Identification of the active site residues in ATP-citrate lyase's carboxy-terminal portion

Article abstract of DOI:10.1002/pro.3708

ATP-citrate lyase (ACLY) catalyzes production of acetyl-CoA and oxaloacetate from CoA and citrate using ATP. In humans, this cytoplasmic enzyme connects energy metabolism from carbohydrates to the production of lipids. In certain bacteria, ACLY is used to fix carbon in the reductive tricarboxylic acid cycle. The carboxy(C)-terminal portion of ACLY shows sequence similarity to citrate synthase of the tricarboxylic acid cycle. To investigate the roles of residues of ACLY equivalent to active site residues of citrate synthase, these residues in ACLY from Chlorobium limicola were mutated, and the proteins were investigated using kinetics assays and biophysical techniques. To obtain the crystal structure of the C-terminal portion of ACLY, full-length C. limicola ACLY was cleaved, first non-specifically with chymotrypsin and subsequently with Tobacco Etch Virus protease. Crystals of the C-terminal portion diffracted to high resolution, providing structures that show the positions of active site residues and how ACLY tetramerizes.

Full text of DOI:10.1002/pro.3708

Article
Protein Science
DOI10.1002/pro.3708
Identification of the active site residues
in ATP-citrate lyase’s carboxy-terminal portion
1
1
2
2,3*
1*
Vinh H. Nguyen , Noreen Singh , Ana Medina , Isabel Usón , Marie E. Fraser
1
Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4 Canada;
2
3
CSIC, Baldiri Reixac, 15, Barcelona, Barcelona, 08028, Spain; ICREA, Pg. Lluís Companys
2
3, 08010 Barcelona, Spain
*
To whom correspondence should be addressed: Phone: 1-403-220-6145. Fax: 1-403-289-9311
frasm@ucalgary.ca; Phone: 93 402 01 98 uson@ibmb.csic.es
Running title: Active sites of C-terminal portion of ATP-citrate lyase
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
1
0.1002/pro.3708
©
2019 The Protein Society
Received: Jun 09, 2019;Revised: Aug 08, 2019;Accepted: Aug 12, 2019
This article is protected by copyright. All rights reserved.

Abstract
ATP-citrate lyase (ACLY) catalyzes production of acetyl-CoA and oxaloacetate from
CoA and citrate using ATP. In humans, this cytoplasmic enzyme connects energy metabolism
from carbohydrates to the production of lipids. In certain bacteria, ACLY is used to fix carbon in
the reductive tricarboxylic acid cycle. The carboxy(C)-terminal portion of ACLY shows
sequence similarity to citrate synthase of the tricarboxylic acid cycle. To investigate the roles of
residues of ACLY equivalent to active site residues of citrate synthase, these residues in ACLY
from Chlorobium limicola were mutated, and the proteins were investigated using kinetics assays
and biophysical techniques. To obtain the crystal structure of the C-terminal portion of ACLY,
full-length C. limicola ACLY was cleaved, first non-specifically with chymotrypsin and
subsequently with Tobacco Etch Virus protease. Crystals of the C-terminal portion diffracted to
high resolution, providing structures that show the positions of active site residues and how
ACLY tetramerizes.
Keywords: CoA-binding, proteolysis, site-directed mutagenesis
5
0-75-word statement
ATP-citrate lyase (ACLY) is an important enzyme to make a key molecule used in lipid
production. Understanding how ACLY functions can aid in drug development to limit lipid
production as a potential treatment for obesity and cancer. Key catalytic residues were identified,
and their importance was determined. The structure of a portion of ACLY was determined that
provides atomic resolution view of how this part of ACLY facilitates catalysis.
This article is protected by copyright. All rights reserved.

INTRODUCTION
In humans, ATP-citrate lyase (ACLY, EC 2.3.3.8) is the cytoplasmic enzyme connecting
energy metabolism from carbohydrates to the production of lipids. Within mitochondria, citrate
synthase (CS) converts acetyl-CoA and oxaloacetate to CoA and citrate as part of the
tricarboxylic acid (TCA) cycle. While acetyl-CoA cannot cross the mitochondrial membrane,
citrate can be exported to the cytoplasm via transporters. In the cytoplasm, ACLY converts
citrate back to acetyl-CoA using CoA and ATP. This acetyl-CoA can be used to synthesize
1
cholesterol or fatty acids in the cytoplasm or to acetylate histones in the nucleus. Due ACLY’s
role in producing cholesterol and fatty acids, the enzyme has been suggested as a target for drug
2
,3
design to treat cancer and obesity.
