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§1§ Structural Basis for the Feedback Regulation of Escherichia coli
Pantothenate Kinase by Coenzyme A[15]* §1§
1. [16]Mikyung Yun[17]‡,
2. [18]Cheon-Gil Park[19]‡,
3. [20]Ji-Yeon Kim[21]‡,
4. [22]Charles O. Rock[23]§[24]¶,
5. [25]Suzanne Jackowski[26]§[27]¶ and
6. [28]Hee-Won Park[29]‡[30]‖
1.
From the Departments of ^‡Structural Biology and ^§Biochemistry, St.
Jude Children's Research Hospital, Memphis, Tennessee 38105 and the
^¶Department of Biochemistry, University of Tennessee, Memphis,
Tennessee 38163
[31]Next Section
§2§ Abstract §2§
Pantothenate kinase (PanK) is a key regulatory enzyme in the coenzyme A
(CoA) biosynthetic pathway and catalyzes the phosphorylation of
pantothenic acid to form phosphopantothenate. CoA is a feedback
inhibitor of PanK activity by competitive binding to the ATP site. The
structures of the Escherichia coli enzyme, in complex with a
nonhydrolyzable analogue of ATP, 5′-adenylimido-diphosphate (AMPPNP),
or with CoA, were determined at 2.6 and 2.5 Å, respectively. Both
structures show that two dimers occupy an asymmetric unit; each subunit
has a α/β mononucleotide-binding fold with an extensive antiparallel
coiled coil formed by two long helices along the dimerization
interface. The two ligands, AMPPNP and CoA, associate with PanK in very
different ways, but their phosphate binding sites overlap, explaining
the kinetic competition between CoA and ATP. Residues Asp^127, His^177,
and Arg^243 are proposed to be involved in catalysis, based on modeling
of the pentacoordinate transition state. The more potent inhibition by
CoA, compared with the CoA thioesters, is explained by a tight
interaction of the CoA thiol group with the side chains of aromatic
residues, which is predicted to discriminate against the CoA
thioesters. The PanK structure provides the framework for a more
detailed understanding of the mechanism of catalysis and feedback
regulation of PanK.
CoA is the predominant acyl group carrier in biology and participates
in a wide variety of biochemical reactions including pyruvate
dehydrogenase, citrate synthase, α-ketoglutarate dehydrogenase, and the
synthesis and β-oxidation of fatty acids. The universal CoA
biosynthetic pathway consists of five enzymatic steps ([32]1,[33]2).
The first step produces 4′-phosphopantothenic acid by phosphorylation
of pantothenic acid. The subsequent formation of 4′-phosphopantetheine
is a two-step process in which 4′-phosphopantothenate and cysteine are
converted to 4′-phosphopantothenoyl-cysteine by formation of a peptide
linkage followed by decarboxylation of the cysteine. In the final two
steps, 4′-phosphopantetheine is adenylated to form dephospho-CoA, which
in turn is phosphorylated at the 3′ position of ribose to form CoA.
The first step of CoA biosynthesis catalyzed by pantothenate kinase
(PanK)^1 is a key regulatory point in CoA biosynthesis ([34]3-9).
Escherichia coli PanK (bPanK) is a homodimer of 36-kDa subunits, and
the amino acid sequence contains an A-type ATP-binding consensus
sequence, GXXXXGKS ([35]10, [36]11). bPanK exhibits highly positive
cooperative ATP binding and mediates a sequential ordered mechanism
with ATP as the leading substrate ([37]11). PanK activity is inhibited
by nonesterified CoA and to a lesser extent by its thioesters, which
competitively interfere with ATP binding ([38]4, [39]12). A detailed
mechanism of PanK inhibition by CoA is not known at the molecular
level. Interestingly, nonesterified CoA is the most potent inhibitor of
theE. coli enzyme ([40]4), whereas acetyl-CoA is a more effective
inhibitor of eukaryotic enzymes ([41]8, [42]9, [43]12).
Eukaryotic PanK cDNAs have recently been cloned, one fromAspergillus
([44]12) and two from mouse ([45]13). ADrosophila PanK has been
identified as encoded by thefumble gene and may play a role in cell
division (GenBank^TM accession number [46]AF221546). Comparison of the
protein sequences predicted from these cDNAs with the bPanK amino acid
sequence points out strong dissimilarity between prokaryotic and
eukaryotic PanKs, although significant homology is found among the PanK
sequences within each class ([47]12). This observation suggests that
targeting the bacterial enzyme would be an effective therapeutic
strategy for development of new anti-infective agents. We investigated
the molecular basis for bPanK catalysis and regulation by determining
the structures of binary complexes of bPanK and AMPPNP, a
nonhydrolyzable analogue of ATP, and CoA.
