X-ray crystallographic and NMR studies of pantothenate
synthetase provide insights into the mechanism of
homotropic inhibition by pantoate
Kalyan Sundar Chakrabarti*, Krishan Gopal Thakur, B. Gopal and Siddhartha P. Sarma
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India
Keywords
competitive inhibition; NMR; pantothenate
biosynthesis; substrate binding; X-ray
crystallography
Correspondence
S. P. Sarma, 207, Molecular Biophysics
Unit, Indian Institute of Science, Bangalore
560012, India
Fax: +91 80 23600535
Tel: +91 80 22932839
E-mail: [email protected]
*Present address
Department of Biochemistry and Howard
Hughes Medical Institute, MS009 Brandeis
University, Waltham, MA, USA
Database
The
1
H
N
,
15
N,
13
C
a

binding to the ATP-binding site induced large changes in structure, mainly
for backbone and side chain atoms of residues in the ATP binding
HXGH(34–37) motif. Sequence-specific NMR resonance assignments and
solution secondary structure of the dimeric N-terminal domain, obtained
using samples enriched in
2
H,
13
C, and
15
N, indicated that the secondary
structural elements were conserved in solution. Nitrogen-15 edited two-
dimensional solution NMR chemical shift mapping experiments revealed
that pantoate, at 10 mm, bound at these two independent sites. The solu-
tion NMR studies unambiguously demonstrated that ATP stoichiometri-
cally displaced pantoate from the ATP-binding site. All NMR and X-ray
studies were conducted at substrate concentrations used for enzymatic
characterization of pantothenate synthetase from different sources [Jonczyk
R & Genschel U (2006) J Biol Chem
281,
37435–37446]. As pantoate bind-
ing to its canonical site is structurally conserved, these results demonstrate
that the observed homotropic effects of pantoate on pantothenate biosyn-
thesis are caused by competitive binding of this substrate to the ATP-bind-
ing site. The results presented here have implications for the design and
development of potential antibacterial and herbicidal agents.
Structured digital abstract
l
MINT-7301221: PS (uniprotkb:P31663) and PS (uniprotkb:P31663) bind (MI:0407)byx-ray
crystallography (

A. thaliana have shown that there is a high degree of
conservation ( 40% sequence identity) among the
enzymes from various sources, with the E. coli and
A. thaliana enzymes sharing 42% [9] sequence identity,
and the M. tuberculosis and A. thaliana enzymes shar-
ing 36% sequence identity [11]. Recently, it has been
shown that, unlike their bacterial counterparts, the
plant PSs are subject to allosteric control, and that this
allosteric control arises from the homotropic effects of
one of the substrates, i.e. pantoate [8,9]. One of the
interesting features of the plant PSs is the presence of
a conserved 24 residue insertion in the sequence [9].
This sequence of amino acids, which is missing in the
sequence of bacterial enzymes, is thought to play a sig-
nificant role in the proposed allostery in plant PSs [9],
through long-range interactions.
All PSs are known to be dimeric, with distinct N-ter-
minal and C-terminal domains in each protomer.
Structural studies have shown that the catalytic site
residues, as well as the dimerization interface, lie in the
N-terminal domain of the bacterial enzyme. Enzymatic
and other biochemical studies have shown that for PS
from M. tuberculosis, pantoate, ATP and b-alanine
have K
m
values of 0.13 mm, 2.6 mm and 0.8 mm,
respectively. Enzymatic studies of plant PSs have indi-
cated that the affinity of these substrates echoes that
observed for the bacterial enzyme. However, pantoate
at concentrations above 5 mm has been shown to inhi-