4
ACLY is a member of the acyl-CoA synthetase (NDP-forming) superfamily. The prototype
for this superfamily is succinyl-CoA synthetase (SCS) and the N-terminal portion of human
2
+
ACLY (hACLY) is similar to SCS (Fig. 1). In the presence of Mg , ACLY catalyzes the
reaction: citrate + CoA + ATP → ADP + P + acetyl-CoA + oxaloacetate, while SCS catalyzes
i
i
the reaction: succinate + CoA + ATP/GTP → ADP/GDP + P + succinyl-CoA. Both SCS and
ACLY have a catalytic histidine that transfers the phosphoryl group between nucleotide
5
,6
triphosphate and organic acid. Structures of the N-terminal portion of hACLY in complex with
7
,8
2+
9
citrate and Mg -ADP have been determined using X-ray crystallography. As in SCS, the
distance between the two binding sites, site I and site II, is large, ~40 Å. In both SCS and ACLY,
8
,10
the active site histidine is part of a loop thought to swing from one binding site to the other.
The phosphohistidine segment of the related acetyl-CoA synthetase has been observed at each of
1
1
the binding sites. Addition of the phosphoryl group to the organic acid makes the compound
susceptible to attack by CoA to form the thioester, succinyl-CoA in the case of SCS, citryl-CoA
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in the case of ACLY and acetyl-CoA in the case of acetyl-CoA synthetase.
The carboxy-terminal (C-terminal) portion of ACLY shows sequence similarity to CS of the
TCA cycle (Fig. 1). CS catalyzes first condensation of oxaloacetate and acetyl-CoA to form
citryl-CoA, then hydrolysis of citryl-CoA to citrate and CoA. The portion of ACLY showing
similarity to CS would be expected to catalyze cleavage of citryl-CoA to oxaloacetate and acetyl-
CoA (site III). CS is multimeric, existing as a dimer or hexamer, where the hexamer is a trimer
1
2
12,13
of dimers. In either form, the active site is at the dimer interface.
Since hACLY exists as a
homotetramer with 1101 amino acid residues in each polypeptide, the C-terminal portion would
be expected to provide at least one of the sites of dimerization. Recent biophysical investigations
14
support this hypothesis.
To investigate the roles of residues of ACLY equivalent to the active site residues of CS (Fig.
S1), these residues were mutated, and the proteins investigated using kinetics assays and
biophysical techniques. ACLY from the green sulfur bacterium Chlorobium limicola was used
for these experiments. C. limicola uses ACLY in the reductive tricarboxylic acid cycle to fix
15
carbon. C. limicola ACLY (ClACLY) is similar to hACLY, but is a heterooctamer with four
1
6
copies of two different subunits, A and B. Subunit B and the N-terminal portion of subunit A
are similar to the N-terminal portion of hACLY (Fig. 1). The C-terminal portion of subunit A is
similar to the C-terminal portion of hACLY and CS. To obtain the crystal structure of the C-
1
7,18
terminal portion of ACLY (prior to the recent publications ), full-length ClACLY was cleaved
specifically with Tobacco Etch Virus (TEV) protease. Crystals diffracted to high resolution,
providing structures that show the positions of active site residues and how ACLY tetramerizes.
RESULTS
Analysis of mutant proteins. Specific activities were measured for ClACLY and the mutant
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proteins, H491A, D543A and the H491A&D543A double mutant. While ClACLY and the
H491A mutant had specific activities of 1.4 ± 0.1 and 0.5 ± 0.1 U/mg, respectively, neither the
D543A mutant nor the double mutant showed activity. Kinetics assays for ClACLY and the
H491A mutant showed only small differences for ATP, CoA and citrate (Table 1).
Thermal stabilities of ClACLY and the three mutants were tested using differential scanning
fluorimetry (DSF). The melting temperature (T
.0 was 48°C. None of the mutants showed significant changes from the T
similar conditions.
m
) for ClACLY measured in 10 mM TrisHCl pH
7
m
of ClACLY under
Isothermal titration calorimetry proved that the double mutant still bound CoA. Figure S2
depicts the plots and Table 2 presents the fitted parameters. Both ClACLY and the double mutant
bind CoA, although the conditions that best showed the binding were somewhat different.