[48]Previous Section[49]Next Section
§2§ EXPERIMENTAL PROCEDURES §2§
§5§ Protein Preparation §5§
The CoaA gene encoding bPanK ([50]10) was subcloned into pET21a
([51]Novagen, Inc.) and transformed intoE. coli B834(DE3)pLysS
([52]Novagen, Inc.). The E. coli cells harboring the pET21-bPanK
plasmid were grown in 4 liters of M9 minimal medium (6 g/liter
Na[2]HPO[4], 3 g/liter KH[2] PO[4], 0.5 g/liter NaCl, and 1 g/liter
NH[4] Cl) containing 0.4% glucose, 1 mm MgSO[4], 0.1 mmCaCl[2], 0.0005%
thiamine, amino acid mixture (0.5 g/liter of each amino acid), 50 μg/ml
of ampicillin, 34 μg/ml of chloramphenicol, and 0.1 g/liter of
methionine at 37 °C. When the culture reached an absorbance of 0.7–0.8
at 600 nm, the cells were harvested and washed with the M9 minimal
medium twice. The cells were resuspended in the M9 minimal medium
containing 40 mg/l of seleno-l -methionine (Sigma). After induction
with 0.4 mmisopropyl-1-thio-β-d-galactopyranoside, the cells were
allowed to grow for another 3 h to be harvested. The harvested cell
pellet was resuspended in 400 ml of disruption buffer (20 mm Tris-HCl,
pH 8.0, 1 mm dithiothreitol, 1 mm EDTA, and 1 mm phenylmethylsufonyl
fluoride) and lysed with sonication. The cell lysate was centrifuged at
14,000 × g for 30 min. The supernatant was purified by Q-Sepharose
anion exchange chromatography in which bPanK was eluted with an
increasing NaCl gradient. Eluted factions containing bPanK were pooled
and concentrated. The concentrated sample was then purified with a
S-200 Superdex gel filtration column for the fast protein liquid
chromatography system (Amersham Pharmacia Biotech). The elution buffer
was 20 mm Tris-HCl, pH 7.6, 200 mm NaCl, 1 mm dithiothreitol, and 1 mm
EDTA, and the flow rate was 60 ml/h. Fractions containing bPanK were
pooled and dialyzed at 4 °C for 15 h against dialysis buffer (20
mmTris-HCl, pH 8.0, 1 mm dithiothreitol, and 1 mmEDTA). The dialyzed
sample was concentrated to 20 mg/ml for crystallization trials.
§5§ Crystallization §5§
Co-crystals of selenomethionine bPanK and AMPPNP were grown by the
hanging drop vapor diffusion method. Before crystallization, AMPPNP was
added to the protein to a final concentration of 2 mm, and the mixture
was incubated overnight at 4 °C. The hanging drops containing the equal
volume of the protein and the reservoir solution were equilibrated at
4 °C against the reservoir solution (11% polyethylene glycol 8,000 and
0.1m HEPES, pH 7.5). The trigonal crystals appeared after several days,
which grew further for about 1 more week. These crystals were
transferred to a cryoprotectant solution containing 40% glycerol, 15%
polyethylene glycol 8,000, 2 mm dithiothreitol, 0.5 mm AMPPNP, 2.5 mm
MgCl[2], and 0.1m HEPES at pH 7.5 for cryo-data collection.
Crystals of bPanK in complex with CoA were grown at room temperature by
the hanging drop vapor diffusion method. To achieve the full occupancy
of bound CoA, bPanK was mixed with CoA at the final concentration of 2
mm, and the mixture was incubated for 24 h at 4 °C. Equal volumes of
protein solution and reservoir solution were combined. The reservoir
solution consisted of 10% polyethylene glycol 4,000, 50 mm Li[2]SO[4,]
2% isopropanol, and 0.1m N-[2-acetamido]-2-iminodiacetic acid at pH
6.5. Rod-like crystals appeared after 1 week and grew for another 2–3
weeks. These crystals were frozen using a cryoprotectant solution
containing 40% glycerol, 12% polyethylene glycol 4,000, 2.5 mm
dithiothreitol, 0.1 mm CoA, 2 mmMgCl[2], 50 mm Li[2]SO[4], and 0.1 m
N-[2-acetamido]-2-iminodiacetic acid at pH 6.5 for cryo-data
collection.
