Shedding X-ray Light on the Role of Magnesium in the Activity of Mycobacterium tuberculosis Salicylate Synthase (MbtI) for Drug Design

Article abstract of DOI:10.1021/acs.jmedchem.0c00373

The Mg2+-dependent Mycobacterium tuberculosis salicylate synthase (MbtI) is a key enzyme involved in the biosynthesis of siderophores. Because iron is essential for the survival and pathogenicity of the microorganism, this protein constitutes an attractiv

Full text of DOI:10.1021/acs.jmedchem.0c00373

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Article
Shedding X-ray light on the role of magnesium in the activity
of M. tuberculosis salicylate synthase (MbtI) for drug design
Matteo Mori, Giovanni Stelitano, Arianna Gelain, Elena Pini, Laurent R.
Chiarelli, josè camilla sammartino, Giulio Poli, Tiziano Tuccinardi, Giangiacomo
Beretta, Alessio Porta, Marco Bellinzoni, Stefania Villa, and Fiorella Meneghetti
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00373 • Publication Date (Web): 12 Jun 2020
Downloaded from pubs.acs.org on June 17, 2020
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Shedding X-Ray Light on the Role of Magnesium in
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the Activity of M. tuberculosis Salicylate Synthase
(MbtI) for Drug Design
a,§
b,§
a
a
b
Matteo Mori , Giovanni Stelitano , Arianna Gelain , Elena Pini , Laurent R. Chiarelli ,
b
c
c,d
e
José C. Sammartino , Giulio Poli , Tiziano Tuccinardi , Giangiacomo Beretta , Alessio
f
g,*
a,*
a
Porta , Marco Bellinzoni , Stefania Villa , Fiorella Meneghetti
a
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, via L.
Mangiagalli 25, 20133 Milano; Italy.
b
Dipartimento di Biologia e Biotecnologie “Lazzaro Spallanzani", Università degli Studi
di Pavia, via A. Ferrata 9, 27100 Pavia; Italy.
c
Dipartimento di Farmacia, Università di Pisa, via Bonanno Pisano 6, 56126 Pisa; Italy.
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d
Sbarro Institute for Cancer Research and Molecular Medicine, Center for
Biotechnology, College of Science and Technology, Temple University, Philadelphia,
PA 19122; USA.
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e
Dipartimento di Scienze e Politiche Ambientali, Università degli Studi di Milano, via G.
Celoria 2, 20133 Milano; Italy.
f
Dipartimento di Chimica, Università degli Studi di Pavia, via T. Taramelli 12, 27100
Pavia; Italy.
g
Unité de Microbiologie Structurale, Institut Pasteur, CNRS, Université de Paris, F-
75015 Paris; France.
KEYWORDS. Tuberculosis, magnesium, divalent cations, antitubercular agents, MST
enzymes, catalytic mechanism, siderophores, X-ray crystal structures, mycobactins,
MbtI conformations.
2+
ABSTRACT. The Mg -dependent salicylate synthase (MbtI) from Mycobacterium
tuberculosis is a key enzyme involved in the biosynthesis of siderophores. Because iron
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is essential for the survival and pathogenicity of the microorganism, this protein
constitutes an attractive target for antitubercular therapy, also considering the absence
of homologous enzymes in mammals. An extension of the SAR of our furan-based
candidates allowed us to disclose the most potent competitive inhibitor known to date
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(
10, K = 4 μM), which also proved effective on mycobacterial cultures. By structural
i
2+
studies, we characterized its unexpected Mg -independent binding mode. We also
2+
investigated the role of the Mg cofactor in catalysis, analyzing the first crystal structure
2+
of the MbtI-Mg -salicylate ternary complex. Overall, these results pave the way for the
development of novel antituberculars through the rational design of improved MbtI
inhibitors.