Structure determination of the C-terminal portion of ClACLY. Initial crystals of chymotrypsin-
cleaved ClACLY grew from solutions containing acetyl-CoA, suggesting that the C-terminal
portion crystallized. Mass spectrometry identified tryptic fragments that included residues 369-
6
08 of subunit A. 369-608 would have a mass of 26.3 kDa, but the polypeptide ran with a
19
relative molecular weight of 28 kDa on SDS-PAGE. PeptideCutter indicated two possible
cleavage sites for chymotrypsin, one between 344 and 345 (29.1 kDa) and one between 354 and
3
55 (28.0 kDa). The shorter fragment was produced by truncating the gene and adding codons
for a histidine-tag at the N-terminus. This fragment could be purified but did not behave well in
crystallization experiments. Interestingly, the C-terminal portion ran as a tetramer on gel
filtration (data not shown).
Crystals of the C-terminal portion of ClACLY cleaved by TEV protease (CterClACLY)
contained residues 347-608 of subunit A. The best crystal diffracted to 1.9 Å-resolution (trigonal
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Table S2) and the second best to 2.05 Å-resolution (triclinic). Four copies of the 28.8 kDa
polypeptide were predicted in the asymmetric unit of the trigonal crystal form and 12 in the
2
triclinic. The initial model consisted of 705 alanine residues in space group P3 . Autobuilding
generated a tetramer with 255, 257, 253 and 248 residues in four chains, plus six additional
residues. Refining and rebuilding resulted in nearly complete chains. In the triclinic crystal, two
2
protomers display disorder. Since the models are similar, the P3 model is described (Table S2).
CterClACLY forms a tetramer with 222 symmetry (Fig. 2). The N-termini lie close to one 2-
fold axis and the C-termini close to another. In one orientation, the tetramer appears elliptic, but
the perpendicular view shows four domains extending from the central core. The P450 domains
2
0
(
CATH Classification 1.10.230.10 ), which extend from 447-555, are swung away from the
core of the structure. The tetramer has five domains, one forming the core and the other four
extending from it.
CterClACLY is predominantly α-helical. However, residues 594-596 form a parallel β-sheet
with residues 355-357 of a different protomer (Fig. 2). The polypeptide extends from residue 596
to the C-terminus, wrapping around the other protomer. In this region and the three equivalent
regions of the tetramer, residues from three different polypeptide chains interact. In the tetramer,
each of the polypeptide chains interacts with all three of the other polypeptides.
Since acetyl-CoA had been included in the crystallization experiment, it was possible that its
binding site could be identified in the electron density maps. Adenine, ribose-3-phosphate and
diphosphate of the 3’-phosphoadenosine end of acetyl-CoA were fitted to two protomers (chains
A and D). The diphosphate groups of the two acetyl-CoA molecules show different
conformations, influenced by crystal-packing interactions. There was insufficient electron
density to fit even the adenine base to chain B, the protomer that has no crystal-packing
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interactions near the CoA-binding site. There was no evidence of binding to chain C. Five
phosphate ions and 16 molecules of glycerol were included in the model.
DISCUSSION
Identification of the active sites. The crystallographic model includes the 3’-phosphoadenosine
end of acetyl-CoA at two active sites. Since acetyl-CoA is a product of the reaction, it could bind
if the concentration were high enough to lead to product inhibition; and the goal was to see the
binding site. Acetyl-CoA was used rather than CoA in case interactions of the acetyl group with
residues of the active site could be seen. Isothermal titration calorimetry had shown that the N-
terminal portion of hACLY does not bind CoA, leading to the conclusion that the CoA-binding
7
site must be in the C-terminal portion. However, a second possibility is that both the N- and C-
terminal portions are needed to form the CoA-binding site. This is what differential scanning
14
calorimetry indicated. In those experiments, CoA stabilized full-length hACLY, but did not
stabilize the individual N-terminal portion nor the C-terminal portion.
The 3’-phosphoadenosine end of acetyl-CoA interacts with the protein primarily through van
der Waals interactions. L538 packs against adenine, while P485 packs near N6A. There is a
weak hydrogen bond (3.2 Å) donated by the amide of V486 to N1A. The only charged
interaction supported by electron density is between K534 and the 3’-phosphate. The
diphosphate of CoA extends out of the active site cleft where electron density disappears,
indicating that the rest of the molecule is disordered.
