Abbreviations ACP, acyl carrier protein; FabG, b-ketoacyl acyl carrier protein reductase; FAS, fatty acid synthase; PfACP, Plasmodium falciparum acyl carrier protein; PfFabG, Plasmodium
Trang 1b-ketoacyl acyl carrier protein reductase responsible
for interactions with Plasmodium falciparum acyl
carrier protein
Krishanpal Karmodiya, Rahul Modak, Nirakar Sahoo, Syed Sajad and Namita Surolia
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, India
The human malaria-causing parasite Plasmodium
falci-parum harbors the type II fatty acid synthase (FAS)
[1,2], which is essential for its sustenance and survival
In contrast to the multifunctional FAS enzyme in the
type I pathway operating in humans [3], the type II
FAS system has discrete enzymes for each step of the
pathway Type II FAS in P falciparum is one of the
pathways specific to its ‘plastid’ and has been validated
as a unique target for developing new antimalarials [4–8]
During the elongation cycle of type II FAS, the growing acyl chain, i.e butyryl–acyl carrier protein (ACP), is elongated successively in each round by two carbon units by the action of four enzymes acting con-secutively First, b-ketoacyl ACP synthase (either FabB
or FabF) elongates the acyl-ACP of the Cn acyl chain
Keywords
fatty acid synthase; fluorescence; malaria;
protein–protein interactions; surface
plasmon resonance
Correspondence
N Surolia, Molecular Biology and Genetics
Unit, Jawaharlal Nehru Centre for Advanced
Scientific Research, Jakkur, Bangalore
560064, India
Fax: +91 80 22082766
Tel: +91 80 22082820-21
E-mail: surolia@jncasr.ac.in
(Received 14 June 2008, revised 23 July
2008, accepted 25 July 2008)
doi:10.1111/j.1742-4658.2008.06608.x
The type II fatty acid synthase (FAS) pathway of Plasmodium falciparum is
a validated unique target for developing novel antimalarials, due to its intrinsic differences from the type I pathway operating in humans b-Ketoacyl acyl carrier protein (ACP) reductase (FabG) performs the NADPH-dependent reduction of b-ketoacyl-ACP to b-hydroxyacyl-ACP, the first reductive step in the elongation cycle of fatty acid biosynthesis In this article, we report intensive studies on the direct interactions of Plasmo-diumFabG and Plasmodium ACP in solution, in the presence and absence
of its cofactor, NADPH, by monitoring the change in intrinsic fluorescence
of P falciparum FabG (PfFabG) and by surface plasmon resonance To address the issue of the importance of the residues involved in strong, spe-cific and stoichiometric binding of PfFabG to P falciparum ACP (PfACP),
we mutated Arg187, Arg190 and Arg230 of PfFabG The activities of the mutants were assessed using both an ACP-dependent and an ACP-indepen-dent assay The affinities of all the PfFabG mutants for acetoacetyl-ACP (the physiological substrate) were reduced to different extents as compared
to wild-type PfFabG, but were equally active in biochemical assays with the substrate analog acetoacetyl-CoA Kinetic analysis and studies of direct binding between PfFabG and PfACP confirmed the identification of Arg187 and Arg230 as critical residues for the PfFabG–PfACP interac-tions Our studies thus reveal the significance of the positively charged⁄ hydrophobic patch located adjacent to the active site cavities of PfFabG for interactions with PfACP
Abbreviations
ACP, acyl carrier protein; FabG, b-ketoacyl acyl carrier protein reductase; FAS, fatty acid synthase; PfACP, Plasmodium falciparum acyl carrier protein; PfFabG, Plasmodium falciparum b-ketoacyl acyl carrier protein reductase; SPR, surface plasmon resonance.
Trang 2to a Cn+ 2, b-ketoacyl form The b-ketoacyl-ACP thus
formed is reduced to b-hydroxyacyl-ACP by an
NADPH-dependent b-ketoacyl ACP reductase (FabG)
The b-hydroxyacyl group is then dehydrated to an
enoyl-ACP by a b-hydroxyacyl ACP dehydratase
(FabZ or FabA) Reduction of the enoyl group by an
enoyl ACP reductase (FabI, FabK or FabL) finally
produces Cn+ 2 acyl-ACP, which can either re-enter
the elongation cycle, or be hydrolyzed to ACO and the
acyl moiety for the synthesis of phospholipids or
sphingolipids, or become diverted for other
modifica-tions [9] All of the enzymes participating in type II
FAS interact, but not much is known about the
resi-dues involved in interactions
Plasmodium falciparumACP (PfACP) is a small
pro-tein, with a flexible conformation, which shuttles the
substrates between the enzymes of the pathway
PfACP is a nuclear-encoded and plastid-targeted
pro-tein of 137 amino acids that includes leader and transit
sequences Mature PfACP consists of 79 amino acids
(residues 58–137) with a preponderance of acidic
resi-dues [10] Two-dimensional NMR [11] has revealed
that PfACP has a defined but flexible tertiary structure
dominated by four a-helices located at residues 4–15
(helix I), 37–51 (helix II), 57–61 (helix II¢) and 66–74
(helix III), all connected by loops with a long
struc-tured turn between helix I and helix II The unusually
mobile structure of ACP can be best represented as a