INTRODUCTION
Nowadays, tuberculosis (TB) ranks among the top ten causes of death worldwide;
therefore, the development of new scaffolds is imperative to sustain the drug pipeline,
considering the issues of the available antitubercular therapies and the increasing
1
emergence of resistant infections. The salicylate synthase (MbtI) from Mycobacterium
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tuberculosis (Mtb, the etiological agent of TB) is the first enzyme involved in the
biosynthesis of mycobactins. These small-molecule siderophores are capable of
chelating iron, a key cofactor involved in several mycobacterium-specific biological
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processes. MbtI has been structurally and biochemically characterized, it is essential
3
for the survival of Mtb under iron-deficient conditions, and, as such, it has been
identified as a promising therapeutic target.4
2+
MbtI belongs to the group of the structurally homologous Mg -dependent
menaquinone, siderophore and tryptophan (MST) enzymes, which transform chorismate
by rearrangement, to generate precursor molecules for the respective biosynthetic
pathways.5,6 In this context, MbtI catalyzes the two-step conversion of chorismate to
salicylate, via isochorismate as intermediate, performing an isomerase activity followed
by a lyase activity (Figure 1).4
COO-
COO-
COO-
OH
MbtI (Mg2+
MbtI (Mg2+
Step 2
O
)
OH
)
+
COO-
O
COO-
Step 1
O
COO-
OH
Figure 1. Reactions catalyzed by MbtI.
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Several strategies have been adopted for the discovery of MbtI inhibitors. In particular,
dicarboxylate substrate analogs (chorismate and isochorismate), transition state
analogs and compounds originated from HTS have been identified.7–10 However, these
compounds, albeit active against the isolated enzyme, show modest effects on the
whole mycobacterial cell.7–10 Notably, Methyl-AMT (IC50 = 11.6 μM), the best MbtI
inhibitor discovered to date not belonging to our furan-based class, exhibited a poor
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minimum inhibitory concentration (MIC ≥ 1 mM), which remained weak (MIC = 792
μM) for its more lipophilic dimethyl ester analog as well.4
Therefore, in order to find improved MbtI inhibitors, we followed an in silico screening
approach, which led to the identification of a new hit.11 While exploring the chemical
space around this compound in the frame of a thorough structure-activity relationship
(SAR) study, we discovered two effective derivatives, I and II (Figure 2), which are, to
our knowledge, the most potent MbtI inhibitors reported to date (IC = 7.6 ± 1.6 μM and
50
13.1 ± 2.0 μM, respectively). This strong inhibition of MbtI is correlated to a lethal effect
on Mtb cultures (MIC99 = 156 μM and 250 μM, respectively).11,12 Furthermore, the
Universal CAS assay confirmed the connection between the antimycobacterial effect of
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these compounds and the disruption of mycobactin biosynthesis, thus highlighting the
importance of this pathway as a target for the development of therapeutic interventions.
Moreover, we demonstrated the possibility of removing one of the carboxylic groups of
Methyl-AMT, without causing a loss in the inhibitory potency and allowing, at the same
time, a better permeability through the cell wall of Mtb.11,12
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O N
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CF3
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OH
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II
Figure 2. Chemical structures of compounds I and II.
4
Despite the promising results obtained in the inhibition of this target, its mechanism
of action is still poorly understood and not supported by conclusive experimental data.
In particular, a robust definition of the role of Mg2+ in the interaction between MbtI and
inhibitors is still lacking. It has been reported that the affinity of the MST enzymes EntC,
PchA and Irp9 for ligands is much higher than that for the metal ion, so the driving
element of the catalytic reaction is the binding of the substrate, followed by interaction
6
with the cofactor. After the conversion of chorismate to isochorismate, a sudden
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change in the affinity for the metal occurs: Mg2+ is retained by MST enzymes with an
extremely high affinity, promoting the subsequent reaction which quickly leads to the
formation of salicylate.6
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Stimulated by these findings, we started to consider if I and II may participate in the
inhibition mechanism by occupying the active site of the free MbtI before the
intervention of the Mg2+ ion, thus blocking the isomerase activity. In this work, we
provide experimental data to explain the role of Mg2+ on the activity and inhibition of
MbtI. Firstly, we expanded our library of furan-based compounds, designing and
synthesizing new meta-derivatives. Among them, the most potent inhibitor identified so
far (the m-cyano compound 10, Table 1) was selected as the most suitable candidate to
deepen our investigations. The determination of its activity, performed at increasing
2+
concentrations of Mg , showed that the metal did not influence the binding of the
compound to the target. Then, we updated our computational model, taking into account
2+
the Mg -independent binding mode, which allowed us to identify a previously
unconsidered key role of some residues. This hypothetical pose was supported by the
obtainment of the crystallographic complex of MbtI with 10, which is here described. As
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a proof of principle, we also solved a crystal structure of the enzyme in complex with
2+
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Mg , obtained at saturating concentrations of the metal. Moreover, the MbtI-Mg
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crystal structure evidenced the presence of salicylate, the product of the enzymatic
reaction. Notably, the presence and the interaction pattern of salicylate at the active site
confirmed the hypothesized catalytic mechanism of MbtI, previously inferred by
similarity to other MST enzymes.
