From the Departments of Biochemistry and
Structural Biology, St. Jude Children's Research Hospital,
Memphis, Tennessee 38105, the § Department of Molecular
Biology, The Scripps Research Institute, La Jolla, California 92037, and the ** Department of Biochemistry, University of Tennessee, Memphis,
Tennessee 38163
Received for publication, September 1, 2000, and in revised form, November 6, 2000
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ABSTRACT |
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The molecular details that govern the specific
interactions between acyl carrier protein (ACP) and the enzymes of
fatty acid biosynthesis are unknown. We investigated the mechanism of
ACP·protein interactions using a computational analysis to
dock the NMR structure of ACP with the crystal structure of
The 4'-phosphopantetheine prosthetic group is a central and
universal feature in the mechanism of fatty acid biosynthesis that
provides two crucial functionalities to the process: a long and
flexible arm that can reach into active sites and a terminal sulfhydryl
group for the attachment of acyl groups through a thioester linkage.
Two types of fatty acid synthase architectures exist in nature, and the
4'-phosphopantetheine moiety operates quite differently in each type.
The type I, or associated system, found in metazoans, consists of a
single large polypeptide containing multiple active centers. In this
system, the prosthetic group with its attached nascent fatty acid
swings between the active sites in the multifunctional complex. This
contrasts with the type II or dissociated system found in bacteria and
plants in which the active centers reside in discrete protein
molecules. Here, the 4'-phosphopantetheine moiety is covalently
attached to acyl carrier protein
(ACP),1 a small protein that
sequentially delivers the lipid intermediates to the active site of
each enzyme in the pathway.
ACP is a small, acidic and highly conserved protein with a molecular
mass of 8847 Da (1). In Escherichia coli, it is
encoded by the acpP gene that is located within a cluster of
other fatty acid biosynthetic genes (2, 3). Biophysical (4) and
solution NMR structural studies (5) show that the protein is an
asymmetric monomer composed of three In bacteria, the involvement of ACP in these various synthetic pathways
means that it must interact with a plethora of functionally different
enzymes. However, analysis of the primary structures of these enzymes
does not reveal a common ACP-binding motif. The interaction must be
specific to enable the prosthetic group to deliver its cargo precisely
to the active site, but it must also be relatively weak to allow rapid
on and off rates for the substrates. It is also anticipated that
conformational changes facilitate the entry and exit of the extended
prosthetic group from the active site. Biophysical (18, 19) and
structural studies (20) point to an interaction between the acyl chain
and the ACP protein. However, crystal structures reveal that the active
sites of the fatty acid biosynthetic enzymes are generally located at
the bottom of hydrophobic clefts or tunnels with varying dimensions
(for two examples, see Refs. 21, 22). Therefore, the fatty acid intermediate must dissociate from ACP and be injected into the active
site cavity. The physicochemical and structural basis of these
interactions is not known.
Materials--
Amersham Pharmacia Biotech supplied the
[1-14C]acetyl-CoA; Sigma supplied the acetyl-CoA,
malonyl-CoA, and ACP; Qiagen supplied the nickel-nitrilotriacetic acid
resin; Promega supplied the molecular biology reagents.
NH2-terminally His-tagged FabD, FabH, and FabB were
expressed in E. coli strain BL21(DE3) (Novagen) and purified by nickel chelation affinity chromatography as described previously (27-29). Protein was stored in 50% glycerol at Modeling of the FabH·ACP Complex--
The program SurfDock,
which is described in detail elsewhere (31, 32), was used to
investigate the FabH·ACP complex. The coordinates of ACP and FabH
were both retrieved from the Protein Data Bank (accession codes 1acp
and 1ebl, respectively), and hydrogen atoms were added using the suite
of programs from Molecular Simulations Inc. The partial charges were
assigned according to the cff91 forcefield (33), and the
solvent-excluded surfaces were created with the MSMS program (34) using
probe radii of 1.5 and 1.56 Å for ACP and FabH, respectively. These
probe radii are slightly larger than normal to avoid the possibility of
creating artifactual tunnels in the protein surfaces, because the
spherical harmonic approximation in SurfDock requires a genus 0 surface (i.e. no holes). At this point, sets of spherical harmonic
surfaces of various resolutions ranging from order 6 to 40 at
approximate intervals of 5 were generated for ACP and FabH. The
electrostatic potentials and hydrophobicities were then mapped to these
generated surfaces. The former were calculated on the basis of a
distance-dependent dielectric constant with no
distance cutoff, and the latter were based on the residue
hydrophobicities (35, 36). The expression used for computing the
dielectric constant as a function of distance, r, is
(37):
In the docking procedure, FabH was considered as the fixed molecule,
and no constraints were placed on the positioning of the mobile ACP
molecule. During the docking calculations, ~60,000 docking pairs were
evaluated by analyzing the interactions at the interfacial surface
between the two proteins. The interfacial surface is defined as the
locus of points equidistant between the interaction surfaces. SurfDock
maps atomic properties onto the surfaces of the interacting molecules
and uses a combination of geometric and chemical criteria to score
putative complexes. In the current implementation, the total score is a
linear combination of the following terms: contact area, geometric
shape, chemical properties, steric overlap, and interface topology. The
initial population size was set to 200, and at every generation during the competition algorithm 50 individuals were kept and 150 new individuals were generated. The process was repeated for 300 generations, at which point all the top scoring complexes were analyzed visually.