dynamic equilibrium between two conformers Highly
mobile portions of PfACP include the loop regions
and helix II
FabG is highly conserved across species, and is the
only known isoform that functions as a ketoacyl
reductase in the type II FAS system Recently, the
crystal structure of P falciparum FabG (PfFabG) has
been solved [12], and suggests that the interactions of
PfFabG with the 4¢-phosphopantetheine moiety of
PfACP are hydrophobic in nature Plasmodium FabG
with a lone tryptophan provides an ideal system with
which to study ligand-induced conformational changes
by monitoring the change in its intrinsic fluorescence
In these studies, we have investigated the interactions
between PfFabG and PfACP using a combination of
computational, biochemical and biophysical methods
We have been able to identify specific surface features
on PfFabG that are critical for these interactions In
PfFabG, Arg187 and Arg230 are located in a
hydro-phobic patch adjacent to the active site entrance of
PfFabG, whereas Arg190 is located away from the
active site Hence, to characterize the role played by
these residues in the interactions of PfFabG and
PfACP, we generated the following mutants: R187E,
R230E, R187A⁄ R230A, R187E⁄ R230E, R190E,
R190A and R230K We also generated R187K, as this
is conservatively substituted throughout the apicom-plexan group (present as Lys187 in other species of Plasmodium)
In the type II FAS pathway, the growing acyl inter-mediates are attached to the terminal sulfhydryl of the 4¢-phosphopentatheine prosthetic group [13], which is attached via a phosphodiester linkage to the Ser37 located at the beginning of helix II The primary gene product is an apoprotein that is converted to holo-ACP (ACP) by the transfer of the 4¢-phosphopen-tatheine moiety of CoA to Ser37 by holo-ACP syn-thase ACP performs two functions: first, it sequesters the growing acyl chain from the aqueous environment; and second, upon binding to one of the type II FAS proteins, it releases its grip on the fatty acid, which is inserted into the active site of the enzyme ACPs from various natural sources share significant primary sequence similarity, particularly at the prosthetic group attachment site, extending to helix II However, the individual ACP-binding partners do not share any common ACP-binding motif
The molecular details that govern the specific inter-actions between Plasmodium ACP and type II FAS enzymes are poorly understood Here, we report subtle aspects of the interactions between PfFabG and PfACP, with emphasis on association constants and number of binding sites with reference to the cofactor NADPH The site-directed mutagenesis studies reveal that both electrostatic and hydrophobic interactions play important roles in PfFabG–PfACP complex formation
Results
Identification of residues putatively involved in the PfFabG–PfACP interaction
Multiple sequence alignment indicates that the FabG sequences from different species, including plants and bacteria, share a high degree of sequence identity (Fig 1A) The crystal structures of FabG enzymes from Escherichia coli [14], Brassica napus [15] and
P falciparum [12] are also homologous, and show FabG to be a tetramer consisting of two homodimers
of monomers arranged in a head-to-tail configuration The crystal structure of FabG from E coli shows that there is a conserved positively charged patch on its surface [14,16] This positively charged patch is posi-tioned at the entrance of the active site and is involved
in recognition of the highly conserved and negatively charged a2 helix of ACP This patch is identical in
E coli FabG, Plasmodium FabG and counterparts
Trang 3from plants Mutagenesis of E coli FabG showed that
two arginine residues (Arg29 and Arg172) present in
this patch are central to the binding of ACP [16]
Multiple sequence alignment of FabG sequences from different species shows that these residues are conserved in PfFabG too (Arg187 and Arg230,
A
Fig 1 Multiple sequence alignment of FabG sequences: (A) P falciparum (P falc) (accession number PFI1125c), Bacillus subtilis (B subt) (accession number AAC44307), Cuphea lanceolata (C lanc) (accession number P28643), B napus (accession number CAC41363), and E coli (accession number NP_415611) (B) P falciparum (P falc) (accession number PFI1125c), Plasmodium berghei (P ber) (accession number PB000052.00.0) and Plasmodium chabaudi (P cha) (accession number PC000242.01.0) *Residues thought to be involved in the FabG–ACP interaction Color scheme: conserved residues blocked in gray, negatively charged residues in red characters, positively charged residues in blue characters, aliphatic residues blocked in yellow, and aromatic residues in green (C) Electrostatic potential surface of the Plasmodium FabG adjacent to the active site entrance Red indicates negative charge, blue indicates positive charge, and white is hydrophobic Arg187 and Arg230 are located adjacent to the active site entrance.