PS
Full-length PS is a dimer of 63 kDa. The protein was
found to aggregate at the concentrations used for the
NMR studies, and did not provide spectra of adequate
quality under a number of different sample conditions
(phosphate buffer, pH 6.8; acetate buffer, pH 5.5; and
Tris buffer, pH 7.5), in the presence of substrates
(10 mm pantoate, ATP, and b-alanine), in the presence
of protein stabilizers such as arginine, glutamate and
proline [14–17], or even in the presence of detergents
(Chaps [18] or dodecylphosphocholine) [17].
Like PS (283 amino acids), nPS (residues 1–176)
exists as a dimer in solution. Evidence for this comes
from gel filtration data and from the experimentally
determined rotational correlation time (s
c
) of 17.25 ns
for nPS, calculated from the measured average
15
N T
1
and T
2
relaxation time constants [19].
The C-terminal domain of PS (cPS; residues 177–
283) showed spectra of poor quality, similar in nature
to that of the full-length PS under similar conditions.
Coconcentration of cPS with nPS did not prevent
aggregation of cPS. It is possible that the C-terminal
Structure and binding studies of nPS K.S. Chakrabarti et al.

13
C
a
,
13
C
b
,
13
C¢ and
15
N
nuclei of the homodimeric nPS was obtained from
transverse relaxation optimized spectroscopy (TROSY)
[20] versions of HNCA, HN(CO)CA, HNCACB,
HN(CO)CACB and HNCO [21–23] experiments. The
resonance assignments leading to the establishment of
sequential connectivity for Glu5–Glu20 are shown in
Fig. S1. The backbone assignment for the amide
1
H–
15
N pairs is 93% complete, whereas the backbone
and side chain
13
C
a
and
13
C¢ and

1
H
N
NOE pattern
[27] that define the secondary structure are shown in
Fig. S2. The 3
10
helices formed by Pro59–Gln61 and
Glu119–Ser122 that are present in the crystal struc-
tures of E. coli and M. tuberculosis PS, are missing in
the solution structure. Helix IX, which is a continuous
helix (16 residues) in the PS crystal structure, is broken
at Thr132 and resumes only at Phe138 in the solution
structure. In the absence of substrates, the crystal
structure of the E. coli PS has shown that the protein
has an extensive dimer interface constructed from
strands formed by Tyr108–Val111 from each subunit
in an antiparallel b-sheet arrangement. Under the con-
ditions of these solution studies, the resonances for
Tyr108 could not be identified. However, we have been
able to identify resonances for Val109, Asp110, and
Val111. Prediction of backbone u and w dihedral
angles from calculated
13
C secondary chemical shifts,
using the program talos [28], indicates that this region
adopts a b-strand conformation under solution condi-
tions. The occurrence of a b-strand is corroborated by
the absence of backbone H
N

tion spectra were conducted by addition of substrates
in the concentration range used for biochemical char-
acterization of PS [7–9]. Changes in chemical shifts
were observed at pantoate concentrations > 5 mm,
indicating weak binding coupled to an exchange rate
that is intermediate on the NMR timescale. Addition
of d-pantoate (10 mm) to nPS caused deviations in
chemical shifts of more than 0.03 p.p.m. for 24 resi-
dues. Addition of ATP brought about further changes
in the heteronuclear single quantum coherence (HSQC)
spectrum of nPS. The changes in chemical shifts in the
HSQC spectra for select residues that lie in the panto-
ate-binding and ATP-binding pocket of nPS are shown
in Fig. 2. The residues that showed deviations in chem-
ical shifts of more than 0.03 p.p.m. upon binding pan-
toate and ATP fell into three categories. Residues in
class I were almost uniquely shifted in the presence of
ATP (Fig. 2, row 1). Residues in class II showed a net
perturbation in chemical shifts in the presence of both
pantoate and ATP (Fig. 2, row 2). Finally, residues in
class III showed deviations in the presence of pantoate
but reverted back to the free protein resonance upon
addition of ATP (Fig. 2, row 3). The backbone atoms
of Val111 and Gly113, which are involved in dimeriza-
tion (see above), were also affected by substrate bind-
ing. However, the backbone of His106, whose side
chain is involved in dimerization, and a residue
Fig. 2. The overlay of selected regions of
1
H–