Crystal Structure Analysis of FabB--
His-tagged FabB was
expressed and purified as described previously (29). Crystals of FabB
in space group P212121 were grown by the hanging drop vapor diffusion method using a well solution that
contained 2.0 M ammonium sulfate, 20% polyethylene glycol 400 and 100 mM Tris, pH 6.5. Data from a single crystal
were processed using HKL (38). The structure was solved by molecular
replacement using the FabF structure (39) and the programs SegMod (40) and AMoRe (41). The final structure was obtained by alternating rounds
of refinement using XPLOR (42) and visual inspection using O (43).
Solvent flattening and NCS averaging using the DM program (44) were
included in all maps. Omit maps were used extensively to avoid model
bias. The stereochemistry was evaluated using PROCHECK (45). Data and
refinement statistics are shown in Table
I.
Construction of FabH Mutants--
Mutations were introduced into
the fabH gene in pET-fabH (27) using an overlap extension
PCR method. All of the FabH mutants were prepared using the same two
outside primers: HNsiFor (5'-CATTTGACGTTGCAGCAGCCT) and HBamRev
(5'-ACGGCCTAGGAATTGCGTCATGT). The internal primers for all the FabH
mutants are listed in Table II. To
construct each mutant, two PCR reactions with pET-fabH as the template
and consisting of one outside primer and the respective inside primer were performed, and the products were then pooled and used as a
template for a second PCR using both outside primers. The 677-bp PCR
product was purified from a 1% agarose gel using the QIAquick gel
extraction kit (Qiagen, Inc.) and ligated into pCR2.1 (Invitrogen). Following transformation into E. coli InvaF', plasmid DNA
was isolated and sequenced. A clone with the sequence containing the desired mutation was digested with NsiI and BamHI
and ligated into pET-fabH, which had been digested with the same
enzymes and dephosphorylated with calf intestinal alkaline
phosphorylase. The resultant plasmids were transformed into competent
E. coli BL21(DE3) cells by electroporation. Expression and
purification was as for the wild type protein.
Circular Dichroism Spectroscopy--
The correct folding of the
mutant proteins was verified by analysis of their CD spectrum between
200 and 280 nm using an AVIV 62A DS spectrometer (22). Protein
concentration was determined by measuring the absorbance at 280 nm,
immediately prior to collecting the spectrum. The extinction
coefficient of His-tag FabH was taken to be 27,493 (M × cm) Coupled Assay of FabH Activity with Malonyl-ACP--
The coupled
assay of FabH, as described previously (25, 27), contained 25 µM ACP, 1 mM Spectrophotometric Assay of FabH Activity with
Malonyl-CoA--
The reaction mixture contained 0.1 mM
acetyl-CoA, 5 mM MgCl2, 50 µg of FabH
protein, 5 mM malonyl-CoA, 0.1 M Tris-HCl
buffer, pH 7.0, in a final volume of 300 µl. The reaction was
initiated by the addition of malonyl-CoA. Surface Plasmon Resonance--
Binding studies were performed on
a Biacore 3000 surface plasmon resonance instrument. ACP was covalently
attached to a carboxymethyl-dextran-coated gold surface (CM-5 Chip,
Biacore). The carboxymethyl groups on the chip were activated with
N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to activate the
carboxymethyl-dextran. The ACP was attached at pH 4.5 to this activated
surface by reaction of the carboxyl groups of the dextran with primary
amines on the ACP to form an amide linkage. Any remaining reactive
sites on the surface were blocked by reaction with ethanolamine. A
reference cell was prepared similarly except that no ACP was added.
Binding was measured by flowing FabH in 10 mM HEPES, 150 mM NaCl, pH 7.4, at a flow rate of 20 µl/min through the
reference and ACP-containing flow cells in sequence. A blank was also
run consisting of buffer only. Following the injection, release of the
bound FabH was measured by flowing only buffer through the flow
cells. Regeneration of the chip surface to remove bound FabH consisted
of allowing the protein to dissociate in buffer for 60 min between
injections. Data reported are the difference in surface plasmon
resonance signal between the flow cell containing ACP and the reference cell.