Trang 4respectively) (Fig 1A) Three residues of PfFabG
selected for the mutagenesis studies, namely Arg187,
Agr190 and Arg230, were highly conserved in all
spe-cies of Plasmodium Analysis of the Plasmodium FabG
crystal structure shows that the conserved residues
Arg187 and Arg230 are located at the surface, near its
active site entrance (Fig 1C) We replaced the
posi-tively charged arginines with glutamates to introduce
electrostatic repulsion between PfFabG and PfACP
and to test whether PfACP associates with PfFabG
over the entire predicted surface We also changed
these positively charged residues to alanines to
deter-mine which of the electrostatic interactions are
impor-tant for promoting the binding to PfACP
Interestingly, within the Plasmodium genus, Arg187 is
substituted (Fig 1B) with a lysine
Expression and purification of PfFabG, PfFabG
mutants and PfACP
The recombinant wild-type PfFabG, its mutants and
PfACP were purified to homogeneity using an Ni2+–
nitrilotriacetic acid affinity column as previously
described [17,18] Figure 2 shows the apparent
electro-phoretic homogeneity of the purified proteins The
purified proteins on SDS⁄ PAGE yielded a monomeric
Mr of 31 000 ± 1000 for PfFabG as well as for
PfFabG mutants
Gel filtration and CD analyses of the mutants
Changes in the overall shape or the quaternary
struc-ture of the molecule, potentially introduced by
muta-genesis, were first probed using size exclusion
chromatography Wild-type PfFabG was eluted as a
single peak at a volume of 13.67 mL on a
Superdex-200 gel filtration column [17] The PfFabG mutants were also eluted at the retention volume of their wild-type counterpart The elution positions of the wild-type and mutants of PfFabG corresponded to a relative molecular mass of 110 kDa (± 10 kDa), indicating that the enzymes are homotetramers and that the mutations did not alter the overall shape or the quaternary structure of PfFabG CD spectroscopy was used to investigate potential perturbations in the secondary and tertiary structure of PfFabG mutants
CD spectra of wild-type PfFabG and the PfFabG mutants were superimposable (Fig S1), suggesting that the relative contents of a-helical and b-sheet secondary structure in the PfFabG mutants are not changed as a result of the individual point mutations
Kinetic analyses of the PfFabG mutants
In order to evaluate the effects of the mutations on the specific activity of PfFabG, we used an ACP-indepen-dent spectrophotometric assay, where acetoacetyl-CoA was used as a substrate in place of acetoacetyl-ACP, and the disappearance of NADPH was monitored at
340 nm As can be seen in Table 1, the kinetic con-stants (Kmand ACP-independent specific activities) of the R187A, R187E, R230A, R230E, R187A⁄ R230A and R187E⁄ R230E mutants remained largely unchanged with respect to wild-type PfFabG Wild-type PfFabG shows less activity with acetoacetyl-CoA than with acetoacetyl-ACP All the mutants exhibited very poor activity in the ACP-dependent spectroscopic assay, but not in the ACP-independent spectroscopic assay, which shows that PfFabG mutants are selec-tively compromised for utilization of the acyl-ACP substrate (acetoacetyl-ACP) The R230E and R187E⁄ R230E mutants had higher Km values of
45
kDa
35
25
Fig 2 Purification of PfFabG mutants by Ni 2 + –nitrilotriacetic acid
chromatography SDS ⁄ PAGE of recombinant PfFabG and PfFabG
mutants Lane 1: purified wild-type PfFabG Lane 2: protein
molecu-lar weight markers (MBI Fermentas) Lane 3: R187A Lane 4:
R187E Lane 5: R230A Lane 6: R230E Lane 7: R187A ⁄ R230A.
Lane 8: R187E ⁄ R230E.
Table 1 Specific activity of wild-type PfFabG and the PfACP binding site mutants in ACP-dependent and ACP-independent spectroscopic assays Enzyme activity was monitored spectro-photometrically at 340 nm as described in Experimental proce-dures Values in brace are presented as percentages.
PfFabG
Km(mM) AcAcCoA
Specific activity ACP-independent spectroscopic assay (UÆmg)1)
Specific activity ACP-dependent spectroscopic assay (UÆmg)1)
Trang 50.49 mm and 0.57 mm, respectively, than wild-type
PfFabG for acetoacetyl-CoA (0.43 mm) [17]
More-over, the point mutation R187K gives similar results
to those for wild-type PfFabG
The ACP-dependent activity assay clearly showed
the involvement of the two surface arginine residues of
PfFabG in the interaction with PfACP In order to
determine the ability of PfACP to function as the
inhibitor, we used a spectrophotometric assay, utilizing
acetoacetyl-CoA, with the indicated concentrations of
PfACP As can be seen in Fig 3, wild-type PfFabG
showed inhibition with increasing concentrations of
PfACP, whereas no inhibition with the R187E, R230E,
R187A and R230A mutants was observed The effect
was more deleterious when arginine was changed to
glutamate than when it was changed to the neutral
res-idue alanine (Table 2)
Interaction of PfFabG mutants with wild-type
PfACP monitored by measuring intrinsic PfFabG
fluorescence
The intrinsic fluorescence of PfFabG decreased when
it was titrated with increasing concentrations of
PfACP As reported earlier [17], binding of PfACP to
PfFabG, as analyzed by quenching of its fluorescence
at 334 nm, gave an association constant of 400 nm)1
with n = 1 The value of Ka was determined for
other mutants using nonlinear least squares fit of the
data, using the Adair equation with one to four
equivalent and independent, as well as equivalent and interdependent, binding sites (n) The Ka values for binding of the R187A and R230A mutants were, respectively, 150 and 82 nm)1 Thus, mutation of Arg187 and Arg230 to alanine decreased the binding affinities by three-fold and five-fold respectively The
Ka values for the binding decreased even more dra-matically to 93 and 9 nm)1, respectively, in the R187E and R230E mutants Apparently, mutation of Arg187 and Arg230 to an acidic residue, glutamate, diminishes the strength of the PfACP–PfFabG inter-action in a relatively more significant manner than their replacement by a neutral alanine residue The effect was more drastic when both the residues were converted to glutamate, there being an 80-fold reduc-tion in associareduc-tion constant (Table 2) The data shown here are in close agreement with those from the ACP-dependent assay
The affinity of wild-type PfACP increased three-fold (Ka= 1.10 lm)1) in the presence of NADPH, and the number of binding sites increased from one to two, whereas in all PfFabG mutants examined except R187K, the number of binding sites remained the same, with decreased binding of the PfFabG mutants
to PfACP in the presence of NADPH (Table 2) The maximum effect was observed on binding of the R230E mutant and the double mutant R187E⁄ R230E,
120
90
60
30
0
ACP conc (µ M )
Fig 3 Inhibition of PfFabG activity by PfACP The ability of PfACP
to function as an inhibitor of the condensing enzyme reaction was
evaluated using the spectrophotometric assay utilizing
acetoacetyl-CoA as described in Experimental procedures with 1 lg of PfFabG
(d) or 5 lg of the mutants R230E (.), R187E ⁄ R230E () and
R187A ⁄ R230A (s) The activities of the mutant enzymes were not
significantly affected by the addition of PfACP, suggesting that the
mutation reduced PfACP binding to PfFabG.