bone H
N
chemical shift reverted back to the resonance
position observed for the free form of the protein. The
observed changes in resonance positions in the HSQC
spectrum upon addition of pantoate followed by ATP
can be reconciled if one takes into consideration that a
molecule of pantoate is bound in the ATP-binding
pocket in addition to its canonical binding site, and
that ATP displaces this molecule of pantoate.
b-Alanine and pantothenate did not bind nPS under
the experimental conditions. Attempts to study the
binding of ATP alone to the protein were unsuccessful,
as the protein precipitated upon addition of ATP in
the absence of pantoate. An important feature of the
NMR study is the fact that a single set of resonances
were observed for nPS in the absence or presence of
substrates. This strongly suggests that binding of pan-
toate and ATP preserves the dimer symmetry in solu-
tion. In an effort to unambiguously identify and
understand the nature of binding of pantoate at the
ATP-binding site, we also structurally characterized
the pantoate-bound form of nPS using X-ray crystal-
lography, as described below.
Solution and refinement of the structure using
X-ray crystallography
The crystal structure of the pantoate–nPS complex was
solved by molecular replacement using phaser [29] in
ccp4 [30]. The first solution with a log likelihood gain of
1995 was obtained using the edited coordinates of

The crystal structure of the nPS–pantoate complex
indicated that it is a dimer in the asymmetric unit. The
backbone structure of the dimeric N-terminal domain
of the E. coli PS determined here is identical to that
determined earlier for the full-length protein [13]. The
A
B
Fig. 3. The absolute value of deviation in chemical shift (p.p.m.) of
residues of nPS upon binding to (A)
D-pantoate as a function of
sequence, and (B)
D-pantoate and ATP as a function of sequence.
Residues that showed a deviation of more than 0.03 p.p.m. were
considered to have a direct interaction with the ligand(s) (see text
for details).
K.S. Chakrabarti et al. Structure and binding studies of nPS
FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS 701
dimer interface in PS is extensive. The dimer interface
is stabilized by 19 hydrogen bonds and two salt
bridges, with a total buried surface area of 1240 A
˚
2
(Table S3). The structure of the complex showed two
molecules of pantoate bound to one monomer
(chain A) and a single pantoate bound to the second
monomer (chain B). Thus, pantoate binding occurs in
the canonical pantoate-binding site (site I) in both
monomers with full occupancy, and at the ATP-bind-
ing site (site II) in one monomer with full occupancy.
Figure 4A shows the dimeric nPS molecule bound to

and M. tuberculosis proteins, and are involved in iden-
tical conserved interactions with pantoate. Further-
more, the pantoate molecule is hydrogen-bonded to
other protein residues via networks of water-mediated
hydrogen bonds. The backbone nitrogen and carbonyl
oxygen of Thr29 and the side chain oxygen of Ser54
are hydrogen-bonded to the O4 atom of pantoate via a
network that consists of two water molecules. The
backbone carbonyl oxygen of Phe56 is also hydrogen-
bonded to the O4 atom of pantoate via one of the
water molecules in the same network. The phenolic
oxygen of Tyr71 is hydrogen-bonded to the O1 atom
of pantoate via a water molecule included in a network
of five water molecules. Also hydrogen-bonded to the
O1 atom of pantoate via this cluster of water mole-
cules is the Ne nitrogen of the imidazole ring of His37.
In addition to the hydrogen-bonding interactions, the
pantoate molecule is also involved in hydrophobic con-
tacts with the protein, as shown in Fig. S3. The Ca
atoms of residues in site I do not exhibit more than
1A
˚
deviations as compared with the substrate-free
form of the protein.
Pantoate bound at site II
The structure of nPS distinctly shows the presence of a
second molecule of pantoate bound at site II in one of
the monomers, albeit at full occupancy. The pantoate
in site II (Fig. 5) has the same orientation as the pan-
toate molecule in site I. In contrast to the pantoate