Computational Docking of ACP and FabH--
The interaction between
FabH and ACP was computationally investigated using the program
SurfDock. This automated protein·protein docking algorithm has been
fully described elsewhere (32, 47) and was used successfully to predict
the structural complex of
Using the NMR structure of ACP (5-7) and the x-ray structure of FabH
(22, 26), we generated an optimal FabH·ACP complex using SurfDock as
described under "Experimental Procedures." The top scoring
complexes from the docking computation could be grouped into two
subsets. In the larger subset, which contained the lowest scores (
Fig. 1 shows an overview of this complex,
and it highlights the matching physicochemical surface properties of
the two molecules that were detected by SurfDock. Table
III lists the favorable amino acid
contacts at the FabH·ACP interface, and Fig.
2 shows a stereo view of the interface
that illustrates these interactions. There are several attractive
features of this model. First, Ser-36 of ACP, the attachment site for
the 4'-phosphopantetheine, is adjacent to the FabH active site entrance
and oriented appropriately for insertion of the prosthetic group into
the tunnel. Second, the interfacial amino acids from both proteins are
highly conserved (Fig. 3). Third, the
closest contact between the proteins is mediated by two Effect of Surface Mutations on the Putative FabH·ACP
Complex--
The model predicted by the SurfDock program was tested by
introducing a number of mutations into FabH that should disrupt FabH·ACP interactions. Five residues were selected that are both integral to the interface (see Fig. 2) and also highly conserved (see
Fig. 3), namely, lysines 214, 256, and 257, Arg-249, and Ala-253. We
replaced the positively charged arginine and lysines with glutamates to
introduce electrostatic repulsion between FabH and ACP and to test
whether ACP associates with FabH over the entire predicted surface. We
also changed these same positively charged residues to alanines to
determine which of the electrostatic interactions are the most
important for promoting the binding to ACP. Finally, we replaced
Ala-253 with a bulky tyrosine residue to test the importance of the
hydrophobic depression on the FabH surface. None of these mutations
introduced significant structural changes into the FabH molecule based
on their CD spectra (data not shown).
Two assays were used to evaluate the effects of these mutations on the
FabH-specific activity. The first was ACP-dependent, and
used malonyl-ACP as the substrate in a standard radiochemical procedure
that was originally developed to evaluate FabH activity (24, 25, 27).
The second assay was ACP-independent and was developed specifically for
this analysis. In this case, malonyl-CoA was used as the substrate
instead of malonyl-ACP, and the formation of the
acetoacetyl-CoA·Mg2+ complex was monitored
spectrophotometrically at 305 nm. One would predict that any mutation
that interferes with the FabH·ACP interaction would adversely affect
the ACP-dependent assay but not the ACP-independent assay.
Table IV lists the FabH mutants that were
produced and their performance in the two assays. It should be noted
that the wild type FabH enzyme was about 40-fold less active with the
malonyl-CoA substrate than with its normal substrate malonyl-ACP. This
result was anticipated, and the reduced activity with the substrate
analogue was compensated for by an increased protein concentration in
the spectrophotometric assay.
With the exception of FabH[A253Y], the mutant proteins did not have
impaired activity with the CoA substrate analogue when compared with
the wild-type protein. This was expected, because none of the mutations
were directed against the known binding site for CoA on FabH (22, 26).
This result also confirmed that the mutations did not significantly
alter the overall structure of the protein (Table IV). In contrast, all
of the mutants exhibited reduced enzyme activity with malonyl-ACP
compared with the wild-type enzyme (Table IV), thereby supporting the
general location of the ACP interaction surface shown in Fig. 3. In the
case of the FabH[A253Y] mutant, the activity with malonyl-CoA was
17% of wild type, which indicates a general structural effect of this
mutation. However, this mutant retained only 0.1% of the wild type
activity in the ACP-dependent assay (Table IV), indicating
that the alanine to tyrosine substitution introduced a change into the
protein that was specific for ACP over CoA substrates. If the overall structure was perturbed randomly, then the activity in both assays should be reduced equally.
A closer analysis of the specific activities of the mutant
proteins in the ACP-dependent assay supports the more
detailed features of the model. The substitution of Ala-253 with the
bulky side chain of tyrosine was predicted to hinder the close approach of the protein molecules, and the specific activity is indeed minimal.
Also, the substitution of negatively charged glutamic acid for the
positively charged arginines or lysines (FabH[R249E], FabH[K256E/K257E], and FabH[K214E]) uniformly dropped the
activities of the mutant proteins, presumably by introducing repulsive
ionic interactions into regions where attractive interactions were
predicted to occur. In contrast, although the FabH[K214A] and
FabH[K256A/K257A] mutants displayed reduced activities, the effects
were less severe. The only mutation that produced an unexpected result
was FabH[R249A]. Like the other alanine mutations, this was predicted
to show a subtle change in activity, but it turned out to be very
defective in the ACP-dependent assay (Table IV).