Table 2 Binding constants (K a ) for interaction of wild-type PfACP with mutant PfFabG, in the absence and in the presence of reduced cofactor NADPH at 20 C, obtained using the changes in protein fluorescence intensity at 334 nm and SPR Experimental details are provided in Experimental procedures n, number of bind-ing sites for the best value of r 2 ; Ka, association constant for the best value of r 2 , determined using protein fluorescence (334 nm);
ND, not determined.
Serial
no Sample
Titrated with:
Fluorescence
n Ka(nM)1)
SPR analysis
Ka(nM)1)
14 R187A ⁄ R230A-NADPH PfACP 1 205.3 ND
15 R187E ⁄ R230E-NADPH PfACP 1 92.3 ND
Trang 6with association constants of 110.4 and 92 nm)1,
respectively
Interaction of the PfFabG mutants with wild-type
PfACP monitored by surface plasmon resonance
(SPR)
The PfFabG–PfACP interaction data were further
ver-ified by direct measurement of binding of these two
proteins by SPR (BIAcore) Wild-type PfFabG and its
mutants were immobilized on the nitrilotriacetic acid
sensor chip surface, following the manufacturer’s
pro-tocol [19] Approximately 330–350 resonance units
(RU) of His-tagged PfFabG was immobilized in each
channel of the sensor chip A continuous flow of
buf-fer was maintained on the chip surface until a stable
baseline was reached All of the binding studies were
conducted at 20C
Various concentrations of His-tag cleaved PfACP
(ranging from 1 to 200 lm) were passed over the
sur-face of the chip The PfFabG immobilized sursur-face
devoid of the PfACP from a previous reaction could
be regenerated by passing the buffer alone A typical
sensorgram for the binding of varying concentrations
of PfACP to immobilized PfFabG is shown in Fig 4A
The occurrence of an enhancement in the RUs is
indic-ative of the increase in mass on the chip surface, which
indicates binding As shown in Fig 4B, wild-type
PfFabG showed strong binding, whereas the R230E
and R187A⁄ R230A mutants did not show any
signifi-cant binding at similar PfACP concentrations The
apparent binding and association constants are shown
in Table 2 There was a three-fold enhancement in
PfACP binding to wild-type PfFabG in the presence of
NADPH PfACP binding to the mutant PfFabGs was
also enhanced in the presence of NADPH There was
a three-fold to 100-fold decrease in the affinity of
bind-ing of mutant PfFabGs to PfACP as compared to
wild-type PfFabG (Table 2) The apparent binding
constants determined by fluorescence quenching and
SPR experiments for PfFabG and PfACP are
essen-tially similar Fluorescence quenching corroborated
with SPR experiments shows the involvement of these
residues in the PfFabG–PfACP interaction
Allosteric binding of NADPH to PfFabG mutants
in the presence of PfACP
The Ka value for the binding of NADPH to PfFabG,
with n = 4, was found to be 40.90 lm)1, and the
bind-ing exhibited negative, homotropic cooperativity (with
a Hill constant of nH= 0.8) [17] The association
con-stant was nearly equal for all of the PfFabG mutants,
suggesting that there was no effect of point mutations
on the NADPH binding (data not shown) Further-more, the affinity (Ka) of PfFabG for its cofactor NADPH determined by fluorescence spectroscopy in the presence of 20 lm PfACP was found to be 48.36 lm)1 and n = 2 Thus, whereas the number of cofactor-binding sites decreased in the presence of PfACP, the affinity of PfFabG for NADPH increased, indicating a negative, heterotropic cooperative effect of PfACP upon binding of NADPH This effect of
A
B
100 80 60
60
[ACP] µ M vs FabG WT [ACP] µ M vs R230E [ACP] µ M vs R187A R230A
Time (s)
40 20
20 0
60
40
20
Fig 4 (A) Sensorgram depicting the binding of PfACP to wild-type PfFabG Varying concentrations of PfACP (5, 10, 20, 50, 100 and
200 lM) in 10 mM Hepes buffer, containing 150 mM NaCl and
50 lM EDTA (pH 7.4), were passed over the immobilized PfFabG at
a flow rate of 20 lLÆmin)1 The dissociation was studied subse-quently by passing the same buffer at a flow rate of 20 lLÆmin)1 (B) Direct binding between PfFabG and PfACP measured by BIAcore The binding results with the fluorescence assay were confirmed with the SPR approach (BIAcore) described in Experi-mental procedures Wild-type PfFabG and mutants were immobi-lized on a nitrilotriacetic acid chip, and wild-type PfACP protein solutions were pumped across the surface Wild-type PfFabG (solid line and solid circles) exhibited a strong binding signal, whereas nei-ther the R230E mutant (dashed line and inverted triangle) nor the R187A ⁄ R230A mutant (dotted line and open circle) exhibited signifi-cant binding to the PfACP chip at similar protein concentrations.