§5§ Data Collection and Structure Determination of AMPPNP-bound bPanK §5§
Multiwavelength anomalous dispersion data were measured from a single
crystal of the AMPPNP-bound bPanK at beamline X12C at the National
Synchrotron Light Source, equipped with a Q4 CCD detector (Brandeis)
operating at 100 K. Three wavelengths near the selenium absorption
edge, 0.95 Å (the remote), 0.9791 Å (the peak), and 0.9794 Å (the
inflection point), were chosen by measuring the x-ray absorption
spectrum of the protein crystal. The data set from each wavelength was
integrated, reduced, and scaled by the programs DENZO and SCAPEPACK
([53]14). The crystals of the AMPPNP-bound enzyme belong to space group
P3[2]21 with unit cell dimensions ofa = 130.1 Å and c = 281.8 Å.
The program SOLVE ([54]15) was used to scale three wavelength data sets
together, to locate selenium positions and to calculate the
multiwavelength anomalous dispersion phases. The statistics of data
collection and phasing are summarized in Table[55]I. The initial
electron density map calculated at 3.0 Å resolution was improved by the
noncrystallographic symmetry averaging method using the program DM (the
CCP4 suite) ([56]16). The atomic model was built using the graphics
programs O ([57]17) and XtalView ([58]18), followed by crystallographic
refinement in the program XPLOR ([59]19). The refinement steps included
rigid body, simulated annealing, conjugate gradient minimization, and
individual B-factor refinement. A solvent mask was calculated with the
bulk solvent correction routine in XPLOR ([60]19). During refinement,
NCS restraints were imposed on the main chain atoms. The final
structure contains four subunits of bPanK (6–316 residues in one
subunit and 8–316 residues in the other three subunits). The refinement
statistics are shown in Table[61]II.
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Table I
Data collection and phasing statistics
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Table II
Refinement statistics
§5§ Data Collection and Structure Determination of CoA-bound bPanK §5§
Complete diffraction data from a single crystal of the CoA-bound bPanK
were also measured at beamline X12C at the National Synchrotron Light
Source. The programs DENZO and SCALEPACK ([66]14) were used to
integrate and scale the data set. The crystals of the CoA-bound enzyme
belong to space group P1 with unit cell dimensions ofa = 62.0 Å, b =
71.2 Å,c = 87.7 Å, α = 102.4°, β = 89.5°, and γ = 93.2°. The data
collection statistics are summarized in Table [67]I.
The structure of the CoA-bound bPanK was determined by the molecular
replacement method using one subunit of the AMPPNP-bound enzyme as a
search model. The cross-rotation and translation functions and
Patterson correlation refinement were calculated using the program
XPLOR ([68]19). Using the data between 15.0 and 4.0 Å, the rotation
function, followed by Patterson correlation refinement, gave four
outstanding solutions that correspond to four subunits of bPanK.
Because any point can be taken as an origin in space group P1, rotation
of the initial search model according to one of the four rotation
function solutions determined the orientation and position of the first
subunit. With fixing this orientation and position of the first
subunit, the orientations of the other three subunits were determined
by applying the corresponding noncrystallographic symmetry operations
to the first subunit. The positions of the other three subunits were
then determined by finding the relative x, y, andz translations of each
of the three subunits with respect to the first subunit. The model
including all four subunits was subjected to rigid body refinement at a
resolution between 6. 0 and 4. 0 Å in the program XPLOR ([69]19). The
high resolution limit, which restricted the maximum shift of structure
in the refinement, was set to 4. 0 Å resolution to provide a possible
large shift of the structure up to 4.0 Å. The low resolution limit was
set to 6.0 Å resolution below which the intensities of reflections were
severely affected by the diffraction of solvent molecules in the
crystal. The Rfactor at this stage was high (51.0%), suggesting that
the conformation of the CoA-bound structure was different from that of
the search model, the AMPPNP-bound structure. To consider the solvent
contribution to the intensities of reflections at low resolution, the
structure factor amplitudes of solvent molecules in the crystal were
calculated using the bulk solvent correction routine in XPLOR ([70]19),
and thus the low resolution limit could be extended to 50.0 Å
resolution. Crystallographic refinement including simulated annealing,
conjugate gradient minimization, and individual B-factor refinement was
then performed at the resolution between 50.0 and 2.5 Å. After the
first round of refinement, the quality of the structure was
dramatically improved with a working R factor of 27.4% and a freeR
factor of 30.1%. The F [o] −F [c] difference map showed continuous
density for CoA, verifying the correct molecular replacement solution.
Several iterations of model building in the program O ([71]17) and the
refinement in the program XPLOR ([72]19) further dropped both R
factors. The final refinement statistics are shown in Table [73]II.
Because of the slight difference between four subunits,
noncrystallographic symmetry restraints were applied to only 89% of
total residues of each subunit. In the final model, residues 1–5 in
subunit 1; residues 1–7 and 211–212 in subunit 2; residues 1–5 and
210–213 in subunit 3; and residues 1–7, 83–85, and 212–214 in subunit 4
are excluded because they are disordered in the crystal.