RESULTS AND DISCUSSION
Synthesis and SAR of compounds 1-10
Inspired by the encouraging activity of our leads I and II,11,12 we decided to further
explore the phenyl-furan scaffold. In particular, with the aim of obtaining additional SAR
data, we focused on exploring the meta position of the phenyl ring using substituents
endowed with different stereoelectronic properties, many of which already employed in
our previous studies in which the ortho and para positions of the phenyl ring were
mainly substituted. Compound 1, bearing only one CF at position 3, was purchased
3
from a commercial source, while 2-10 were synthesized.
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Compounds 2-4,6-10 were synthesized by a Suzuki-Miyaura reaction,11 either
between methyl 5-bromofuran-2-carboxylate and the suitable boronic acid, or between
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(
5-(methoxycarbonyl)furan-2-yl)boronic acid and the appropriate bromo-derivative. The
so-obtained esters were hydrolyzed in basic-conditions to yield the free carboxylic acids
(
Scheme 1). Finally, compound 5 was synthesized from 5-(3-nitrophenyl)furan-2-
carboxylate (23) through the reduction of its nitro group with tin(II) chloride,13 followed
by the hydrolysis of the ester function (see Scheme S1 in Supporting Information, SI).
Scheme 1. Synthesis of compounds 2-4, 6-10.
R
R
R
Br
Br
O
O
a
O
O
b
c
+
O
O
89%
OH 25-76%
O
81-94%
O
OH
OCH3
B
OH
OCH3
OH
12
13 R = Cl
2
R = Cl
1
1
1
4 R = OH
5 R = CONH2
6 R = CN
3
6
R = OH
R = CONH2
d
75%
10 R = CN
R
R
OH
R
e
c
B
O
O
HO
+
O
O
O
32-77%
84-88%
O
OCH3
Br
OCH3
OH
17
18 R = CH3
19 R = CONHCH3
4 R = CH3
7 R = CONHCH3
2
0 R = SO NH2
8 R = SO NH2
2
2
21 R = COOCH3
9 R =COOH
Reagents and conditions: a) conc. H SO , MeOH, reflux, 24 h; b) Pd(PPh ) Cl , 2M
2
4
3 2
2
Na CO , 1,4-dioxane, 90 °C, overnight, N ; c) NaOH, EtOH/THF 1:1, reflux, 5 h or
2
3
2
LiOH∙H O, THF/H O 1:1, 20 °C, 2 h. d) 1. bis[2-(N,N-dimethylamino)ethyl] ether, 2M i-
2
2
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PrMgCl, THF, 20 min. 10-15 °C – 30 min. r.t., N ; 2. B(OCH ) , 0 °C, 10 min., N ; e)
2
3 3
2
Pd(PPh ) Cl , 2M Na CO , 1,4-dioxane, 60 °C, 90 min., MW, N .
3
2
2
2
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2
The activity of compounds 1-10 was tested against recombinant MbtI (Table 1),
prepared and assayed as previously reported.12 Compound 1 showed a promising
activity, while the replacement with a halogen (2) or with electron-donating functions like
the hydroxyl (3), methyl (4) or amino (5) groups abolished or significantly weakened the
activity. The insertion of a strong electron-withdrawing group capable of forming a
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localized negative charge, like the NO function, previously published (inhibitory activity:
2
5
0%, at a concentration of 100 μM),11 did not lead to an improvement of the activity.
Conversely, the amide group of 6 allowed for a significant enhancement of the
inhibition, as shown by the IC value of 31 µM. While the methylation of the amide in
50
compound 7 led to a strong decrement of the activity, the substitution of the amide with
classical bioisosteres (sulfonamide, 8; carboxylic acid, 9) allowed for a retention of the
inhibitory effect. Interestingly, the substitution with a nitrile in 10 afforded the best
compound of the series, characterized by an IC50 value of about 6 µM; this inhibitor
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proved to be slightly better than the previous candidates I and II, thus becoming our
improved lead compound.
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4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. m-Substituted derivatives 1-10. Inhibitory effect is expressed as percentage of
residual enzymatic activity (at 100 μM ligand concentration) for all compounds and Half-
Maximal Inhibitory Concentrations (IC , μM) only for the most active candidates
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
(residual activity ≤ 25%).