FabH·ACP Binding Studies--
The activity assays provide
indirect support for the validity of the FabH·ACP docking model.
However, to further test the model, we used two methods to directly
monitor the interaction between the proteins. For the first method, we
predicted that the binding of ACP to FabH would inhibit the FabH
reaction using malonyl-CoA as substrate (the second assay described
above). When bound to FabH, ACP and its prosthetic group should occlude
the active site tunnel and prevent the entry of malonyl-CoA. This effect should not be observed with a FabH mutant that does not bind
ACP. The results of these experiments using FabH[R249A] are presented
in Fig. 4, and clearly show that this
mutant protein was indeed significantly compromised in its ability to
interact with ACP. Thus, in the absence of ACP, wild type FabH and
FabH[R249A] show identical activities of 0.1 nmol/min/mg in the
spectrophotometric assay. However, ACP exhibited an IC50 of
about 25 µM with the wild type enzyme, but FabH[R249A]
was refractory to ACP inhibition. The second approach we used to
monitor the FabH·ACP interaction was surface plasmon resonance
(BIACORE). In these experiments (see "Experimental Procedures") the
ACP molecule was covalently attached to the chip (the so-called
"ligand"), and FabH was flowed across the chip and monitored for
binding (the so-called "analyte"). The important results are shown
in Fig. 5. Wild type FabH produced a
robust signal at less that 1.0 µM, and elicited fast off
and on kinetics consistent with its function as a rapidly dissociating substrates. However, FabH[R249A] showed no evidence of binding ACP
even at 133 µM. These data are consistent with Arg-249 as a critical residue in the interaction between FabH and ACP.
Features of ACP Binding Sites--
The surface of FabH surrounding
the active site tunnel is generally electropositive, in contrast to the
rest of FabH, which is highly electronegative (Fig.
6A). However, ACP is an acidic protein, and it is reasonable to suppose that ACP is attracted to this
general locale on the FabH surface and away from the rest of the
protein. Accordingly, the top scoring complexes from SurfDock all
involve ACP bound to the electropositive FabH surface. The specificity
determinants of the interaction cannot easily be determined from the
models, because the precise orientations of the side chains at the
interface are not known. However, the mutagenesis experiments that
replaced the lysine and arginine residues in this region of FabH with
oppositely charged glutamates support the idea that ionic interactions
are important docking determinants and that ACP interacts with each of
these residues.
To eliminate the ability of lysines 214, 256, and 257 to form ionic
bonds with ACP, we mutated them to alanines and demonstrated only a
moderate effect on FabH catalytic activity (Table IV). In contrast,
when Arg-249 was changed to an alanine, the mutant FabH was severely
impaired in the ACP-dependent assay (Table IV) and
refractory to ACP inhibition (Fig. 4). To confirm the importance of
Arg-249, we used surface plasmon resonance to directly monitor the
interaction between FabH and ACP. Wild type FabH clearly bound to ACP,
but FabH[R249A] showed no detectable interaction even at a relative
100-fold excess (Fig. 5). These data point to Arg-249 as the most
important electropositive residue involved in FabH·ACP docking, and
it is therefore significant that Arg-249 is the only residue on this
surface that is completely invariant in FabH proteins (Fig. 3).
Although Arg-249 appears to make a strong electrostatic interaction
with Glu-41 of ACP, it is also likely that the guanidinium moiety and the proximal elements of the side chain form more extensive interactions with ACP.
A magnified view of the ACP-binding electrostatic surface of FabH (Fig.
6B) reveals the positive patches from Arg-249 and the other
basic residues, but these are interspersed with additional hydrophobic
regions that presumably contribute to the binding specificity. Ala-253
is a highly conserved residue located in a hydrophobic depression on
the surface, and it is predicted to interact with a sister alanine of
ACP. The importance of this depression was tested by introducing a
bulky side chain in place of Ala-253. This modification effectively
attenuates the interaction between ACP and FabH. Thus, we suggest that
the small hydrophobic alanines in the FabH·ACP interface are critical
for allowing the close approach of the two proteins.
Our model predicts that other ACP-binding proteins will also contain a
conserved arginine (lysine) residue in a hydrophobic/electropositive patch adjacent to their active sites. The crystal structures of five
other ACP-binding proteins are known: FabA, FabD, FabF, FabI, and LpxA.
FabB is highly homologous to FabF, and we determined its crystal
structure to 2.5-Å resolution using molecular replacement methods (see
"Experimental Procedures"). While this analysis was underway, the
FabB structure was reported elsewhere (50), and we refer the reader to
this citation for a more detailed description of the FabB structure.