Phosphate ions are bound to all active sites. Phosphate is neither a substrate nor a product of
the reaction catalyzed by CterClACLY. Its negative charge leads to binding to positively charged
R568 and, in the P1 crystal form, a second phosphate ion is detected, bound to H415, H491 and
R502 of nine of the protomers. Some residual electron density in the active site cleft was fitted
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with molecules of glycerol.
Comparison of ClACLY with citrate synthases of the TCA cycle. ClACLY shows quaternary
structure that has not been observed in structures of CSs. When one protomer of the
2
1
CterClACLY is superposed on chicken CS (Protein Data Bank (PDB) rcsb.org identifier (ID):
2
2
6
CSC ), ClACLY appears to be missing certain α-helices of CS (Fig. S3). In the ClACLY
tetramer, α-helices from a second protomer superpose with these α-helices. A sequence
alignment based on superposing the α-helices in the two structures highlights the
pseudosymmetry within CS (Fig. S1). The C-terminal copy has residues of both the CS domain
and the smaller P450 domain, residues 271 to 388. The N-terminal copy has only residues of the
CS domain: it is missing the P450 domain.
In ClACLY, two pairs of protomers dimerize to form the tetramer in a similar way that the
two protomers of CS dimerize. While the CS dimer has two active sites, the C-terminal ClACLY
tetramer has four. This is because the N-terminal portion of CS lacks the P450 domain required
to form an active site.
2
3
The hexameric form of CS found in E. coli is allosterically inhibited by NADH. The
structure of NADH in complex with mutated E. coli CS shows the allosteric site at the interface
of pairs of dimers, but NADH is predominantly bound to residues of the N-terminal portion of
2
4
CS . When a CoA analogue was soaked into the same crystal form, it bound at the same site,
25
rather than at the active site. This allosteric site is located where the second P450 domain is
missing from CS (Fig. S3). CS could have lost this second P450 domain to allow allosteric
regulation. However, dimeric forms of CS are not known to be regulated by allostery.
In the crystal structure of CterClACLY, the adenine base of CoA lies near the loop equivalent
to where it binds in CS (Fig. 3). The specific interactions that orient adenine in the CoA-binding
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site of CS do not occur in the crystal structure of the C-terminal portion of ClACLY. While the
diphosphate of CoA extends out of the active site cleft, CoA bound to CS has a bent
conformation extending into the active site.
CS adopts both an open and a closed conformation. When oxaloacetate and acetyl-CoA are
bound, CS closes to catalyze the condensation to form citryl-CoA and the subsequent hydrolysis.
In structures that show the closed conformation of CS, CoA analogues bind to the P450 domain,
but also interact with residues of the second protomer in the dimer. The structure of
CterClACLY is in the open conformation, despite the addition of acetyl-CoA and the binding of
phosphate ions in the active site. Residues that bind these phosphate ions, H415, H491, R502 and
R568, are equivalent to residues that interact with citrate in the complex of chicken CS with
2
2
trifluoroacetonyl-CoA and citrate (PDB ID 6CSC ): H238, H320, R329 and R401. A third
arginine residue in chicken CS, R421’, also interacts with citrate, the prime indicating that it is
from the other protomer in the CS dimer. The equivalent arginine in CterClACLY, R588, adopts
two conformations, one hydrogen bonding to D591 of the same chain and the second hydrogen
bonding to D362 of a third protomer. It does not interact with either phosphate ion.
Recent publications also showed ACLY in an open conformation with CoA bound, and two
structures in a closed conformation one with malate and acetyl-CoA bound and one with citrate
1
7,18
bound.
No structure had been determined with only the addition of acetyl-CoA in the absence
of an organic acid. The open conformation in the presence of acetyl-CoA proves that binding of
an appropriate organic acid is what induces a closed conformation, regardless of whether acetyl-
CoA is bound.
Site-directed mutagenesis of the catalytic residues. H491 and D543 were predicted to be
catalytic residues based on sequence comparisons with CSs. These residues are equivalent to
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H320 and D375 of chicken CS (Fig. S1), two residues important in binding substrates and
1
3
catalyzing the condensation of oxaloacetate and acetyl-CoA to form citryl-CoA. H320
2
6
hydrogen bonds to the carbonyl of oxaloacetate (PDB ID: 1CSH ) or the hydroxyl of citrate
2
2
(
PDB ID: 6CSC ) and donates a proton to the carbonyl oxygen that becomes the hydroxyl of
2
7
citrate. In the complex of CS with the acetyl-CoA analogue amidocarboxymethyldethia
2
6
coenzyme A (PDB ID:1CSH ), D375 accepts a hydrogen bond from the amide nitrogen. D375
2
7
acts as a base to remove the proton from the acetyl group in the enolization of acetyl-CoA.