R
O
O
OH
Code R
% Residual activity
42.0 ± 6.3
IC (μM)
50
1
2
3
4
5
6
7
8
9
1
3-CF3
-
3-Cl
101.6 ± 17.8
70.4 ± 21.8
103.9 ± 4.8
65.8 ± 9.6
-
3-OH
-
3-CH3
-
3-NH2
-
3-CONH2
3-CONHCH3
3-SO NH
20.9 ± 4.3
31.4 ± 10.3
84.0 ± 9.1
-
28.6 ± 6.8
-
2
2
3-COOH
27.2 ± 4.5
-
0
3-CN
3.1 ± 1.0
6.3 ± 0.9
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Biological assays
Compound 10 underwent an in-depth biological evaluation (Figure 3), aimed at
characterizing its activity on MbtI and confirming the correlation with a lethal effect on
the whole mycobacterial cell. A kinetic analysis confirmed 10 as a competitive inhibitor
1
1
1
1
1
1
1
1
1
1
2
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5
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0
1
2
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0
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9
0
1
2
3
4
5
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7
8
9
0
of MbtI, with a K value of 4.2 ± 0.8 M (Figure 3A). Additional tests were performed to
i
1
4
ensure that it was not a PAIN: the addition of BSA and Triton X-100 did not influence
the IC (6.1 ± 0.9 μM and 5.8 ± 1.2 μM, respectively), suggesting that it does not form
50
aggregates with the target. Similarly, the addition of DTT did not impact on the activity of
1
0 (IC50 7.2 ± 1.1 μM), showing that the ligand does not interact with the cysteine
residues of the protein (Figure 3B). The antimycobacterial activity of 10 was tested on
M. tuberculosis H37Rv, providing an MIC99 value (250 µM) similar to those of the
previous inhibitors I and II.11 Moreover, to ascertain that the effects of the compound
were due to mycobactin inhibition, 10 was assayed against the non-pathogenic M. bovis
BCG strain, whose siderophores closely resemble Mtb mycobactins,15 in iron-limiting
conditions, using the chelated Sauton’s medium. The compound showed an MIC99
value of 250 μM, and the Universal CAS liquid assay and quantification of the
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mycobactins16 in treated cultures demonstrated that siderophore concentration
decreased at higher concentrations of the compound (Figure 3C and 3D). This
observation confirmed that the inhibitory effect towards mycobacterial growth was due
to mycobactin biosynthesis inhibition. Finally, 10 was screened, following the previously
published procedure,12 against human MRC-5 fibroblasts to evaluate its cytotoxicity,
revealing an IC > 100 μM, thus indicating a low level of toxicity.
0
1
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0
1
2
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9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Global reciprocal plot of data from MbtI steady-state kinetics analysis towards
chorismic acid, at different concentrations of 10 (A). IC50 plot of 10 in the presence of
BSA (red), Triton X-100 (green) and DTT (blue) (B). Universal CAS assay performed on
M. bovis BCG, grown at different concentrations of 10 (C). Determination of
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mycobactins in the abovementioned cells. Bars represent mean and standard
deviations of three independent experiments (D).
0
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9
0
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9
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9
0
1
2
3
4
5
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8
9
0
2+
Investigations around the Mg ion
6
Meneely et al. demonstrated that, in iron-limiting conditions, the affinity of the MST
2+
enzymes EntC, PchA and Irp9 for Mg is rather low; hence, the ligand is the first driving
2+
element of the catalytic reaction. Subsequently, the binding of the Mg caps the active
site, thus promoting the initiation of the biosynthetic process. After the conversion of
chorismate to isochorismate, a sudden change in the affinity for the metal occurs: the
2+
enzyme tightly binds Mg , preventing the release of the intermediate and allowing the
reaction to quickly evolve towards the formation of salicylate.6
According to this theory, a ligand endowed with inhibitory properties should therefore
be able to bind to the active site of an MST enzyme, without having to interact with or,
2+
much less, be oriented by the Mg ion. On these bases, we undertook an investigation
to verify if such a deduction could be applied to MbtI and to our compounds.