Trang 7PfACP was not observed with different PfFabG
mutants, as there was no increase in the affinity (Ka)
and also the number of NADPH-binding sites
remained at four, corresponding to the number of
subunits (Table 3)
Discussion
Our site-directed mutagenesis, kinetic analysis and
binding studies provide evidence that Arg187 and
Arg230 of PfFabG are involved in the interaction with
PfACP These studies demonstrate that the highly
conserved second a-helix of PfACP recognizes
electro-positive residues on the surface of PfFabG Whereas
the consequences of mutating the corresponding
resi-dues in E coli FabG involved in the binding of E coli
ACP have been investigated earlier, the number of
binding sites and ACP-induced cooperativity for the
interactions of the cofactor NADPH for these mutants
have been characterized for the first time in studies
reported here for Plasmodium FabG We have used
ACP-dependent and ACP-independent
spectrophoto-metric assays and a fluorescence assay to estimate the
association constants and number of binding sites of
different mutants of PfFabG and PfACP, as well as
the interactions in the presence of the cofactor
NADPH
Studies with E coli FabG have identified a
con-served, positively charged patch on its surface [14,16]
The surface of FabG surrounding the active site tunnel
is electropositive This positively charged patch is
posi-tioned at the entrance of the active site and is thought
to be involved in the recognition of the highly
con-served, negatively charged a2 helix of PfACP PfFabG
would therefore be expected to also contain a
posi-tively charged region at the active site entrance capable
of interacting with the negatively charged region of
PfACP The sequences of PfFabG were compared for similarity with FabGs from other organisms, using clustalw [20] As shown in Fig 2A, the sequence of PfFabG is most similar to those of plants and bacteria, consistent with its evolutionary linkage to a photosyn-thetic bacterium and its location in the apicoplast of the parasite Plasmodium FabG also has the character-istic sequence motif formed by a triad of Ser196, Tyr209 and Lys213 residues that is involved in the cat-alytic mechanisms of the enzymes of the short-chain alcohol dehydrogenase⁄ reductase family This provides
a strong indication of involvement of Arg187 and Arg230 of PfFabG in its interaction with PfACP, as these two residues are located at the entrance of the active site tunnel (Fig 1C) [12] The sequence align-ment of FabG from different Plasmodium species, however, shows the presence of Lys187 instead of Arg187, so the effects of mutations of these residues
on the kinetics and the ACP-induced cooperativity for the binding of NADPH were also examined
To eliminate the ability of Arg187, Arg190 and Arg230 to form ionic bonds with PfACP, we mutated them to alanine or glutamate and demonstrated their moderate effect on PfFabG specific activities (Table 1)
In contrast, when Arg187 and Arg230 were changed to
an alanine or glutamate, the PfFabG mutants were severely impaired in the ACP-dependent spectrophoto-metric assay, with a decrease in activity of more than 90% (Table 2) and refractoriness to PfACP inhibition (Fig 3) To confirm the importance of Arg187 and Arg230, we used a fluorescence assay to directly monitor the interactions between PfFabG and PfACP The PfFabG mutants show three-fold to 80-fold less binding when compared to wild-type PfFabG (Table 2) These data suggest that Arg187 and Arg230 are the most important electropositive residues involved in PfFabG– PfACP interactions Furthermore, the effect was more deleterious when Arg230 was mutated as than when Arg187 was mutated for interactions with PfACP as shown by the direct binding assay (Table 2), which might be the reason for the greater conservation of Arg230 during evolution (Fig 1)
In conclusion, our results provide compelling sup-port for the hypothesis that the highly conserved a2 helix of PfACP recognizes an electropositive⁄ hydro-phobic surface feature adjacent to the active site entrance on PfFabG Two surface residues, Arg187 and Arg230, located in a hydrophobic patch at the active site entrance on the PfFabG make contact with PfACP The hydrophobic residues on PfACP also make contacts with the hydrophobic patches on PfFabG, and in turn help in the proper positioning of PfACP near the active site
Table 3 Binding constants (K a ) of reduced cofactor NADPH for
PfFabG mutants in the presence of PfACP at 20 C, using the
changes in protein fluorescence intensity at 334 nm Experimental
details are provided in Experimental procedures n, number of
bind-ing sites for the best value of r2; K a , association constant for the
best value of r 2 , determined using protein fluorescence (334 nm).