[74]Previous Section[75]Next Section
§2§ RESULTS §2§
§5§ Monomer Structure §5§
In the structures of the AMPPNP-bound and the CoA-bound bPanKs, four
identical subunits are found in an asymmetric unit. When the four
subunits occupying an asymmetric unit of each structure are compared,
the average RMSD of Cα atoms for the AMPPNP-bound and the CoA-bound
enzymes are 0.46 and 0.34 Å, respectively. These values are consistent
with estimated errors in their atomic coordinates. A subunit of bPanK
adopts a mononucleotide-binding fold ([76]20): a seven-stranded β-sheet
(strands, 2, 3, and 8–11) is flanked by α-helices (D and E on one side
and G and J on the other side) (Fig.[77]1). An intervening loop between
strand 2 and helix E, known as the P-loop, contains most of the
residues that interact with phosphate oxygens of the AMPPNP. There are
four small antiparallel β-strands (strands 4–7) that are not part of
the main β-sheet (Fig. [78]1 a). Residues from helices H and I
including their intervening loop are involved in binding CoA (Fig.[79]1
a). The N-terminal region including strand 1, helices A and D, and a
loop between helices A and B forms the major part of the dimer
interface (Fig. [80]1 a).
[81]Figure 1
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Figure 1
A subunit structure of the AMPPNP-bound bPanK. a, schematic
representation. Helices are shown in red, strands are in yellow, and
other secondary structural elements are in green. The N-terminal region
involved in dimerization is shown in blue. AMPPNP (ball-and-stick
representation) is shown in yellow. Figs.[85]1 a, [86]2, [87]3 b, [88]4
b, and [89]5 were prepared using InsightII (Molecular Simulations). b,
topology representation. Cylinders and arrows represent α-helices and
β-strands, respectively. c, the sequence alignment of bPanK with
prokaryotic PanKs fromStreptococcus pyogenes, Enterococcus
faecalis,Pasturella multocida, Hemeophilus influlenzae,Actinobacillus
actinomycetemocomitans, Salmonella typhi, Klebsiella pneumoniae,
Yersinia pestis, Vibrio cholerae, Mycobacterium tuberculosis,
Corynebacterium diptheriae, andStreptomyces coelicolor. The sequence
numbering and secondary structure assignment is according to the bPanK
sequence. Amino acid residues identical in 12 of 13 sequences are shown
inred. Residues implicated in catalysis are marked withasterisks, and
Lys^101 involved in binding of ATP and CoA is marked with a triangle.
The Dali server ([90]21) was used to search for known proteins
structurally similar to bPanK. Applied to one subunit of bPanK, the
best matches occurred with proteins containing a mononucleotide binding
fold,i.e. phosphoribulokinase from Rhodobacter sphaeroides (Protein
Data Bank code [91]1a7j, RMSD 1.9 Å, 185 residues),
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from rat (Protein
Data Bank code [92]1bif, RMSD 1.7 Å, 96 residues), and adenylate kinase
from Bacillus stearothermophilus (Protein Data Bank code [93]1zin, RMSD
2.1 Å, 110 residues). In light of the wide distribution of the
mononucleotide binding fold, these findings are not surprising.
However, the remarkable similarity of bPanK to R. sphaeroides
phosphoribulokinase is unexpected because their sequence homology is
limited to the P loop. Phosphoribulokinase catalyzes the ATP-dependent
phosphorylation of ribulose 5-phosphate to ribulose 1,5-bisphosphate in
the Calvin cycle ([94]22).
§5§ Dimer Structure §5§
Four subunits found in an asymmetric unit of the AMPPNP-bound enzyme
are assembled to form two dimers, consistent with the fact that bPanK
functions as a dimer in solution ([95]11). The dimerization interface
occurs between two long α-helices from each of the subunits that form
an extensive antiparallel coiled coil (Fig.[96]2 a). The interface
contact including this coiled coil buries 4118 Å^2 of accessible
surface area of each subunit. The physical makeup of the interface
contact is slightly hydrophobic: 57% of nonpolar and 43% of
polar/charged residues. By comparison, the dimer-dimer interface is not
extensive. About 1790 Å^2 of the accessible surface area of each dimer
is buried. Most of the residues involved in this contact are
predominantly hydrophilic: 27% nonpolar and 73% polar/charged residues.