Considering that the presence of Mg2+ has been one of the assumptions of most
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computational studies regarding MbtI, including ours, we realized that unravelling the
binding mechanism of our inhibitors would have a particular significance. In order to
1
1
1
1
1
1
1
1
1
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0
1
2
3
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9
0
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9
0
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9
0
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9
0
1
2
3
4
5
6
7
8
9
0
2+
ascertain the likelihood of a Mg -independent binding mode for our compounds,
docking experiments, combined with molecular dynamics (MD) simulations, were
carried out. The predicted dispositions of 10 into the catalytic site of MbtI was calculated
by docking in the absence of the Mg2+ ion and then refined through 100 ns of MD
simulation with explicit water molecules (see Experimental Section for details). The
results of these studies are shown in Figure 4, which represents the minimized average
structures of 10 within the MbtI binding site. The carboxylic group of the ligand shows
interactions with the hydroxyl group of Tyr385 and the backbone nitrogen of Arg405.
The furan oxygen forms a H-bond with Lys438, whereas the benzonitrile fragment
shows lipophilic interactions with Ile207, Leu268, Thr361 and an H-bond with Lys205.
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9
0
1
2
3
4
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6
7
8
9
0
Figure 4. Minimized average structure of 10 within MbtI binding site (PDB ID 3VEH) in
2+
absence of the Mg ion.
2+
Hence, our modeling approach showed that a Mg -independent binding mode was
indeed possible, although the exact pose of the ligand could hardly be predicted by a
docking/MD simulation study. Therefore, further experimental studies were carried out
to characterize the binding mode of our inhibitor.
Biochemical investigations on 10
Firstly, we acquired further biochemical data on 10, testing its activity at different
2+
concentrations of Mg , with the aim of defining the role of the cofactor in the interaction
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between MbtI and the inhibitor. With this strategy, we envisaged that we would observe
differences in the inhibitory effect of our ligand in the presence of varying amounts of
1
1
1
1
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0
2+
Mg , if the ion were necessary for its binding and correct orientation in the active site.
As expected, MbtI was found to be completely inactive in the absence of its cofactor,
reaching the maximal activity only at 2 mM MgCl . This result confirmed that the ion is
2
essential for enzymatic activity, although the affinity seems rather moderate. Despite
Ferrer et al.17,18 reported on the ability of MbtI to act as chorismate mutase in the
absence of the metal, leading to the synthesis of prephenate, our data corroborate the
19
hypothesis formulated by Ziebart and Toney that such activity is likely due to a faulty
2+
purification of the enzyme. Moreover, the use of increasing concentrations of Mg (> 50
mM) showed inhibitory effects, as the enzyme activity significantly decreased (Figure 5).
Conversely, the enzyme no significant decrease in specific activity when assayed at
2+
concentration of KCl up to 500 mM, confirming that inhibition is specific to Mg , and not
due to increased ionic strength.
2+
The determination of the activity of 10, performed at different concentrations of Mg ,
showed that the metal did not strongly influence the binding of the compound to the
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target. However, in the presence of very high Mg2+ concentrations, the performance of
10 was slightly reduced. This observation further supported the hypothesis that at
0
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0
2+
moderate concentrations of Mg , the ligand preferentially binds (and inhibits) MbtI,
2+
while the metal has a low affinity for the enzyme. At high Mg concentrations, our data
suggest that the ion nevertheless binds to the active site, occluding the catalytic region
and blocking the access of the inhibitor. These data might also indicate that our
2+
compound could prevent the binding of Mg in a competitive fashion, becoming slightly
less effective when the concentration of the metal reaches the higher millimolar range
(Figure 5).
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+
Figure 5. MbtI enzyme activity as a function of [Mg ] (A). Profiles of the IC of 10,
50
2+
obtained at different concentrations of Mg (B).
1
1
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0
Synthesis and activity of a probe (11)
To date, most MbtI inhibitors reported in the literature, including our candidates, share
a common carboxylate motif that is supposedly responsible for functional sequestration
of the metal ion within the active site.20 However, according to our hypothesis, this
interaction may not be necessary neither for the binding of the ligand, nor for the
resulting inhibitory effect. In order to further verify this theory, we designed and
2+
synthesized a probe molecule, introducing a chemical modification on 10. The Mg ion
is characterized by a small ionic radius, high charge density and tendency to bind water
molecules in the inner coordination sphere, rather than bulkier ligands. Being a “hard”
ion, it prefers to bind “hard” oxygen-containing ligands, such as carbonyls, carboxylates,
phosphates, hydroxyls, and water.21 Therefore, the carboxylate pharmacophore motif
was converted to an amide function, affording the analog 11 (see Scheme S2 in SI). Its
2+
docking into MbtI in the absence of Mg suggested that a very similar binding mode to
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that proposed for 10 was likely (Figure S62). The biological tests on compound 11
confirmed a very good inhibitory activity (IC50 = 17.3 ± 1.9 μM), thus supporting our
hypothesis that the metal chelation should not be considered an essential
pharmacophore feature to develop inhibitors of MbtI.