Each of these six proteins has a region adjacent to its active site
that incorporates the important features of the putative FabH
ACP-binding site (Fig. 7). In all cases, the active site entrance is adjacent to a positively
charged/hydrophobic patch that would be predicted to dock the incoming
acyl-ACP substrate. The distances between the positively
charged/hydrophobic patches and the active site entrances varied from
10.1 Å for FabH to 14.9 Å for LpxA (Fig. 7). Compelling support for
the generality of the model comes from examination of the recent
crystal structure of the AcpS·ACP complex (51). In this case, a salt
bridge between Glu-41 of ACP and Arg-221 of AcpS is a critical
interaction that determines the binding of ACP to AcpS.
Features of ACP that Support the Model--
A comparison of the
primary structures of ACPs from a variety of species (Fig.
3B) reveals that residues 32 to 50 are highly conserved, in
contrast to the rest of the protein where the primary structure is
quite variable. This pattern of sequence conservation supports our
model, because the conserved region encompasses the entire interface
with FabH. Specifically, it includes the whole of helix II, which is
predicted to pack against helix C
The structural studies of ACP using NMR spectroscopy have included an
analysis of the molecule's conformational flexibility (52), and the
protein is known to contain a number of mobile segments. The most
flexible region is the extended loop between helices I and II spanning
residues 15 to 35. NMR studies of the complex of ACP with FabA (53),
the smallest of the ACP-binding enzymes (9), reveals chemical shift
perturbations of the ACP side chains in the complex. Perturbed residues
include Asn-25, Ser-36, Asp-38, glutamates 41, 47, 53, and 60, Val-40,
and Ala-45, indicating that these residues on the ACP surface mediate
its interaction with FabA. In addition, changes in solvent
accessibility were measured to monitor conformational movements within
the protein, and these revealed that the mobile extended loop between
helices I and II unfolds during the binding process. These NMR data are entirely consistent with the docking model generated by SurfDock. Specifically, the majority of the ACP residues that experience chemical
shift changes are located on the face of helix II within the relatively
rigid three-helix bundle. This is precisely the interface region
predicted by SurfDock in the highest scoring FabH·ACP complex
described here. Also, the apparent conformational change in the ACP
loop would not disrupt our predicted FabH·ACP complex, and it would
provide a reasonable mechanism whereby the prosthetic group attached to
Ser-36 could be injected into the active site tunnel.
A Model for the Interaction of ACP with Its Target Proteins--
A
crucial requirement of any proposed FabH·ACP complex is that the
4'-phosphopantetheine prosthetic group attached to Ser-36 must be able
to deliver the acyl group on its terminal sulfhydryl to the active
site. As already noted, the orientation of Ser-36 in our model is
ideal, and one reason for this may be the proposed ion pair between
Arg-249 and Glu-41 (Table III). This interaction between two completely
conserved residues is directly adjacent to Ser-36 on helix II. It is
tempting to speculate that this ion pair has a critical role in
orienting the prosthetic group relative to the active site tunnel, and
this may be a crucial interaction in all the complexes that ACP forms
with the enzymes of fatty acid biosynthesis. Although the orientation
of Ser-36 is ideal in the docking model, it is not close enough to
allow the attached prosthetic group to deliver the substrate to the
FabH active site. We estimate that a movement of some 10 Å would be
required in the ACP molecule to bridge this gap. Because the NMR data
show that ACP has conformational flexibility in this region, we propose that this is a required feature of the interaction. Biochemical evidence suggests that the lipid molecule attached to ACP interacts with and modifies the ACP structure (18, 54). Therefore, the required
dissociation of the lipid during the binding process may help to
promote this proposed conformational change in the ACP molecule.
Thus, we envisage three discrete steps in a dynamic ACP-binding
process. The first step involves a weak but specific interaction between the target protein and the rigid three-helix bundle of ACP.
This is the interaction found by SurfDock for FabH, and effectively aligns Ser-36 and the prosthetic group with respect to the active site
entrance. In the second step, a conformational change in ACP injects
the substrate attached to the prosthetic group into the active site.
These movements include an unfolding of an extended flexible loop, and
might be driven by the release of the acyl group from its interactions
with ACP after the initial binding step. Finally, the modified
substrate is removed from the active site by a reversal of the
conformational changes, and the ACP structure is stabilized through
interactions with the new lipid intermediate. This mechanism has two
attractive features. First, it accommodates the various architectures
of the target ACP-binding proteins in which the distance from Ser-36 to
the active site is not constant. Second, it provides a way of easily
introducing and extracting the prosthetic group from the active site
openings without forming a tight complex between the two proteins. The FabH·ACP model forms the basis for strategies to stabilize the interaction with a view to growing diffraction quality crystals of this
and other complexes.