H491 and D543 of ClACLY would be expected to serve similar roles to H320 and D375 in
catalysis of the cleavage of citryl-CoA to form acetyl-CoA and oxaloacetate.
The specific activity measured for ClACLY, 1.4 ± 0.1 U/mg, is similar to values of 1.1 U/mg
2
8,29
and 1.30 U/mg previously measured.
Mutation of H491 to alanine led to a significant loss of
app
M
activity (Table 1). K
for ATP and citrate remain the same, as expected since both binding
7
,9
app
M
sites are distant from the mutation. K
for CoA increased, but not significantly, showing that
the protein folded correctly and loss of activity is due to loss of the side chain. Mutagenesis of
3
0
the equivalent H in pig CS to G, R, N or Q led to a decrease of greater than 99% in kcat. While
CS uses H to bind oxaloacetate, this interaction would be less important in ClALCY, which
binds citryl-CoA and releases oxaloacetate. The interaction with the hydroxyl of citryl-CoA
could be replaced by interaction with water or a different residue of the H491A mutant protein.
Mutation of D543 to A led to no measurable activity, so the side chain of D543 is more
critical than that of H491 for ClACLY. The result is consistent with neither of the D375N nor
3
1
D375Q mutants of pig CS being active. Since N and Q can still hydrogen bond but neither has
a dissociable proton, the acid-base property of D375 was concluded to be most important. This
interpretation was supported by the higher activity of D375E mutant CS, at 0.2% that of wild-
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type. Since the D375G mutant protein had 0.06% activity, it was thought that the lack of a side-
chain allowed a water molecule access to the active site to act as a general acid-base in catalysis.
The total loss of activity when D543 of ClACLY is mutated to alanine indicates that neither
water nor another residue of the mutant protein substitutes for the side chain of D543.
Since kinetic analyses could not be performed on either D543A ClACLY or the
H491A&D543A double mutant, isothermal titration calorimetry was used to test the ability of
the double mutant to bind CoA. Both ClACLY and the double mutant bound CoA (Fig. S2), but
the magnitude of the binding enthalpy was lower for the double mutant (Table 2). Surprisingly,
only two binding sites were detected. Since ClACLY is a tetramer of heterodimers, it has four
active sites and would be expected to bind four molecules of CoA, one at each active site. This
stoichiometry was seen for hACLY, where 4.06 ± 0.04 binding sites and a binding enthalpy of -
-1
7
7
.19 ± 0.09 kcal mol were measured under similar conditions.
Tests of thermal stability by DSF showed that the three mutants had the same T
indicating that they were correctly folded. This contrasts with results obtained with pig CS by
m
as ClACLY,
3
2
circular dichroism where mutation of D375 increased or decreased stability. However, pig CS
has a higher T , 55.7°C. The lower T of 48°C for ClACLY could be due to unfolding of any
domain, not specifically the CS domain.
m
m
Catalytic residues in the crystals. In the crystal structure, H491 and D543 adopt similar
conformations to H320 and D375 of CS. ND1 of H491 accepts hydrogen bonds from the
backbone nitrogen atoms of residues 493 and 494. If H491 is neutral, NE2 would be protonated.
There is residual electron density in the active site, between H491 and R502. In the H491A
mutant protein, either a water molecule or the amino group of K494 could interact with the
hydroxyl of citryl-CoA or the carbonyl of oxaloacetate. In CS, the residue equivalent to K494 is
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L323. This residue could not form a hydrogen bond through its side chain, so could not substitute
for the imidazole side chain in the same way K494 could. This could be why the mutation of H
3
0
to A has a less drastic effect on catalysis by ClACLY than by CS. In the crystal structure of
CterClACLY, D543 interacts with a molecule of glycerol.