0
1
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8
9
0
Structure of MbtI in complex with 10
In the context of our attempts to elucidate the structure of complexes between MbtI
and 5-phenylfuran-2-carboxylic acid-based inhibitors, we obtained cocrystals with the
lead compound 10: structural data and refinement statistics are summarized in Table 2.
These experiments were undertaken to definitively characterize the binding mode of
our candidates, thus providing a reliable means to verify the results of the computational
simulations, as well as our previous observations. Furthermore, considering both the
limited size of the compounds and the high plasticity of the active site, we were
convinced that structural studies would assume a particular significance in the
identification of the key enzyme-inhibitor interactions.12
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Among the 3D structures of MbtI available in the PDB, two different conformations
2
have been reported, which have been described as “open” (e.g. PDB ID 2G5F) and
1
1
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7
“
closed” (e.g. PDB ID 3LOG) , depending on the relative position of two mobile loops
with respect to the active site. In the closed form, the flexible sequences (residues 268-
93 and 324-336)20 are bent over the binding pocket, while in the open form they are
2
tilted upward. A comparative analysis of previously published structures allowed us to
identify the intermolecular bonds responsible for this conformational shift. In particular,
the closed state seems to be determined by the formation of a H-bond between a
suitable moiety of the ligand and the NH of Gly270 and/or the hydroxyl group of Thr271:
these contacts effectively drag the first mobile loop towards the active site. In turn,
Thr271 establishes a bond with His334, thus pulling the second mobile loop in the same
direction. The link between the flexible regions is further stabilized by additional
interactions, formed by adjacent amino acids. Overall, this movement determines the
capping of the active site. The chemical entity responsible for the interaction with
Gly270 or Thr271 is a carbonate anion (or an ordered water molecule) in 3LOG, and a
carboxyl group in PDB IDs 3ST6 and 3VEH (chains A, B, C). Interestingly, in PDB IDs
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3
RV7, 3RV8, 3RV9, the absence of this H-bond induces an open conformation, despite
the orientation of the ligands does not differ significantly compared to the one observed
in 3VEH. In chain D of 3VEH, the increased distance between the COOH and Thr271
0
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(3.21 Å vs 2.70-2.89 Å) determines an intermediate conformation. Another intermediate,
but overall open state can be observed in 3RV7 chain B, in which a contact between
Thr271 and Ser331 induces a slightly more closed configuration compared to the other
chains. The peculiar binding mode of the bulkier inhibitor of PDB ID 3RV6 determines
an unprecedented conformation, in which the opening of the mobile loops is
characterized by a particularly wide angle. Notably, these mobile regions are
characterized by high B-factors and partial lack of supporting electron density,
especially when the conformation is open, despite some chains show stabilized
conformations, mostly in the 324-336 loop, due to different crystal packing interactions
involving these regions.
In the complex MbtI-10, the enzyme conformation can be defined as an open state for
the ensemble of the four molecules in the ASU, despite the partially incomplete electron
density of the mobile loops (unresolved regions for chain A: 271-276; chain B: 270-290
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and 328-334; chain C: 275-282; chain D: 274-277). The adoption of this conformation is
due to the fact that 10 does not possess a suitable functional group in the correct
orientation to establish a H-bond with Gly270 or Thr271. Consequently, the
arrangement of the flexible loops of MbtI-10 is similar to that of 2G5F, 3RV7, 3RV8, and
1
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3RV9, with root-mean-square deviations in the range 1-4 Å (calculated for residues 325-
335).