-ketoacyl-ACP synthase III (FabH) and experimentally tested the
model by the biochemical analysis of FabH mutants. The activities of
the mutants were assessed using both an ACP-dependent and
an ACP-independent assay. The ACP interaction surface was defined by
mutations that compromised FabH activity in the
ACP-dependent assay but had no effect in the
ACP-independent assay. ACP docked to a positively charged/hydrophobic patch adjacent to the active site tunnel on FabH, which included a
conserved arginine (Arg-249) that was required for ACP docking. Kinetic analysis and direct binding studies between FabH and ACP confirmed the identification of Arg-249 as critical for FabH·ACP interaction. Our experiments reveal the significance of the positively charged/hydrophobic patch located adjacent to the active site cavities
of the fatty acid biosynthesis enzymes and the high degree of sequence
conservation in helix II of ACP across species.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices packed into a bundle
with an extended and flexible loop at one end (6, 7). The fatty acid
intermediates are attached to the terminal sulfhydryl of the
4'-phosphopantetheine prosthetic group, which in turn, is covalently
attached to serine 36 via a phosphodiester linkage (8). In addition to
fatty acid biosynthesis, ACP also supplies acyl groups for the
synthesis of a variety of bacterial biomolecules, including
glycerolipids (9), polyamino acid antibiotics (10), lipid A (11),
quorum sensing compounds (12, 13), polyketides (14), and
poly-
-hydroxyalkanoates (15). In mitochondria, an ACP-like protein
is a subunit of NADH-ubiquinone oxidoreductase (16, 17).
-Ketoacyl-acyl carrier protein synthase III (FabH) is a condensing
enzyme that catalyzes the initial step in the elongation of fatty
acids. Its two substrates, acetyl-CoA and malonyl-ACP, bind
sequentially in a ping-pong enzyme mechanism to produce acetoacetyl-ACP (23-25). We and others have recently determined the crystal structure of FabH (22, 26) and have identified the active site and the specific
binding site for CoA. The active site is at the base of a 20-Å-deep
tunnel, and a bound CoA molecule in the crystal structure reveals that
the interactions with the 4'-phosphopantetheine moiety are mainly
hydrophobic in character. The weak interaction between FabH and ACP
thioesters (ca. 5 µM) (27) and the flexibility of the ACP
molecule (4) have made it difficult to crystallize the FabH·ACP
complex. As an alternative strategy, we have investigated the
interaction between the two proteins using a combination of computational, biochemical, and structural methods. Using this approach, we have identified specific surface features on both proteins
that are critical to their interaction and propose a model by which ACP
can deliver and extract substrates from FabH and other fatty acid
biosynthetic enzymes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. Protein concentration was determined using the Bradford method with
-globulin as the standard (30). All other reagents were of the
highest available purity.
In this equation, B = (Eo
(Eq. 1)
A), Eo is the dielectric
constant of bulk water at 25 °C (74.4), and A (
8.5525),
(0.003627 Å
1), and k (7.7839) are
parameters to partially account for the fact that, in solution, a
protein encloses a microscopic region of low permittivity where the
effective shielding may be dampened relative to the shielding in the
solvent-accessible regions.
Statistics of the final FabB model
Inner primers used to construct FabH mutants
1 using the Biopolymer calculator (available on the
Web), and the measured ellipticity was converted to molar values
for direct comparison of the mutants.
-mercaptoethanol, 65 µM malonyl-CoA, 45 µM
[1-14C]acetyl-CoA (specific activity 60 µCi/µmol),
2.64 µg of purified FabD, 0.1 M sodium phosphate buffer,
pH 7.0, and 1 ng to 0.1 µg of FabH protein in a final volume of 40 µl. The ACP,
-mercaptoethanol, and buffer were preincubated at
37 °C for 30 min to ensure the complete reduction of ACP. The
reaction was initiated by the addition of FabH. After incubation at
37 °C for 15 min, 35 µl of reaction mixture was removed and
dispensed onto a paper filter disc (Whatman 3MM filter paper). The disc
was washed successively with ice-cold 10%, 5%, and 1%
trichloroacetic acid with 20 min for each wash. The filter discs were
dried and counted for 14C-isotope in 3 ml of scintillation fluid.
-Ketobutyryl-CoA, the
final product of this reaction, formed a complex with Mg2+,
which absorbs at 305 nm (46). Increase in the absorbency at 305 nm was
recorded for 2 min. The ability of ACP to inhibit either FabH or
FabH[R249A] activity in the spectrophotometric assay was tested by
incubating ACP with the protein at room temperature for 5 min before
the addition of malonyl-CoA to initiate the reaction.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactamase with its inhibitory protein
RTEM (31) and the ternary TF·VIIa·Xa complex (32). SurfDock is a
hierarchical docking program that performs a rigid-body six-dimensional
search to dock two proteins of known three-dimensional structure.