Unique reflections 43 231 (5676)
Completeness (%) 96.2 (87.5)
I ⁄ r(I) 12.8 (2.0)
R
merge
(%) 5.1 (46.4)
Redundancy 2.5 (2.4)
Refinement
R
cryst
(%) 18.7
R
free
(%) 24.4
rmsd from ideal bond
length (A
˚
) ⁄ angles (°)
0.024 ⁄ 2.03
No. of protein residues 332
No. of water molecules 279
No. of ligand molecules 3
Wilson B-factor (A
˚
2
) 20.3
Ramachandran plot (%)
Most favored regions (%) 99.1
Allowed regions (%) 0.9
a

and O4 atoms of pantoate occupy the positions of the
A
B
Site I
Site II
Site I
N
N
C
Gln61
Gln155
Asp152
Lys151
Glu150
Tyr71
Phe56
Ser54
Thr29
His37
Lys39
Val175
Phe56
Ser54
Thr29
His37
Lys39
Val175
Glu150
Lys151
Asp152

bone atoms in this region are small. Significant
changes are observed for residues that line the site II
pantoate-binding pocket. The residues that are shifted
by more than 1 A
˚
upon pantoate binding are listed in
Table 2.
Figure 7 shows a superposition of chain A of E. coli
PS (1IHO.pdb) on the A-chain and B-chain of nPS in
the region of the HXGH (34–37) motif (where
X = Asp for E. coli PS and Glu for M. tuberculosis
and A. thaliana PS). As compared with the substrate-
free form, the imidazole rings of His34 in the A-chain
and B-chain of nPS move by  4A
˚
and 5.5 A
˚
,
whereas the backbone Ca atom moves by only
 0.9 A
˚
and  0.5 A
˚
, respectively. The backbone Ca
atom of Asp35 moves by as much as 1.62 A
˚
and
Fig. 6. An expanded view of the super-
posed substrate-binding sites of nPS (green)
and M. tuberculosis PS (magenta). In nPS,

Phe56
Tyr71
Fig. 5. Protein–ligand and water-mediated protein–ligand hydrogen bond interactions (broken lines) that stablize the pantoate molecules in
site I and site II of nPS. Oxygen atoms of pantoate and water are colored magenta and ochre, respectively. Direct protein–ligand hydrogen
bonds are colored magenta, and water-mediated hydrogen bonds are colored red. Several networks of water-mediated hydrogen bonds can
be observed.
Structure and binding studies of nPS K.S. Chakrabarti et al.
704 FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS
0.34 A
˚
in the A-chain and B-chain of nPS. Finally, for
His37, the A-chain Ca atom is displaced by 0.72 A
˚
and the B-chain Ca atom deviates by 0.23 A
˚
, concomi-
tant with a very minor deviation in the positions of
the imidazole rings.
Comparison of side chain torsion angles for these
residues between the free and the liganded forms shows
that the v
1
torsion angle for His34 changes from )64°
in the former to 164° and )169° in the A-chain and B-
chain of nPS, respectively. The v
1
torsion angle for
Asp35 changes from ) 75.20° to )171.65° and )89.25°
in the A-chain and B-chain of nPS, and in the case of
His37, the v

His34 in NMR spectra. In the presence of ATP, the
His34 resonance reverts back to the position of the
Table 2. The residues that show > 1 A
˚
displacement upon binding
pantoate at both site I and site II.
Residue Displacement (A
˚
)
Arg18 1.06
His34 1.10
Asp35 1.75
Gly36 1.74
Arg64 1.89
Pro65 1.32
Glu66 1.57
Asp67 1.45
Arg70 1.10
Tyr71 1.02
Pro72 1.06
Thr74 1.17
Leu75 1.13
Gln76 1.41
Glu77 1.19
Lys96 1.38
Glu97 1.27
Tyr99 1.07
Pro100 1.57
Fig. 7. Superimposition of the A-chain of nPS (green), the B-chain
of nPS (cyan), and the substrate-free protein (1IHO.pdb; magenta).