Serial
Titrated
Ka (lM)1)
Trang 8Experimental procedures
Materials
Acetoacetyl-CoA, b-hydroxybutyryl-CoA, b-NADPH, NADP+,
imidazole, kanamycin, chloramphenicol and SDS⁄ PAGE
reagents were obtained from Sigma Chemicals (St Louis,
MO, USA) Protein molecular weight markers were from
MBI Fermentas GmbH (St Leon-Rot, Germany)
His-bind-ing resin and anti-His-tag horseradish peroxidase
conju-gates were obtained from Novagen (Darmstadt, Germany)
Media components were obtained from Difco (Franklin
Lakes, NJ, USA) Hi-Trap desalting and Superdex 200
col-umns were from Amersham Biosciences (Uppsala, Sweden)
All other chemicals used were of analytical grade
Strains and plasmids
FabG was cloned into the pET-28a(+) vector (Novagen),
and BL21 (DE3) codon plus (Novagen) was used for the
expression of PfFabG
Construction of PfFabG mutants
Mutations were introduced into the PfFabG gene in pET–
FabG [17], using an overlap extension PCR method All
the PfFabG mutants were prepared using the same two
outer primers: FabG-NcoI-F, 5¢-CATGCCATGGGAAAA
GTTGCTTTAGTAACAG GTGCAGGA-3¢; and FabG
-XhoI-R, 5¢-CCGCTCGAGAGGTGATAGTCCACCGTCT
ATTACGAAAACTCG-3¢ (with NcoI and XhoI sites
underlined) The internal primers for all the PfFabG
mutants are listed in Table 4 To construct each mutant,
two PCR reactions with pET–FabG as the template, consisting of one outer primer and the respective internal primers, were performed, and the products were then pooled and used as a template for a second PCR using the outer primers The PCR products were purified from a 1% agarose gel using the QIAquick gel extraction kit (Qiagen, Hilden, Germany) and cloned in the NcoI and XhoI sites of the pET-28a(+) vector (Novagen) The clone thus obtained was confirmed by DNA sequencing Expression and purifi-cation was as for the wild-type protein as reported earlier [17]
CD spectroscopy
The correct folding of the mutant proteins was verified by analysis of their CD spectrum between 200 and 250 nm (2 nm bandpass) using a JASCO J-810 (Tokyo, Japan) spectropolarimeter at 20C and a 0.1 cm path length quartz cuvette The protein concentration was determined
by measuring the absorbance at 280 nm, immediately prior
to collecting the spectrum The extinction coefficient of His-tagged PfFabG was 17 690 cm)1Æm)1, using the ‘peptide properties calculator’ (http://www.basic.northwestern.edu/ biotools/proteincalc.html) The measured ellipticity was converted to molar values for direct comparison of the mutants with the wild-type protein
Expression and purification of PfACP
PfACP was purified as described previously [18]
ACP-dependent spectrophotometric assay of PfFabG activity
PfFabG activity was tested in an ACP-dependent assay
by analyzing the formation of b-hydroxybutyryl-ACP, measuring the disappearance of its cofactor NADPH, spectrophotometrically, at 340 nm The gel reconstitution assay reported for E coli [16] could not be used for mea-suring ACP-dependent PfFabG activity, as the substrate acetoacetyl-ACP and product b-hydroxybutyryl-ACP were not separable by electrophoresis under nonreducing condi-tions The reaction mixture contained 25 lm PfACP,
1 mm b-mercaptoethanol, 65 lm malonyl-CoA, 45 lm ace-tyl-CoA, 200 lm NADPH, 1 lg of purified E coli FabD, 0.5 lg of purified PfFabH in 0.1 m sodium phosphate buffer (pH 7.0), and 0.2–1 lg of PfFabG (or PfFabG mutant) in a final volume of 95 lL The PfACP, b-mer-captoethanol and buffer were preincubated at 37C for
30 min to ensure the complete reduction of PfACP The substrate for the PfFabG reaction was generated by using
E coli FabD to transfer the malonyl group from CoA to PfACP to produce malonyl-ACP, and subsequently PfFabH to condense acetyl-CoA and malonyl-ACP to
Table 4 Overview of the oligonucleotide primers used to generate
PfFabG mutants The mutation sites are underlined.
Primer Sequence (5¢- to 3¢)
FabGF CATGCCATGGGAAAAGTTGCTTTAGTAACAGGTGCAGGA
FabGR CCGCTCGAGAGGTGATAGTCCACCGTCTATTACGAAAA
CTCG
R187AF TAATAATGCGTATGGTCGAATAATTA
R187AR GACCATACGCATTATTAATCATTCTT
R187EF TAATAATGAATATGGTCGAATAATTA
R187ER GACCATATTCATTATTAATCATTCTT
R187KF AATAATAAATACGGCCGAATAATTA
R187KR TAATTATTCGGCCGTATTTATTATT
R230AF AGCTTCAGCCAATATAACTGTAAATG
R230AR CATTTACAGTTATATTGGCTGAAGCT
R230EF AGCTTCAGAAAATATAACTGTAAATG
R230ER CATTTACAGTTATATTTTCTGAAGCT
R190AF TATGGTGCCATAATTAATATTTCAAGT
R190AR ACTTGAAATATTAATTATGGCACCATA
R190ER TATGGTGAAATAATTAATATTTCAAGT
R190ER ACTTGAAATATTAATTATTTCACCATA
Trang 9form b-ketobutyryl-ACP The reaction was initiated by
the addition of NADPH A decrease in the absorbance
at 340 nm was recorded for 2 min The initial rate was
used to calculate the enzyme activity
ACP-independent spectrophotometric assay of
PfFabG activity
The activity of PfFabG was assayed at 25C by
monitor-ing spectrophotometrically the decrease in absorbance at
340 nm due to the oxidation of NADPH to NADP+