Briefly, surface properties such as hydrophobicity and electrostatic
potential are abstracted onto the molecular surfaces of the interacting proteins and represented as expansions of spherical harmonics functions. By varying the number of terms used in the expansion function, the resolution of the surface and the property representation may be controlled (48). During the docking procedure, we moved progressively from low resolution to high resolution representations, which increased the details in the shape of the surface and also the
resolution of the physical properties. This hierarchical strategy is
efficient for protein·protein docking problems, because many complexes have good complementarity of geometrical and physical properties at a resolution significantly lower than the precision of
the atomic coordinates (47). An evolutionary programming technique, a
stochastic optimization procedure inspired by biological evolution, was
used to explore the configurational search. This technique determines a
population of candidate solutions, which then undergo mutation,
competition, and selection to find the global optimum of a given
function. The population of docked models was scored using a
pseudo-energy function to determine the quality of the complex.
524
to
400), the ACP preferred to dock at the entrance to the FabH active
site. This is significant, because the ACP docking procedure was
completely unconstrained, and the result was consistent with the
proposed substrate binding site based on the surface electrostatic
potential of the FabH crystal structure (22, 26). In the smaller
subset, which contained the next lowest scoring group (
380 to
250),
ACP was predicted to dock in a cavity at the dimer axis of FabH. We
describe in detail only the top scoring complex (score =
524),
which has a number of features that are consistent with the
requirements of the interaction and with protein·protein interactions
in general.
-helices
(helix II from ACP and helix C
2 from FabH) that are oriented at a
relative angle of
60°, which is the optimal angle for this type of
protein·protein interaction (49). Fourth, two alanine residues
(Ala-45 from ACP and Ala-253 from FabH) permit the close approach of
the two
-helices. Fifth, the contacting surfaces are oppositely
charged, and there are many possible ionic interactions between them
(Table III). We emphasize "possible" because SurfDock does not
determine the orientations of the surface side chains in the complex
and their conformations in the complex will certainly differ from their
orientations observed in the free proteins. Sixth, the basic patch on
FabH is quite extensive and actually comprises two nonoverlapping
binding sites, one for ACP described here and another for CoA
characterized earlier (22, 26). The fact that the two sites are
adjacent but distinct supports our model of the FabH·ACP interaction.
Finally, the conserved and unusually exposed Phe-213 of FabH forms a
hydrophobic interaction with the conserved Met-44 of ACP.
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Fig. 1.
A surface rendition of the FabH·ACP complex
predicted by SurfDock. Left panel, a surface
rendition of the complex in which the FabH and ACP surfaces are
colored according to their hydrophobic/hydrophilic
properties. ACP is on the left differentiated from FabH by
the net surface. Red to blue indicates the shift
from hydrophobic to hydrophilic character. Most of the FabH
surface is hydrophilic, with only a few hydrophobic patches evident.
Right panel, a ribbon diagram of the modeled
complex showing the interaction of the two molecules and the location
of the active site entrance.
Favorable amino acid contacts in the top scoring FabH·ACP complex
predicted by SurfDock
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Fig. 2.
A stereo view showing the important
interactions at the interface of the FabH·ACP complex predicted by
SurfDock. Left panel, overview of the interacting
helices on ACP helix II (chocolate brown) and FabH C 2
(blue). The location of Ser-36 of ACP, which is the
attachment site for the 4'-phosphopantetheine prosthetic group, is
indicated in relation to the active site entrance. Right
panel, a magnified stereo view of the predicted interaction
surface. The amino acid residue pairs (see also Table III) that
interact are color-coded as follows (ACP/FabH):
orange, Glu-13/Lys-256; blue, Glu-41/Arg-249;
purple, Met-44/Phe-213; green, Ala-45/Ala-253;
yellow, Glu-47 and Glu-48/Lys-214; cyan,
Glu-49/Lys-257. The interacting
-helices at the interface, helix II
from ACP and C
2 from FabH, are highlighted in
chocolate brown and blue, respectively. For
clarity, some of the peripheral interactions listed in Table III have
been omitted from the figure. Arg-249 from FabH and Glu-41 from ACP are
located just above Ser-36 of ACP and are proposed to form a crucial
ionic interaction adjacent to the active site entrance. The figure was
produced using MOLSCRIPT (55) and rendered with Raster3D (56).
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Fig. 3.
Conservation of residues on FabH and
ACP. A, primary sequences of FabH enzymes from bacteria
and plants are compared, and the region containing the residues
proposed to interact with ACP (highlighted) is shown.
Conserved residues are shown in uppercase, and the His in
the active site is marked with an asterisk.