the enzyme is not clear. Kinetic and structural studies
of E. coli PS [3,8,13] and M. tuberculosis PS [7,11,12]
suggest that active sites of the dimer are independent
of each other, and that reactions occur at both sites
simultaneously. In plant PSs, dimerization has been
shown to have a very different effect on the kinetic
properties of the enzyme [9]. A study of the sequence
(Fig. 9) and the modeled structure of A. thaliana PS
(Fig. S5 and Table S1) shows that it has a large loop
at the dimer interface and fewer energetically favorable
N-terminal and C-terminal interdomain interactions,
such as the hydrophobic, hydrogen bond and ionic
interactions, than E. coli PS. Thus A. thaliana PS is
expected to be structurally more open than E. coli PS.
Importantly, the residues at the active site are highly
conserved, and thus the nature of the interactions
between enzyme and substrate are expected to be iden-
tical. M. tuberculosis PS has a significantly higher
number of such energetically favorable interactions
that stabilize the ‘closed’ conformation. For PS from
L. japonicas, the reported dissociation constants for
the first and second molecules of pantoate are
0.042 mm and 5.33 mm, respectively; that is, the sec-
ond pantoate molecule binds with 100-fold lower affin-
ity than the first. Thus, the substrate inhibition by
pantoate is consistent with the finding that pantoate
binds to the ATPase site as well, and is displaced from
this site at equimolar concentrations of ATP.
On the basis of kinetic studies, an enzymatic scheme
has been proposed [9] in which the second molecule of

site on the N-terminal domain of PS [9]. A kinetic
scheme to explain this observation is shown in Fig. 10.
Such competitive inhibition by one of the substrates
of a reaction has also been observed in the case of ade-
nylosuccinate synthetase, one of whose substrates,
IMP, binds to the GTP-binding site and inhibits the
reaction [37–39]. The binding of pantoate to the
ATP-binding site also reveals the origin of the negative
cooperativity with b-alanine [9] at high pantoate con-
centrations. The model of A. thaliana PS (Fig. S5)
shows that there is a 24 residue insertion between
Thr105 and His106 of the E. coli sequence [9]. The
HETWIRVER(131–139) motif, which is part of the 24
residue insertion, and is present in all plant PSs and
absent in bacterial PSs, has been shown to be impor-
tant for the observed allosteric behavior [9] of plant
PSs. This insertion sequence is rich in Gly residues
N-terminal to the HETWIRVER(131–139) motif,
suggesting that this loop may adopt an extended
conformation in A. thaliana PS. Mutations in or dele-
tion of this stretch of residues at the dimer interface are
known to affect the catalytic properties of A. thaliana
PS [9], as a consequence of disruption of long-range
allosteric interactions. Gly102, which is N-terminal to
the insertion and completely conserved in E. coli,
M. tuberculosis and A. thaliana, is virtually unaffected
by substrate binding (Fig. 2). His106 in E. coli PS,
which is located at the end of the insertion sequence in
A. thaliana PS, is also unaffected by substrate binding.
His106 is involved in intersubunit interactions via its

NH
4
Cl,
13
C
6
H
12
O
6
,
13
C
6
2
H
5
1
H
7
O
6
,
12
C
6
2
H
5
1

tidine tag construct was a kind gift from C. Abell and
coworkers (University of Cambridge, UK).
Cloning of nPS
Specific primers were designed (forward primer, 5¢-GAC
CAGCTTCATGTGGCCATCGTGCAGGTT-3¢; reverse
primer, 5¢-CACGATGGCCACATGAAGCT-3¢) to PCR-
amplify the coding regions of these domains in the panC
gene from E. coli genomic DNA [44]. The amplified prod-
uct was ligated into an appropriately digested pet21a plas-
mid vector between Nde1 and EcoR1 restriction sites, to
obtain the clone of the N-terminal domain of PS. She gene
starts with an unusual codon, GTG. The C-terminal
domain of PS was cloned as a cytb5-based fusion protein.
The gene corresponding to the C-terminal domain of PS
was cloned downstream of the cytb5 gene, between BamH1
and EcoR1 restriction sites [45].
Protein expression and purification
All proteins were expressed using E. coli BL21(DE3) as
host strain, and purified using the protocol described
below.
Fig. 10. A model for substrate inhibition under conditions of high
pantoate concentration. In this model, a second pantoate molecule
binds to the enzyme–substrate complex to form a catalytically inert
E.Pt.Pt* species, where Pt is a pantoate bound to the canonical
pantoate-binding site, and Pt* is a second molecule of pantoate
bound to the ATP-binding site. ATP at high concentrations can
displace Pt*, and the reaction goes to completion.
K.S. Chakrabarti et al. Structure and binding studies of nPS
FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS 707
Purification of the N-terminal domain of PS

