase
23 | P a g e
under cryogenic conditions after transferring the crystal into crystallization solution containing
10% v/v glycerol. Intensities were indexed and integrated using HKL2000 (HKL Research Inc).
Data were scaled with the CCP4 program suite (CCP4, 1994). Phases were determined using the
program autoSHARP (Vonrhein et al., 2007). Refinement was carried out using REFMAC5
(Murshudov et al., 2011) and rebuilding was done with Coot (Emsley et al., 2004).
Isothermal Titration Calorimetry (ITC) experiments were performed using a high-precision VP-
ITC titration calorimeter system (Microcal Inc.). The buffer of the protein samples was replaced
by 20 mM HEPES 300 mM NaCl pH 7.5 using a desalting column (HiTrap G-25, GE HealthCare
Inc.). Inhibitors and substrates used in these experiments were dissolved in the same buffer.
ITC experiments using PBS and PIPES showed similar ∆H values, indicating that no H+s were
released or bound during the reaction. Titration experiments consisted in the addition of 25
injections of 10 µl of concentrated inhibitor into the protein solution contained in the cell (1.38
ml) with 300 to 450 sec equilibration between injections. Data were analyzed with Origin 5.0
software (Microcal Software, Inc., Northampton, MA) with a single site model. Imipenem
monohydrate and meropenem were purchased from SIGMA Inc., and ertapenem was purchased
from Merck Sharpe and Dhome-Chibret.
Initial rate measurements were used to determine the steady state kinetic parameters of the β-
lactamase activity against nitrocefin —3-(2,4-Dinitrostyryl)-(6R, 7R)-7-(2-thienylacetamido)-
activity was measured by following the absorbance of the product produced by opening the
24 | P a g e
lactam-ring at 486 nm using a molar extinction coefficient of 20500 M-1cm-1 (O'Callaghan et al.,
1972) in metal free buffer of 20 mM HEPES at pH 7.5 and 300 mM NaCl. Nitrocefin was added at
concentrations ranging from 12.5 to 200 µM. The kinetic parameters, Km and kcat were fitted by
a nonlinear least-squares fit of Michaelis-Menten equation the to the initial rate measurements.
Enzyme-acylation by imipenem was measured by quantifying the amount of reacted
imipenem at a given time, following the formation of the open penem-ring product by
monitoring the absorbance at 299 nm. The decrease in absorbance values was converted into
concentration units using the difference between molar extinction coefficients of the open and
closed penem-ring of -7100 M-1cm-1 (Triboulet et al., 2011).
25 | P a g e
Use of beam line X4A and X4C at Brookhaven National Laboratoty NSLS and the technical
support of John Schwanof and Randy Abramowitz are acknowledged. Use of the Advanced
Photon Source at Argonne National Laboratory during early stages of this crystal structure
determination was supported by the U. S. Department of Energy, Office of Science, and Office of
Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. Eli Lilly & Company, who
operates the facility, provided the use of the SGX Collaborative Access Team (SGX-CAT)
beamline at Sector 31 of the Advanced Photon Source. This work was supported by NIH grant
1R56AI087749 and DP2OD008459 to GL.
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31 | P a g e
Figure 1. Peptidoglycan linkages and structure of LdtMt2. (A) 4?3 and 3?3 linkages. The C-
terminal residues from the γ-D-Glu of donor and acceptor peptidoglycan stems are depicted in
different colors, γ-D-Glu (red), m-A2pm (blue), and D-Ala (cyan). Residue numbers of the
acceptor stem are primed. (B) Cartoon representation of the overall structure of ex-LdtMt2 with
elements of secondary structure labeled. Two pink arrows mark the beginning and end of the
CTSD. The peptidoglycan fragment bound to the active site is also shown in a stick
representation. (C and D) Views of the solvent accessible surface of ex-LdtMt2 colored by
electrostatic surface-potential (from blue positive to red negative). (C) side view (same
orientation as part (B). The stick representation of peptidoglycan-fragment bound to the active
site is shown. Oxygen atoms are colored in red, nitrogen atoms in blue, sulfur atoms in yellow
and carbon atoms in green. (D) top view of the outer cavity (rotated 90o around a horizontal
axis from B). The surface was rendered transparent and the peptidoglycan fragment removed to
show residues and secondary structure elements lining the outer cavity.