[97]Figure 2
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Figure 2
A comparison of the dimer structures of the AMPPNP-bound bPanK (a) and
of the CoA-bound bPanK (b). The dimer is viewed down the
noncrystallographic two-fold axis. AMPPNP and CoA (ball-and-stick
representation) are shown in yellow and cyan, respectively. c, a
space-filling diagram showing the superposition of one subunit of the
CoA-bound enzyme onto that of the AMPPNP-bound enzyme. Both subunits of
the AMPPNP-bound enzyme including helix D are shown in gray. The
superimposed subunit of the CoA-bound enzyme is not shown, whereas the
other subunit is shown inblue and its helix D is shown in red. Helical
axes are indicated by solid lines.
There are also two dimers in the asymmetric unit of the CoA-bound
bPanK. However, the dimerization interface of the CoA-bound enzyme is
not identical to that of the AMPPNP-bound enzyme resulting in a
conformational change. The dimerization interface, depicted parallel to
helix D of both subunits, shows a helical break where a couple of turns
of helix D of one subunit, involving residues 68–73, are unwound (Fig.
[101]2 b). This helical break is observed in both dimers. Consequently,
the last two turns of helix D of the first subunit are shifted closer
to helix D of the second subunit. This shift disturbs the dimerization
interface to bring about an 18° rotation of the second subunit with
respect to the first subunit, resulting in movement of the second
subunit of as much as 15 Å (Fig.[102]2 c). The altered interface
contact buries 3004 Å^2 of accessible surface area of each subunit. The
two dimers also interact by burying of 2174 Å^2 of each dimer surface.
§5§ ATP-binding Site §5§
The electron density of AMPPNP is clearly defined in both dimers
occupying an asymmetric unit of the AMPPNP-bound bPanK (Fig. [103]3 a).
AMPPNP is bound in a groove formed by residues from the P-loop, the
connecting loop of helices B and C, and the connecting loop of strands
10 and 11. AMPPNP with residues lining the binding site are shown in
Fig.[104]3 b. The adenine base is sandwiched by the side chains of
Asn^43 and His^307, the latter side chain further interacting with the
side chain of Trp^239. The N6 nitrogen of the adenine base interacts
via water-mediated hydrogen bonds with the hydroxyl groups of Tyr^55
and Thr^104 and the amino group of Lys^303, whereas its N1 nitrogen
forms a water-mediated hydrogen bond to the amide nitrogen of Ser^47.
The ribose is hydrogen-bonded to the carboxyl group of Asp^45. The
α-phosphate interacts with the hydroxyl group and the amide nitrogen of
Thr^103. The β- and γ-phosphates interact with the amino group of
Lys^101 and the β-phosphate interacts with the amide nitrogens of
Gly^100, Lys^101, and Ser^102, whereas the γ-phosphate interacts with
the amide nitrogen of Ala^98 and the guanidinium group of Arg^243. A
magnesium ion is coordinated by β- and γ-phosphates on one side and the
carboxyl group of Glu^199 and the hydroxyl group of Ser^102 on the
other side.
[105]Figure 3
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Figure 3
Stereoview of AMPPNP binding. a, the F [o] −F [c] simulated annealing
omit map of the ATP binding site of one subunit showing the bound
AMPPNP. The map was calculated from 50.0 to 2.6 Å resolution and
contoured at 3.0 ς. Figs. [109]3 A and [110]4 A were prepared using
XtalView ([111]18). b, hydrogen bonds are indicated by solid lines.
Carbons are shown in yellow for AMPPNP andgray for bPanK. Nitrogens are
shown in blue, oxygens are in red, phosphorus is in green, and Mg^2+ is
in cyan. Asn^43 involved in ATP recognition by stacking interaction
with the adenine base of AMPPNP is not labeled for clarity.
§5§ CoA-binding Site §5§
The well defined electron density for CoA is seen in both dimers
occupying the asymmetric unit of the CoA-bound bPanK (Fig. [112]4 a).
CoA is bound in the enzyme in a bent conformation in a deep pocket
lined by residues from helix H, the P loop, the connecting loop of
strands 5 and 6 and the connecting loop of helices H and I (Fig. [113]4
b). Because CoA is a competitive inhibitor with respect to ATP and
these two ligands share the ADP moiety, it was suggested that both
ligands bind to the same site ([114]4, [115]11). Unexpectedly, the
adenine base of CoA is inserted between the side chains of His^177 and
Phe^247, whereas the adenine-interacting residues in the AMPPNP-bound
enzyme are Asn^43 and His^307 (Fig.[116]4 b). The α-phosphate of CoA is
salt bridged to the guanidinium group of Arg^243 and the amino group of
Lys^101 that also interacts with the β-phosphate of CoA (Fig. [117]4
b). The 3′-phosphate of CoA is hydrogen-bonded to the amide nitrogen of
Ile^42 and the hydroxyl group of Ser^102 and salt bridged to the
guanidinium group of Arg^106 (Fig. [118]4 b). These interactions of the
3′-phosphate group may be essential for the inhibitory effect of CoA
because dephospho-CoA is a significantly less potent inhibitor of bPanK
activity ([119]4). By comparison, the same 3′-phosphate group in
several CoA-binding proteins shows no interaction with the protein and
is exposed to solvent (for review see Ref. [120]23). The exceptions are
acyl-CoA binding protein ([121]24) and the surfactin synthetase
activating enzyme Sfp ([122]25), in which the 3′-phosphate interacts
with the protein residues such as His and Lys.