An electron density compatible with the presence of the ligand is detectable in all four
chains of the ASU, with the furan-2-carboxylic acid region always well-defined, and
weaker electron density for the phenyl portion. The analysis of the binding mode of the
compound revealed the presence of H-bonds between its carboxylic group and Tyr385,
Arg405, and an ordered water molecule; the oxygen of the furan interacts with Arg405,
while the phenyl ring forms a cation-π interaction with Lys438 and a Van der Waals
contact with Thr361. Finally, the cyano group forms a H-bond with Lys205, a key amino
acid involved in the first step of the catalytic reaction (Figure 6). This residue is likely
responsible for the activation of a water molecule for nucleophilic attack (S 2”) on C2 of
N
chorismate, which leads to the release of the hydroxyl group at position 4, after its
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protonation mediated by Glu252, thereby affording isochorismate. One may speculate
that the absence of an interaction with the pivotal Lys205 in isochorismate-based
derivatives accounts for their slightly weaker inhibitory activity with respect to 10. The
superposition of MbtI-10 with 3RV7 (Figure S54) shows that the arrangement of the
amino acid side chains around the ligands is similar, with the aromatic carboxylic group
of the AMT derivative occupying the same position as the acid moiety of 10. The most
significant difference is represented by the ability of the nitrile group of 10 to form a H-
bond with Lys205, thereby hindering its physiological functions.
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Figure 6. Ribbon diagram of the MbtI-10 structure (PDB ID 6ZA4): the interactions of the
ligand with the side chains (in sticks) and the water molecules (blue spheres) are
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represented as dashed lines. The blue mesh represents the electron density around the
ligand (contoured at 1σ).
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2+
Despite our experiments were carried out in the presence or in the absence of Mg in
2+
the crystallization buffer (2-5 mM of different Mg salts), we never observed any bound
Mg2+ in the MbtI-10 cocrystal structure. The orientation of the ligand is substantially in
2+
agreement with the predicted binding mode of 10 in the absence of Mg , as shown by
the overlay of the two models (Figure S55). The binding poses in the two complexes
exhibit a root-mean-square deviation of 1.6 Å and similar interactions. The main
differences are related to the orientation of the side chain of Arg405, which was not
predicted to interact with the ligand, as well as the side chains of Thr271 and His334,
which are shifted away from the binding site. As previously mentioned, the absence of
interactions between the ligand with the amide group of Gly270 or the hydroxyl group of
Thr271 seems to favor the outward movement of the backbone in the two mobile loops,
thereby leading to an open conformation. Such shift could hardly be predicted by
computational simulations, as those were based on a closed form of the enzyme. As for
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Arg405, the analysis of electron density indicates a high flexibility of its side chain,
which is otherwise modeled, where supporting density is present, as different rotamers.
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2+
Structure of MbtI with the Mg ion
Although the role of the Mg2+ cofactor in the catalytic activity of MbtI has been the
4
subject of a number of publications, its role remains elusive. Our numerous attempts to
2+
obtain a structure of MbtI in ternary complex with 10 and Mg failed, invariably leading
to the cocrystal described above. Notably, the only available structure of MbtI showing a
bound Mg2+ ion was obtained by Chi and co-workers (3RV6). However, as stated by
the authors, its binding mode under those conditions may not necessarily reproduce the
physiological situation: the ion was found to be hexacoordinated by water molecules,
interacting with the enzyme and the inhibitor only through the surrounding waters,
inconsistently with both its supposed role in catalysis18 and the authors’ predicted
binding mode. Therefore, we decided to further pursue the investigation of the binding
and coordination of Mg2+ into the active site of MbtI, in order to clarify its role in the
enzymatic mechanism.
20
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Considering the inherent difficulty in obtaining MbtI-Mg2+ cocrystals, as well as the
results of our biochemical assays, we hypothesized that we could compensate for the
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2+
rather low affinity of MbtI for its cofactor by using saturating concentrations of Mg .
Interestingly, 2.1 Å resolution data were collected from a non-isomorphous crystal
grown under these conditions (Table 2).
The crystal structure derived from these data presents four molecules in the ASU, with
a well-defined electron density in all regions, including the previously observed mobile
loops that are now stabilized into a closed conformation, superimposable with the one
observed in 3LOG or 3ST6.7,20 The analysis of the active site evidenced electron
2+
density fully consistent with the presence of a Mg ion, though its coordination sphere
2+
is slightly different in the various chains. In chain A (Figure S56), the Mg interacts with
the oxygen of Gly421, with four ordered water molecules, and with a sulfate anion.
Through two of the water molecules, it also contacts Glu431 and Glu434. Chain C does
not show the presence of the ion; in its absence, Glu431 binds a water molecule and
there is no clear electron density for the side chain of Glu434. The coordination pattern
2+
of Mg in chains B and D is analogous (Figure 7): the ion could be modeled as directly
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