(Jasco V-530 UV–visible spectrophotometer) The standard
reaction mixture in a final volume of 100 lL contained
50 mm sodium phosphate buffer (pH 6.8) containing
0.25 m NaCl, 200 lm NADPH, 0.5 mm acetoacetyl-CoA
and 0.2–0.8 lg of PfFabG [17] The assay mixture was
preincubated for 5 min at room temperature before the
reaction was initiated by the addition of substrate or
enzyme Reactions with appropriate blanks were also
per-formed The kinetic parameters were determined by
non-linear regression analyses The data were also evaluated by
double reciprocal plots The ability of PfACP to inhibit
PfFabG activity in the spectrophotometric assay was tested
by incubation of varying concentrations of PfACP with
PfFabG protein at room temperature for 5 min before the
addition of acetoacetyl-CoA to initiate the reaction
Fluorescence titration of PfFabG–PfACP binding
Equilibrium binding of various ligands to PfFabG was
measured by fluorescence titration [17] at 20C using a
Jobin-Yvon Horiba spectrofluorimeter (bandpass of 3 and
5 nm for the excitation and emission monochromator,
respectively) The fluorescence spectrum of PfFabG was
studied by exciting the samples at 280 nm and recording
the emission spectrum in the range 300–400 nm, with an
emission maximum at 334 nm, due to its lone
trypto-phan Aliquots of 3 lL of PfACP (from the stock
solu-tions of 100 lm) were added to 0.5 lm PfFabG in 3 mm
Hepes (pH 7.5), 100 mm NaCl, 2 mm b-mercaptoethanol
and 10% glycerol The solution was mixed after the
addi-tion of each aliquot, and the fluorescence intensity in the
range 300–400 nm was recorded as the average of three
readings Samples were excited at 280 nm The effect of
NADPH on PfACP binding to PfFabG was studied by
titration of NADPH (3 lL) into 0.5 lm PfFabG (in
3 mm Hepes, pH 7.5, 100 mm NaCl, 2 mm
b-mercapto-ethanol and 10% glycerol), from a stock solution of
5 mm, and the corresponding decrease in the fluorescence
intensity of its lone tryptophan was monitored at
334 nm A double reciprocal plot of the fluorescence
intensities and ligand concentrations, from the data
obtained by titration of a fixed concentration of PfFabG
with ligand, gave the fluorescence intensity at infinite
ligand concentration (Fa)
Correction for the inner filter effect was performed according to the equation
Fc¼ F antilog½ðAexþ AemÞ=2
where Fcand F are the corrected and measured fluorescence intensities, respectively [21], and Aex and Aem are the solu-tion absorbance values at the excitasolu-tion and emission wave-lengths, respectively
The fluorescence data were fitted by the Adair equation [22], with number of sites n = 1 to 4, K being the association constants: for a single site, Y = K[X]⁄ (1 + K[X]); for two equivalent and independent sites, Y = (K1[X] + 2K1K2[X]2)⁄ (1 + K1[X] + K1K2[X]2); for two equi-valent and interdependent sites, Y = (2K1[X] + K1K2[X]2)⁄ (1 + K1[X] + K1K2[X]2); for three equivalent and indepen-dent sites, Y = (K1[X] + 2K1K2[X]2+ 3K1K2K3[X]3)⁄ (1 +
K1[X] + K1K2[X2] + K1K2K3[X]3); for three equivalent and interdependent sites, Y= (3K1[X] + 2K1K2[X]2+
K1K2K3[X]3)⁄ (1 + K1[X] + K1K2[X]2+ K1K2K3[X]3); for four equivalent and independent sites, (K1[X] + 2K1K2[X]2 + 3K1K2K3[X]3+ 4K1K2K3K4[X]4)⁄ (1 + K1[X] + K1K2[X]2 + K1K2K3[X]3+ K1K2K3K4[X]4); and four equivalent and interdependent sites, Y = (4K1[X] + 3K1K2[X]2 + 2K1K2K3[X]3+ K1K2K3K4[X]4)⁄ (1 + K1[X] + K1K2[X]2 +
K1K2K3[X]3+ K1K2K3K4[X]4) All calculations were carried out with sigma plot 2000 software
Analysis of PfFabG–PfACP interaction by SPR
Biospecific-interaction analysis was performed using a BIA-core 2000 biosensor system (Amersham Pharmacia Biotech, Uppsala, Sweden) The immobilization of PfFabG on the flow cell of a nitrilotriacetic acid sensor chip involved regen-eration of the sensor surface with a 1 min flow of 350 mm EDTA in 10 mm Hepes (pH 8.3), 150 mm NaCl and 10% glycerol at a rate of 20 lLÆmin)1on the sensor surface This was followed by injection of 500 lm NiCl2in 10 mm Hepes (pH 7.4), 150 mm NaCl, 10% glycerol and 50 lm EDTA for
5 min at a rate of 5 lLÆmin)1on the sensor surface Nearly
70 RU were coupled Wild-type and mutant PfFabG were immobilized on different flow cells of the activated sensor chip by injecting 125 nm protein in 10 mm Hepes (pH 7.4),
150 mm NaCl, 10% glycerol and 50 lm EDTA at a rate of
2 lLÆmin)1 Nearly 350 RU were coupled, where 1 RU cor-responds to an immobilized protein concentration of approx-imately 1 pgÆmm)2 One flow cell was kept as the reference surface The reference surface was treated in the same way as the ligand surface, except that the PfFabG was not passed over this surface This is important to normalize the chemis-tries between the two flow cells All measurements were car-ried out in 10 mm Hepes (pH 7.4), 150 mm NaCl, 10% glycerol and 50 lm EDTA (HBS buffer) [23] In order to determine the association rate constant for the binding of PfACP to the immobilized PfFabG, PfACP without His-tag was passed over the surfaces at various concentrations at