Geisbrecht et al. [43].
Separation of the C-terminal domain of PS from
the fusion host
The cleaved C-terminal domain of the PS was separated
from the fusion host as well as the TEV protease by immo-
bilized metal ion affinity chromatography (Ni
2+
) chroma-
tography. Apo-cytb5 and TEV protease bind to the
column, and the protein of interest is collected in the
flow-through. The protein was dialyzed against 20 mm
phosphate buffer (pH 6.8) containing 20 mm NaCl, 2 mm
dithiothreitol, and 0.01% sodium azide.
Purification of the full-length PS
The cell lysate containing the full-length PS was passed
over a Q-Sepharose column. The protein was eluted with a
linearly increasing gradient of NaCl. Fractions containing
PS were pooled, and passed over a Ni
2+
-nitrilotriacetic acid
column. Protein was eluted from the metal affinity column
with a linearly increasing gradient of imidazole. The PS
was further purified in the ATP-bound form by passage
through a Sepharose blue column that is crosslinked with
cibacron blue dye [46], and then eluting the protein with a
buffer containing ATP.
Crystallization and data collection
For protein crystallization, the purified protein was
exchanged in a dilute buffer of 20 mm Tris (pH 8.0) con-
taining 50 mmd-pantoate [47]. The concentration of the

domain (residues 1–176) of subunit A of E. coli PS
(1IHO.pdb) was used as a model for molecular replacement.
Preparation of isotopically enriched samples of
nPS
Uniformly
2
H,
13
C,
15
N enriched ILV - methyl protonated
samples of nPS were prepared following protocols described
previously [50,51]. All perdeuterated samples of nPS were
subjected to unfolding–refolding. The levels of isotopic
enrichment for the different samples described above were
ascertained using electrospray MS.
Samples for NMR spectroscopy
All of the NMR samples (0.6–0.8 mm) were prepared in
20 mm phosphate buffer, 20 mm NaCl, 2 mm dithiothreitol,
and 0.01% NaN
3
(pH 6.8).
Structure and binding studies of nPS K.S. Chakrabarti et al.
708 FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS
NMR spectroscopy
NMR spectra were acquired on a Bruker-Avance spectrom-
eter operating at a proton frequency of 700 MHz, equipped
with a 5 mm triple-resonance cryoprobe fitted with a single
(z-axis) pulsed field gradient accessory. The
15

in the
15
N dimension. Shifts were calculated using the
equation
D ¼½Dd
2
HN
þð0:1Dd
N
Þ
2

1=2
where D is the cumulative chemical shift deviation, Dd
HN
is
the change in chemical shift of the proton, and Dd
N
is the
change in the chemical shift of the nitrogen [56].
Modeling and comparison of the structures
of PSs
dali [57] was used to superimpose the crystal structures of
PSs from E. coli and M. tuberculosis (1IHO.pdb and
1MOP.pdb) for structural alignment. The sequence align-
ment of PSs from E. coli, M. tuberculosis and A. thaliana
was achieved using clustalw2 [58]. Finally, modeller [59]
was used to model the A. thaliana PS structure, using the
structures of PSs from E. coli and M. tuberculosis.
Structural analyses

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Supporting information
The following supplementary material is available:
Fig. S1. Sequential connectivity walk along the protein
backbone for Glu5–Glu20 of nPS.
Fig. S2. Secondary structure distribution of nPS in
solution along with the sequence, short-range NOE
pattern, and secondary chemical shifts of

from supporting information (other than missing files)
should be addressed to the authors.
Structure and binding studies of nPS K.S. Chakrabarti et al.
712 FEBS Journal 277 (2010) 697–712 ª 2010 The Authors Journal compilation ª 2010 FEBS