The catalytic H274 of CS does not align with a histidine residue in ClACLY, but with F450
(
Fig. S1). In CS, H274 interacts with the thioester and the enolate, donating a proton to the
27
carbonyl of the acetyl group of acetyl-CoA to stabilize the enol intermediate. F450 could not
serve this role, but the crystal structure shows that the amide nitrogen of G451 could. The
peptide bond between F450 and G451 is flipped relative to that in CS (Fig. 4). The carbonyl
accepts a hydrogen bond from G447, which adopts a different conformation than G271 of CS
because of insertion of A270 (Fig. S1). The amide nitrogen is directed to the substrate-binding
site. It interacts with a glycerol molecule in the active site of chain A and water molecules in the
2
active sites of the other protomers in the P3 crystal form. It would interact with the carbonyl of
the thioester in citryl-CoA.
Delivery of citryl-CoA from the N-terminal portion of ACLY to the C-terminal portion.
Identification of the binding site for CoA and active site residues H491 and D543 in the C-
terminal portion leads to the question of how the two portions of ACLY transfer citryl-CoA from
site I to site III. We propose that the adenosine end of CoA binds to the C-terminal portion of
ACLY as seen in the crystal structure. CoA swings the free thiol into site I in the N-terminal
portion (Fig. 1), where citrate and phosphohistidine also bind. The active site histidine residue
would have been phosphorylated by ATP at site II, also in the N-terminal portion. When the
citryl-CoA thioester bond has formed at site I, the citryl end swings into site III in the C-terminal
portion of ACLY. Once citryl-CoA has bound, the active site closes to catalyze cleavage to
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oxaloacetate and acetyl-CoA at site III. This proposal is consistent with the recently published
1
7,18
structures of hACLY and Methanothrix soehngenii ACLY.
The location of the N-terminal
portion with respect to the C-terminal portion shows that the citryl end of citryl-CoA swings 35
Å from site I to site III.
The locations of the four N-termini of CterClACLY indicate that site III is not located in the
same polypeptide chain as sites I and II. Figure 2 shows that the N-terminus of one polypeptide
is closer to a different polypeptide’s active site III. The tetramer needs to be formed before the
enzyme is active.
MATERIALS AND METHODS
Expression and purification of ClACLY. Genes for subunits B and A of Chlorobium limicola
ATP-citrate lyase (ClACLY) and the intergenic region were synthesized by Genscript. Codons
were chosen for optimal production in E. coli and a His
subunit B. NdeI and AvrII sites was included at the 5'- and 3’-ends for insertion in the pET-
2b(+)plasmid (Novagen). After sequencing (University Core DNA Services), the expression
8
-tag was added to the C-terminus of
4
plasmid was transformed into E. coli BL21(DE3), and 100 mL Luria-Bertani broth containing 30
µg/mL kanamycin was inoculated with a single colony. After overnight culture at 37ºC with
shaking at 225 rpm, 50 mL were used to inoculate 1 L Terrific Broth (12 g tryptone, 24 g yeast
2 4 2 4
extract, 4 mL glycerol, 2.31 g KH PO , 12.54 g K HPO per litre) (TB). This culture was grown
until the optical density at 600 nm reached 1.6. Expression was induced with 0.1 mM isopropyl-
β-D-thiogalactoside and the temperature was dropped to 21°C. After ~16 h, cells were harvested
by centrifugation (20 min, 4000 × g) at 4°C and stored at -80°C.
The protein was purified by affinity, gel filtration and anion exchange chromatography. Cells
were thawed in lysis buffer (50 mM sodium phosphate, 300 mM NaCl, 15% (v/v) glycerol, 10
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mM imidazole, pH 8.0) and lysed by sonication. The lysate was separated by centrifugation (30
min, 11000 × g) and loaded on a Ni-NTA Agarose (Qiagen) column. Bound protein was washed
with buffer containing an additional 10 mM imidazole and eluted with buffer containing 100 mM
imidazole. Protein was precipitated in 80% saturated ammonium sulfate and stored at 4°C. Prior
to gel filtration on a 120-mL Superdex 200 PG (GE Healthcare) column, the precipitate was
collected by centrifugation (60 min, 7800 × g) and dissolved in a minimal volume of running
buffer (150 mM NaCl, 50 mM tris(hydroxymethyl)aminomethane hydrochloride (TrisHCl) pH
8
.0, 15% glycerol, 10 mM 2-mercaptoethanol (2-ME)). Fractions containing protein of the
expected size were pooled and the protein was precipitated and stored at 4°C. The precipitate
was collected, dissolved in low salt buffer (20 mM TrisHCl, 15% glycerol, 10 mM 2-ME, pH
8
.0), then injected onto a 60-mL Sephadex G-25 (GE Healthcare) column connected to a 1-mL
HiTrap Q HP column (GE Healthcare). Protein was eluted with a salt gradient (20 mM Tris
HCL, 15% glycerol, 10 mM 2-ME, 1 M KCl, pH 8.0). The protein was concentrated using an
Amicon Ultra-30 centrifuge filter, exchanging the buffer to 10 mM TrisHCl pH 8.0, 10 mM 2-
ME. The sample was flash frozen in 20 µL aliquots and stored at -80°C. Typical yield from 1 L
TB was 7 mg ClACLY, calculated using its predicted absorbance 0.1% at 280 nm, i.e. 0.913.