Figure 2. Sequence alignment of LdtMt2, the L,D-transpeptidase of E. faecium (1ZAT) and other
Mtb LdtMt2 homologues. Top: domain distribution of LdtMt2. The ex-LdtMt2 protein construct used
in this work is indicated by a dashed red box. Bottom: sequence alignment among amino acids
138 to 408 of LdtMt2, LdtMT1, Ldtfm and other LdtMt2 homologues in Mtb. The observed secondary
structure is indicated above the corresponding residues. Identical residues are white with red
al., 1988) are boxed with red letters. Residues involved in binding and catalysis are labeled: the
unit. Final refinement 2DFo – mFc electron density map contoured at 1 sigma around Cys354. (C)
32 | P a g e
conserved motif is indicated with green marks and the rest with pink marks. Stars label main
catalytic residues and ovals label substrate recognition residues. The figure was made using the
web server ESPript (http://espript.ibcp.fr/ESPript/ESPript/).
Figure 3. Comparative Modeling of Mtb homologues. (A) Overlay of Cα-trace of the modeled
structures of the Mtb homologues with the crystal structure of ex-LdtMt2 (green). (B) Overlay of
the modeled catalytic sites. Residues participating in the interactions with the substrate are
displayed; carbons atoms are colored with the same color as the corresponding Cα-trace: Green,
LdtMt2; yellow, MT1477; pink, MT0501; magenta, LdtMt1; and cyan, MT0202. Non-carbon atoms
are colored as Fig. 1.
Figure 4. Electron densities at the catalytic site of LdtMt2. (A) Final refinement 2DFo – mFc map of
the co-crystallized peptidoglycan fragment contoured at 0.5 σ (see below) around the γ-D-Glu-
m-A2pm fragment at the active site of ex-LdtMt2. The fitted fragment, binding residues and the
catalytic cysteine are shown. Hydrogen bond interactions with conserved residues are shown
with pink dashed lines. As the two binding sites in the non-crystallographic dimer occur side-by-
side, overlapping density for two equivalent peptidoglycan fragments was observed. The density
was interpreted as two equivalent ligands related by the non-crystallographic two fold and
refined as alternative orientations with occupancy of 0.5. (See also Fig. S2 for a stereo view of an
omit map). (B-C) Modifications of Cys354 observed in native forms. (B) β-mercapto-ethanol
covalently bound to Cys354 (2.0 Å bond distance). The rms deviation between this structure
(native form) and the refined (PIP derivative) is 0.176 Å for 520 Cα-atoms of the asymmetric
Oxidized Cys354 (suphenic acid form). Atoms are colored as Fig. 1.
Plot of the concentration of imipenem reacted versus the concentration of enzyme used (10, 20,
33 | P a g e
Figure 5. Isothermal titration calorimetry experiments. The chemical structures of (A) Imipenem
azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid), and (B) meropenem (3-[5-(dimethylcarbamoyl)
carboxylic acid) are shown. Titrations of (C) imipenem and (D) meropenem binding to LdtMt2 are
shown in the upper portion of the panels. The lower portion of the panel displays plots of the
total heat released as a function of the molar ratio between each carbapenem and LdtMt2. The
solid line represents the non-linear least square fit of the data using a single-site binding model.
Thermodynamic parameters of binding to ex-LdtMt2 --KD, ∆H and ∆S-- are tabulated at the
bottom of the figure.
Figure 6. C354A mutant. (A) ITC of Imipenem binding to the C354A mutant. Upper panel:
Titration of the C354 mutant with imipenem. Lower panel: plot of the total heat released as a
function of total ligand concentration for the titration shown in the upper panel. No heat of
binding is detected, only heat of dilution. (B) Refined electron density of the C354A mutant
around the mutated residue. Final refinement 2DFo – mFc map contoured at 1 σ around Ala354
of the mutant. Carbons atoms are colored in cyan, the rest as Fig. 1.
Figure 7. Enzymatic activity of LdtMt2. (A) and (B) Irreversible inhibition by iminopenem. (A) three
selected concentrations of enzyme (10, 35 and 60 µM) reacting with 100 µM of imipenem. (B)
35, 40, 50 and 60 µM). (C) Initial-rate experiments of the ex-LdtMt2 β-lactamase activity using
the peptidoglycan fragment with residues of the catalytic site are shown with dashed lines.