[123]Figure 4
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Figure 4
Stereoview of coenzyme A binding. a, the F [o] −F [c] simulated
annealing omit map of the CoA binding site of one subunit showing the
bound CoA. The map was calculated from 50.0 to 2.5 Å resolution and
contoured at 3.0 ς.b, hydrogen bonds are indicated by solid lines. The
thiol group of coenzyme A is shown in light green, and other atom
colors are as described in the legend to Fig.[127]3 b. His^177 involved
in CoA recognition by stacking interaction with the adenine base and
Asn^282forming a hydrogen-bond with the β-mercaptoethylamine moiety are
not labeled for clarity.
Hydrophobic atoms of the pantothenate moiety of CoA form van der Waals'
contacts with residues Leu^130, Tyr^175, and Ile^281 (Fig. [128]4 b).
The carbonyl oxygen of the pantothenate moiety near the
β-mercaptoethylamine moiety is hydrogen bonded to the hydroxyl group of
Tyr^240 and to the amide group of Asn^282 (Fig. [129]4 b). The amide
nitrogen of the β-mercaptoethylamine moiety is hydrogen-bonded to the
hydroxyl group of Tyr^180 (Fig. [130]4 b). Strikingly, the thiol group
of the β-mercaptoethylamine moiety is tightly sealed from water
molecules by interacting with four aromatic residues, Phe^244, Phe^252,
Phe^259, and Tyr^262 and also by intra-molecular hydrogen bonding to
the amino group of the adenine base (Fig. [131]4 b). The thiol group
approaches the face of Phe^259 at the distance of 3.8 Å between the
sulfur atom and the ring centroid. The thiol group also approaches the
edges of Phe^244, Phe^252, and Tyr^262, and the distances between the
sulfur atom and the ring carbons of Phe^244, Phe^252, and Tyr^262 are
3.8, 3.5, and 4.1 Å, respectively. These sulfur-aromatic interactions
are weakly polar interactions ([132]26-30) that are stronger than van
der Waals' interactions between nonpolar atoms ([133]31). These types
of sulfur-aromatic interactions are involved in protein stability and
function ([134]31, [135]32). The structure of another regulatory enzyme
in the CoA biosynthetic pathway, phosphopantetheine
adenylyltransferase, exhibits a dinucleotide-binding fold ([136]33).
The C-terminal loops of five parallel β-strands bind the product
dephospho-CoA. As in the CoA-bound bPanK, the thiol group of
dephospho-CoA in phosphopantetheine adenylyltransferase is shielded
from water molecules by interacting with the side chains of hydrophobic
residues.
§5§ The Specific Binding of Coenzyme A Compared with AMPPNP §5§
Structural analysis of the AMPPNP-bound and the CoA-bound bPanKs
provides the basis for understanding the CoA inhibition compared with
the substrate ATP. Superimposed structures of the AMPPNP-bound and the
CoA-bound enzymes (an average RMSD of 1.48 Å) shows that both the α-
and β-phosphates of CoA and the β- and γ-phosphates of AMPPNP occupy
the same space and interact with the residue Lys^101 (Fig. [137]5).
This suggests that inhibition by CoA and its thioesters is achieved by
occluding ATP binding to Lys^101. Mutation of this residue abolishes
the binding of ATPγS, a nonhydrolyzable ATP analogue, as well as CoA to
bPanK ([138]11), supporting the involvement of Lys^101 in binding of
both ligands.
[139]Figure 5
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Figure 5
Close-up stereoview of the overlapping site of two ligands, AMPPNP and
CoA. The binding pockets of AMPPNP and CoA are shown in yellow and
cyan, respectively. CoA (magenta) and AMPPNP (black) are shown inlines.
a, residues of the AMPPNP-bound enzyme are shown in brown, and residues
of the CoA-bound enzyme are not shown for clarity. Note that the
carboxyl group of Glu^249of the AMPPNP-bound enzyme coincides with the
pantetheine moiety of CoA. b, residues of the CoA-bound enzyme are
shown ingreen, and residues of the AMPPNP-bound enzyme are not shown
for clarity. Note that the carboxyl group of Glu^44 of the CoA-bound
enzyme coincides with the adenine base of AMPPNP.