Numbering is for E. coli FabH. B, the
ACP proteins from representative prokaryotes and eukaryotes (minus
leader sequences) were compared. The identical residues are in
uppercase, whereas those identified as interacting with FabH
by SurfDock are highlighted. The region from Leu-32 to
Phe-50 is highly conserved and includes helix II and the conserved loop
containing the prosthetic group attachment site (Ser-36; indicated with
an asterisk). Numbering is for E. coli ACP and
residues proposed to interact with fabH are highlighted.
Abbreviations: H. influenza, Haemophilus
influenzae; Aq. aeolicus, Aquifex aeolicus;
P. purpurea, Porphyra purpurea; A. thaliana, Arabidopsis thaliana; C. wrightii,
Cuphea wrightii, C. lanceolata, Cuphea
lanceolata.
Specific activities of FabH and ACP docking site mutants in
ACP-dependent and ACP-independent assays
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Fig. 4.
ACP inhibition of FabH and
FabH[R249A]. The ability of ACP to function as an inhibitor of
the condensing enzyme reaction was evaluated using a spectrophotometric
assay utilizing malonyl-CoA as described under "Experimental
Procedures" and the indicated concentration of ACP and either 50 µg
of FabH ( ) or 50 µg of FabH[R249A] (
).
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Fig. 5.
FabH·ACP binding studies using surface
plasmon resonance. ACP was immobilized to the surface of a CM-5
chip, either FabH or FabH[R249A] was injected, and the change in the
relative refractive index was measured using the Biacore 3000 as
described under "Experimental Procedures." The refractive index
change with FabH at 0.83 µM protein is indicative of
binding to the immobilized ACP, whereas a binding signal was not
detected with FabH[R249A] at 133 µM. An apparent
binding constant of 2 ± 1 µM was calculated by
determining the relative extent of FabH binding at seven different
protein concentrations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 6.
The positively charged/hydrophobic region of
FabH that is predicted to be the binding site for ACP.
A, the region in the context of the whole molecule. Note
that the surface of FabH is generally electronegative. The active site
tunnel entrance is flanked on one side by the ACP-binding site and on
the other side by the known coenzyme A binding site. The two sites are
contiguous but distinct. B, a magnified view of the ACP
binding surface. The locations of the residues mutated in this study
are indicated. The extreme ranges of red
(negative) and blue (positive) represent electrostatic
potentials of < 9 to >+9 kbT,
where kb is the Boltzmann constant and
T is the temperature. The figure was calculated using the
GRASP program (57).
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Fig. 7.
Comparison of the electropotential surfaces
of six ACP-binding enzymes. The figure was made using the known
coordinates for FabA (21); FabB (our coordinates were deposited in the
Protein Data Bank, accession code 1G5X); FabD (58); FabF (39); FabI
(59); and LpxA (60). Each protein has a basic/hydrophobic patch
(X) suitable for binding ACP that is adjacent to the active
site ( ). The distances between these zones are: FabH, 10.1 Å; FabA,
11.9 Å; FabB, 13.7 Å; FabD, 11.7 Å; FabF, 13.3 Å; FabI, 13.3 Å;
and LpxA, 14.9 Å. The extreme ranges of red
(negative) and blue (positive) represent electrostatic
potentials of <
9 to >+9 kbT,
where kb is the Boltzmann constant and
T is the temperature. The figure was calculated using the
GRASP program (57).
2 of FabH, and the loop segment
immediately preceding helix II that contains the important Ser-36 (Fig.
2). Significantly, all of the interacting ACP residues listed in Table
III are almost completely conserved. Two residues deserve particular
mention. Glu-41, which is predicted to interact with the crucial
Arg-249 of FabH, is actually conserved in all 49 ACP sequences from the
data base. Ala-45 is only replaced with a glycine residue, and this is
consistent with the suggested role of the amino acid in creating a
depression in the ACP surface that allows the close approach of the
target protein.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank William Lewis for assistance with the surface plasmon resonance experiments, and Suzanne Jackowski for invaluable discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM34496 (to C. O. R.), GM44973 (to S. W. W.), and Cancer Center (CORE) Support Grant CA 21765 and by the American Lebanese Syrian Associated Charities.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1G5|ga) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ Current address: GlycoDesign Inc., 480 University Ave., Ste. 900, Toronto M5G 1V2, Canada.
To whom correspondence should be addressed: Dept. of
Biochemistry, St. Jude Children's Research Hospital, Memphis, TN
38105. Tel.: 901-495-3491; Fax: 901-525-8025; E-mail:
charles.rock@stjude.org.
Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.M008042200
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ABBREVIATIONS |
---|
The abbreviations used are:
ACP, acyl carrier
protein;
FabB, -ketoacyl-ACP synthase I;
FabF,
-ketoacyl-ACP
synthase II;
FabH,
-ketoacyl-ACP synthase III;
PCR, polymerase chain
reaction;
bp, base pair(s).
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REFERENCES |
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