34 | P a g e
nitrocefin as substrate (structural formula show in the Figure). Solid lines are the non-linear fit
of the initial rate to the Michaelis-Menten equation. (Note that pH 7 and 8.5, and 6.5 and 9
overlap.) (D) Plot of the log of kcat for nitrocefin as a function of pH. See also Table S2. (E-F)
Progress of the reaction of nitrocefin (100 µM) and LdtMt2 (500 nM). (E) Initial phase of the
reaction (controlled by binding and acylation rates). (F) Later phase of the reaction (controlled
by de-acylation rate). The amount of product (open ring) is monitored by its absorption at 486
Figure 8. Structural comparison among L,D transpeptidases from B. subtilus, E. faecium and M.
tuberculosis. (A) Side by side ribbon diagrams of the crystal structures of the L,D-
transpeptidases: ykuD (PDB id. 1Y7M) in pink (rms deviation of 0.97 Å between 104 Cα-atoms
aligned with ex-LdtMt2), Ldtfm (PDB id. 1ZAT) in cyan (1.0 Å rms deviation between 116 Cα-atoms
aligned) and LdtMt2 in green. In the E faecium and Mtb case, the regions that apparently fix the
domains orientation are colored in dark green. The catalytic domains of the three enzymes
were used to align the structures. (B) Comparison of the catalytic domains. The catalytic
cysteine residue is depicted in a CPK representation with carbon atoms purple and sulphur atom
yellow. (C) Catalytic site residues of the three enzymes. View them from the outer cavity; the
catalytic site residues are shown as side-chains or main-chains according to their expected
function. The catalytic site triad is labeled with pink letters. The γ-glutamyl-meso-
diaminopymelic acid (γ-D-Glu-m-A2pm) is shown only for ldtMt2 in a stick representation with
magenta colored carbon atoms. Non-carbon atoms are colored as Fig. 1. H-bond interactions of
35 | P a g e
Figure 9. Proposed mechanism of the L,D-transpeptidation reaction and structural model of the
acyl-acceptor and donor-peptidoglycan stems bound to the outer cavity of ex-LdtMt2. (A) Steps 1
– 6 of the mechanism of (3,3) L,D-transpeptidation by LdtMt2. (B) The figure represents step 4 of
the mechanism in part A, after the cleaved D-Ala4 has left and a thioester intermediate is formed
between acyl-donor (carbon atoms colored pink) and the enzyme (cyan). Non-carbon atoms are
colored as Fig. 1. Only the last two residues of the donor stem and the m-A2pm (light gray) of
the acceptor stem are shown. The peptidoglycan stem of the acyl-acceptor binds to the site with
its A2pm3’ side chain (D chiral-center) near the thioester bond (marked with an *). (B) Solvent
accessible surface at the outer cavity. The surface is colored by electrostatic surface-potential
(from blue positive to red negative). The two substrates of the bi-substrate reaction are shown.