A significant difference between the two structures is found at the
structural motif containing helix B and its flanking loops (Fig.
[143]5). Residues from this structural motif interact with the adenine
base of ATP in the AMPPNP-bound enzyme (Figs. [144]3 b and [145]5 a).
In the CoA-bound enzyme, the C-terminal flanking loop of helix B
including the residues 41–44 moves toward the binding pocket for the
adenine base of ATP, which is vacant because there is no bound ATP
(Fig. [146]5 b). This movement orients the carboxyl group of Glu^44
into the empty adenine-binding pocket (Fig.[147]5 b). Consequently, the
carboxyl group of Glu^44forms a salt bridge with the amino group of
Lys^303 and a hydrogen bond to the imidazole group of His^307
(Fig.[148]5 b). In the AMPPNP-bound enzyme, these two interacting
residues define part of the boundary of the adenine-binding pocket
(Fig. [149]3 b). The movement of the C-terminal flanking loop of helix
B also brings the amide nitrogen of Ile^42 within a hydrogen bonding
distance of the 3′-phosphate of CoA (Fig.[150]4 b).
Other differences between the two structures are found at the binding
pocket for the pantetheine moiety of CoA (Fig. [151]5). In the
AMPPNP-bound enzyme, Glu^249 is not a part of helix H, and its carboxyl
group occupies the pantetheine-binding pocket (Fig.[152]5 a). Upon
binding of CoA, the main chain of Glu^249 moves by 12.3 Å to become a
part of helix H (Fig.[153]5 b). Because of this movement, its carboxyl
group no longer occupies the pantetheine-binding pocket and is exposed
to solvent. Instead, the aromatic side chain of Phe^252 moves closer to
the pantetheine-binding pocket to interact with the thiol group of CoA
(Fig. [154]5 b).
[155]Previous Section[156]Next Section
§2§ DISCUSSION §2§
The structure of AMPPNP-bound bPanK illustrates the ATP binding site in
the enzyme and the specific interactions between the enzyme and ATP. We
predict that the reaction proceeds through deprotonation of the
hydroxyl group of pantothenate followed by its nucleophilic attack on
the γ-phosphate of ATP to produce the pentacoordinate transition state,
that breaks down to phosphopantothenate and ADP. This mechanism
requires a general base to activate the hydroxyl group of pantothenate.
There are two residues near the γ-phosphate of ATP that could
potentially act as the general base. ADP:vanadate mimics the
pentacoordinate transition state in ATP hydrolysis byDictyostelium
myosin ([157]34). We therefore superimposed the coordinates of
ADP-vanadate (Protein Data Bank code [158]1vom) onto the γ-phosphate
binding pocket of the AMPPNP-bound enzyme to gain insight into the
potential mechanism. The carboxyl side chain of Asp^127 is located 3.3
Å from the apical oxygen of the modeled vanadate. Because the position
of the apical oxygen approximates the hydroxyl group of the substrate
pantothenate during catalysis, this observation supports the
involvement of Asp^127 in the nucleophilic activation of the substrate
hydroxyl group. A mutagenesis study of the equivalent aspartate in
phosphoribulokinase, a structurally homologous protein ofR.
sphaeroides, demonstrates the importance of this aspartate residue in
catalysis ([159]35). The imidazole side chain of His^177 is located 5.0
Å from the apical oxygen of the modeled vanadate and is an alternative
choice as a general base. The average B-factor of the loop containing
H177 is 52.8 Å^ 2, whereas that of the entire protein is 43.4 Å^2,
indicating the flexibility of this loop and opening the possibility
that His^177 could move closer when pantothenate is bound to the
enzyme. Both candidate residues are completely conserved among the
known prokaryotic PanKs (Fig. [160]1 c).
Positively charged residues are important to stabilize the
pentacoordinate transition state of γ-phosphate in nucleotide
monophosphate kinases ([161]36-38), phosphosugar kinases ([162]39-41),
and G-proteins ([163]42). In the AMPPNP-bound bPanK, the side chain of
R243 neutralizes the negative charge on the γ-phosphate oxygen via a
salt bridge. The superposition of ADP:vanadate and the γ-phosphate
binding pocket of AMPPNP-bound enzyme illustrates that the guanidinium
group of Arg^243 could form a hydrogen bond with one of the equatorial
oxygens in the trigonal bipyramid of vanadate, suggesting that Arg^243
stabilizes the transition state of the γ-phosphate. The involvement of
Arg^243 in catalysis is further supported from a mutagenesis study that
changed the equivalent arginine in R. sphaeroides phosphoribulokinase,
resulting in a reduction of the enzymatic activity ([164]43). Arg^243
is also completely conserved among the known prokaryotic PanKs
(Fig.[165]1 c).