Trang 1020C in Hepes buffer at a flow rate of 20 lLÆmin)1 For the
determination of dissociation rate constant, the same buffer
was passed at a flow rate of 20 lLÆmin)1 To study the effect
of NADPH on the PfFabG–PfACP interaction, PfACP was
diluted to the desired concentrations in HBS buffer
contain-ing 200 lm NADPH, and the bindcontain-ing studies were performed
in the same buffer with NADPH
Evaluation of kinetic parameters
The association (k1) and dissociation (k)1) rate constants
are obtained by nonlinear fitting of the primary sensorgram
data using bia evaluation software version 3.0 The
sensor-grams were fitted globally to the 1 : 1 Langmuir
dissocia-tion model (Eqn 1) to obtain the k)1values:
Rt¼ Rtoek1 ðtt 0 Þ ð1Þ where Rtis the response at time t, and Rt0is the amplitude
of the initial response The measured k)1 values were used
to determine the k1values using the 1 : 1 Langmuir
associa-tion model (Eqn 2):
Rt¼ Rmax½1 eðk1 Cþk 1 Þðtt 0 Þ ð2Þ
where Rmaxis the maximum response, and C is the
concen-tration of the analyte in the solution The ratio of k1and k)1
yields the value of the association constant Ka(k1⁄ k)1) v2
and residual values were used to evaluate the quality of fit
between the experimental data and the binding model [24]
Acknowledgements
We thank the Department of Biotechnology (DBT),
Government of India for their financial support to
N Surolia K Karmodiya acknowledges the CSIR,
Government of India, for a senior research fellowship
References
1 Surolia N & Surolia A (2001) Triclosan offers protection
against blood stages of malaria by inhibiting enoyl-ACP
reductase of Plasmodium falciparum Nat Med 7, 167–173
2 Waller RF, Keeling PJ, Donald RG, Striepen B,
Hand-man E, Lang-Unnasch N, CowHand-man AF, Besra GS,
Roos DS & McFadden GI (1998) Nuclear-encoded
proteins target to the plastid in Toxoplasma gondii and
Plasmodium falciparum Proc Natl Acad Sci USA 95,
12352–12357
3 Witkowski A, Ghosal A, Joshi AK, Witkowska HE,
Asturias FJ & Smith S (2004) Head-to-head
arrange-ment of the subunits of the animal fatty acid synthase
Chem Biol 11, 1667–1676
4 Ramya TNC, Surolia N & Surolia A (2002) Survival
strategies of the malarial parasite Plasmodium
falcipa-rum Curr Sci 83, 101–108
5 Surolia A, Ramya TNC, Ramya V & Surolia N (2004) FAS’t inhibition of malaria Biochem J 383, 1–12
6 Sharma S, Ramya TNC, Surolia A & Surolia N (2003) Triclosan as a systemic antibacterial agent in a mouse model of acute bacterial challenge Antimicrob Agents Chemother 47, 3859–3866
7 Ramya TN, Mishra S, Karmodiya K, Surolia N & Surolia A (2007) Inhibitors of nonhousekeeping functions of the apicoplast defy delayed death in Plasmodium falciparum Antimicrob Agents Chemother
51, 307–316
8 Ramya TN, Karmodiya K, Surolia A & Surolia N (2007) 15-Deoxyspergualin primarily targets the traffick-ing of apicoplast proteins in Plasmodium falciparum
J Biol Chem 282, 6388–6397
9 White SW, Zheng J, Zhang YM & Rock CO (2005) The structural biology of type II fatty acid biosynthesis Annu Rev Biochem 74, 791–831
10 Gardner MJ, Tettelin H, Carucci DJ, Cummings LM, Aravind L, Koonin EV, Shallom S, Mason T, Yu K, Fujii C et al (1998) Chromosome 2 sequence of the human malaria parasite Plasmodium falciparum Science
282, 1126–1132
11 Sharma AK, Sharma SK, Surolia A, Surolia N &
Sar-ma SP (2006) Solution structures of conforSar-mationally equilibrium forms of holo-acyl carrier protein (PfACP) from Plasmodium falciparum provides insight into the mechanism of activation of ACPs Biochemistry 45, 6904–6916
12 Wickramasinghe SR, Inglis KA, Urch JE, Muller S, Van Aalten DM & Fairlamb AH (2006) Kinetic, inhibi-tion and structural studies on 3-oxoacyl-ACP reductase from Plasmodium falciparum, a key enzyme in fatty acid biosynthesis Biochem J 393, 447–457
13 Rock CO, Cronan JE Jr & Armitage IM (1981) Molec-ular properties of acyl carrier protein derivatives J Biol Chem 256, 2669–2674
14 Price AC, Zhang YM, Rock CO & Stephen WW (2001) Structure of beta-ketoacyl-[acyl carrier protein] reduc-tase from Escherichia coli: negative cooperativity and its structural basis Biochemistry 40, 12772–12781
15 Fisher M, Kroon JTM, Martindale W, Stuitje AR, Slabas AR & Rafferty JB (2000) The X-ray structure of Brassica napus beta-keto acyl carrier protein reductase and its implications for substrate binding and catalysis Structure 8, 339–347
16 Zhang YM, Wu B, Zheng J & Rock CO (2003) Key residues responsible for acyl carrier protein and beta-ketoacyl-acyl carrier protein reductase (FabG) interac-tion J Biol Chem 278, 52935–52943
17 Karmodiya K & Surolia N (2006) Analyses of co-opera-tive transitions in Plasmodium falciparum b-ketoacyl-ACP reductase upon co-factor and acyl carrier protein binding FEBS J 273, 4093–4103