TT TT TT
320 330 340 350 360 370
Y G H G GC G V
T V T I S V V SA S TN S LNV AQ FY V VE VNSPNG .R D DWA Q SY F PW VGAQ ...H T H SPSN W DH KR DI
Y G H G GC G V
L A V V S V V SA S AN S INL AA YF V IE LNSSDG .L T HYA R TW Y PW VNSQ ...Y V H SPDN W DA TV DP
Y G H G GC G V
S V M I T V I DS Q TR S INT MK LF V VL ...... .E P NYW P DW G DW PEYG DLWK G H PPSV E GM EK TP
Y G H G GC G V
L V V I S N V SA S RN T INL AK FY F VV VNSAQG .K T SDA R DN F PW VADQ ...K V H SPAN W DN GS DP
Y G H G GC G V
L V V I R L V SA A EN S ISL AE YY V VI VDDPDG .R S DYA R TS Y PW LPAL ...L V H SRED W NA DI DP
Y G H G GC G V
I E V I N E I AN S SN T INL AE YY A VE .....G SH H RWA R SN F PM AGAQ ...N V N STEN Q RS VY DP
140 150 160 170 180 190 200
EV V VAIRF E I R AAEKAI I TTRQLTFQTSSPAHLTMPYVMPGD VG GEP D N A.......D G K T NPPVE AF
AV V VVVTF T V R AVERSI I T...............VASVSPAN VG AHP T P T.......D R R S PHNTT HF
KV L QVRQY T L N TKFKST R E.................WLTYND D. DTE V D G.TKYNTST D K G VTVPV TY
AM V IVINF V I R MAESAI I S GQDPTSFVG.PPPFRPPTFNPVD VG AKP A P A.......D A H S IPPVP KF
QV V VVVTF A I R AAERAV V S ...............VASVLPTR VG AHP S P TN....PAN H E K TPAMT KF
QT I VIIQF S I K AVERAL V TPVAGKFTTVAPVKTINAGFQLAD VG AAP D P S.......D A T T DPPVE GW
210 220 230 240 250 260
L EV R F TAV V V QT I VI TA NTY NNR.... RW PEH WKP...G D A NTYGVDLGEGMFGEDNV HFT GDE A DD KI
V VV V Y TRV V V TE T LI VA SAE ASN.... RW PHR WPP...H S G ..............QEL GFE GDA G SI HT
T ET L I TRS I Q TA L TY EV ENS IQTDS.. EA KKA LAG.QDF P V ..............GGT DHP IED I DL QH
M QV R F TAV I A KS T LV TA ATY SPT.... RW PFE WPA...N N D ..............AGT SFR GDS A DD HQ
L VV V F STV L V SS T VV VA SQE DND.... QW PDR WPA...H E S ..............GSL DFK GPA G SI HT
L RV R Y TTV V A SL I QV KA SSA PDEAQGA HW PRE YPA...G D D KLYGLPFGDGAYGAQDM HFQ GRR V EV HR
270 280 290 300 310
G T G G
V V G V S I V SR K I M SSLT R N ..................E VKSMPT M KDS...TP AN YI G Y H I D TY. VP
G T G G
V R G V S S A SK R V M SRFT S N ..................E LRTMPA L KPS...RP PI FH M E T V D TI. IP
G T G G
Y K G V V V V NK E A L GTMW Y D ..................K ALETDI S KPT...TP PA FY W E D T K N.. TP
G T G G
I R G V S T V EK A V M SS MT T N ..................V QKTFPM M MVSGG.HQ PN YY L F T V D TY. VP
G T G G
V I G E S S V SK R V M SSFT S D VEEGPPPPLPAPHHRVHFG DGVMPA M RPE...YP PV YT L E S I D SV. IP
G T G G
V T A V S I V EK S F M SNIQ V D ..................G IMDFPC Y EADLARNV RN HV T Y D Y . PAA ..
380 390 400
β1 β2β3 α1 β4
β5 β6β7 β8β9 β10β11
β12 η1 β13 β14β15 η2
β16β17 β18β19 α2 β20
1 100 120 150 250
kcal/mole of injectant
Protein Conc. 10 µM 25 µM
Ligand Conc. 150 µM 1 mM
Stoichiometry (N) 1:1 1:1
KD (nM) 54.8 ± 8.8 909 ± 148
ΔH (cal/mol -9,300 ± 92 3,650 ± 75
ΔS (cal(mol K)-1) 2.23 39.6
kcal/mole of injectant
kcal/mole of injectant
010 20304050 60
List of tables
Table 1. Data collection statistics of the PIP complex used to refine the structure and
of the SeMet crystal used for phase determination. See also Table S1 for statistics of
other crystal forms.
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Table 1. Data collection statistics of the PIP complex used to refine the structure and
of the SeMet crystal used for phase determination. See also Table S1 for statistics of
other crystal forms.
a, b, c (Å)
α, β, γ (°)
(Last shell) (Å)
Bond lengths (Å)
Bond angles (º)
PIP (Refinement) SeMet (phase determination)
0.96785 Å 0.97939 Å
119.1, 120.8, 122.8
90.0, 90.0, 90.0
118.3, 121.1, 122.5
90.0, 90.0, 90.0
50.0-2.45(2.49-2.45) 50.0-2.54 (2.58-2.54)
90.0, 90.0, 90.0
90.0, 90.0, 90.0
50 – 1.72(1.765-172)