Our structures provide the framework for understanding the detailed
mechanism for the competitive inhibition of bPanK by CoA with respect
to ATP ([166]11). A surprising finding in this study is that ATP and
CoA bind to bPanK in very different orientations and that the adenine
moieties bind via a nonoverlapping set of residues. However, both
molecules do occupy a portion of the same space in the active site by
utilizing Lys^101 to neutralize the charge on their respective
phosphodiesters. This interaction explains the finding that the
bPanK[K101M] mutant is catalytically inactive and fails to bind both
ATP and CoA ([167]11). A comparison of the AMPPNP- and CoA-bPanK
structures identifies His^177, Phe^247, and Arg^106 as residues
involved in CoA recognition that are not required for ATP binding.
His^177 and Phe^247 are implicated in stacking interactions with the
adenine base of CoA, and Arg^106 forms a salt bridge with the
3′-phosphate of CoA (Fig. [168]4 b).
bPanK is more potently inhibited by nonesterified CoA than by its
thioesters including acetyl-, succinyl-, malonyl-, acetoacetyl-, and
palmitoyl-CoA ([169]4), suggesting that the enzyme distinguishes
various CoAs by the length of the acyl chain. In the CoA-bound enzyme,
the binding of the thiol group is associated with the side chains of
several aromatic residues by the weak electrostatic interactions that
block access of the thiol to solvent. More importantly, the tight fit
of the thiol group with surrounding aromatic residues allows only
nonesterified CoA to optimally bind. If acetyl-CoA is docked at the CoA
binding site, the sulfur atom of acetyl-CoA would still interact with
Phe^244, Phe^259, and Tyr^262. However, the interaction with Phe^252
would be no longer possible because of a bulky acetyl group. A
conformational change involving a rearrangement of the side chain of
Phe^252and/or a movement of the Phe^252-containing loop between helices
H and I would be required to accommodate the acetyl group. The same
steric hindrance principle can be applied to explain why the
inhibitions of other CoA thioesters containing even longer acyl groups
than acetyl-CoA are less effective than that of nonesterified CoA.
The complex of bPanK with AMPPNP provides a first look into the
structure and catalytic residues of bPanK, the first step in the CoA
biosynthetic pathway. Additionally, the specific complex with CoA
provides a structural model for feedback regulation that describes the
unique mechanism for the competitive inhibition of ATP binding by CoA.
Furthermore, these two structures will be valuable tools in the search
for novel anti-bactericides because PanK is an essential protein in
bacteria ([170]44) and bears no resemblance in primary sequence to the
mammalian enzyme ([171]13).
[172]Previous Section[173]Next Section
§2§ ACKNOWLEDGEMENTS §2§
We thank Drs. Robert Sweet and Anard Saxena for help and advice with
multiwavelength anomalous dispersion diffraction data collection on
beamline X12C. We also thank Drs. Stephen White and Allen Price for
help with data collection. We thank Brent Calder for excellent
technical assistance.
[174]Previous Section[175]Next Section
§2§ Footnotes §2§
* [176]↵* This work was supported in part by National Institutes of
Health Grants GM 45737 (to S. J.) and GM 34496 (to C. O. R.),
Cancer Center Support Grant P30 CA21765, and the American Lebanese
Syrian Associated Charities. Research was carried out in part at
the National Synchrotron Light Source, Brookhaven National
Laboratory, which is supported by the United States Department of
Energy, Division of Materials Sciences and Division of Chemical
Sciences.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 and ) have
been deposited in the Protein Data Bank, Research Collaboratory for
Structural Bioinformatics, Rutgers University, New Brunswick, NJ
([177]http://www.rcsb.org/).
* [178]↵‖ To whom correspondence should be addressed: Dept. of
Structural Biology, St. Jude Children's Research Hospital, 332 N.
Lauderdale, Memphis, TN 38105. Tel.: 901-495-3838; Fax:
901-495-3032; E-mail: hee-won.park@stjude.org.
* Published, JBC Papers in Press, June 21, 2000, DOI
10.1074/jbc.M003190200
* Abbreviations:
PanK
pantothenate kinase
bPanK
E. coli PanK
AMPPNP
5′-adenylimido-diphosphate, lithium salt
RMSD
root mean square deviation
ATPγS
adenosine 5′-O-(thiotriphosphate)
*
+ Received April 13, 2000.
+ Revision received June 15, 2000.
* The American Society for Biochemistry and Molecular Biology, Inc.
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