Site-directed mutagenesis of ClACLY. Two residues, H491 and D543 of subunit A, were
mutated to A. Mutagenesis followed the Quikchange Lightning protocol (Agilent) with the
3
3
modification from Wang et al. Primers are listed in Table S1.
Specific activities of wild-type ClACLY and the mutants were measured using a coupled-
3
4
enzyme assay with malate dehydrogenase (MDH). MDH catalyzes reduction of oxaloacetate by
NADH. The assay solution contained 3.3 U MDH, 0.2 mM NADH, 2 mM sodium citrate, 50 μM
CoA, 1 mM ATP, 10 mM MgCl
2
, 10 mM dithiothreitol (DTT), 100 mM TrisHCl, pH 8.4. For
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the kinetics assays, the concentration of citrate, CoA or ATP was varied, with the other
substrates and MDH maintained at 2 mM sodium citrate, 50 µM CoA and 1 mM ATP.
Experiments were performed in triplicate and analyzed using GraphPad Prism.
DSF was performed to test whether the mutants displayed similar thermal stability to wild-
type ClACLY. The conditions mimicked those used during purification: 10 mM either TrisHCl,
pH 7.0, 7.5, 8.0, 8.5 or 9.0 or HEPES, pH 6.8, 7.3, 7.8 or 8.3; 0 mM, 100 mM or 200 mM NaCl;
0
%, 10% or 20% glycerol; 2 μL 100X SYPRO® Orange Protein Dye (Life Technologies) and 1
μM protein in 40 μL total volume. Temperature was increased every 5 s in 0.2°C increments
from 25 to 90°C with measurements every 5 s at 470 and 555 nm (Qiagen Rotor-Gene Q). Three
m
replicates were analyzed using GraphPad Prism to calculate an average T with an ANOVA
two-factor statistical analysis.
Isothermal titration calorimetry was used to test whether the double mutant bound CoA. Wild-
type ClACLY and the double mutant were purified by affinity chromatography and dialyzed
2
overnight at 4°C against 10 mM MgCl , 10 mM sodium citrate, 4 mM 2-ME and 10 mM
potassium phosphate, pH 7.5. 10 mM CoA was prepared using the dialysis solution. Both
solutions were centrifuged to remove aggregates (10 min, 14 000 × g) prior to loading protein in
the cell and CoA in the syringe of a MicroCal VP-ITC isothermal titration calorimeter
(
Malvern). A titration consisted of a single 1-μL injection of CoA followed by 59 4-μL injections
at 5-min intervals at 30°C. Once appropriate concentrations had been determined, three replicates
were done and the thermodynamic parameters for binding CoA to ClACLY and to the double
mutant were determined using the MicroCal VP-ITC Origin-7™ software.
Crystallization of the C-terminal portion of ClACLY using chymotrypsin. The first crystals
were grown by cleaving full-length ClACLY with chymotrypsin. Chymotrypsin was added to the
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crystallization experiment and crystals grew in hanging drops using 17.5-20% PEG3350, 100
mM TrisHCl pH 7.0-8.5, 125–180 mM lithium citrate or 150–175 mM sodium potassium
tartrate. The protein solution contained 10 mM TrisHCl pH 8.0, 5 mM acetyl-CoA and 5 mg/mL
ClACLY and was mixed in a 1:1 ratio with the precipitant solution. To identify what portion
crystallized, crystals were dissolved in 20 μL loading buffer for SDS-PAGE. After SDS-PAGE,
the band was excised and tryptic peptides were analyzed by mass spectrometry (Southern
Alberta Mass Spectrometry Facility).
Introduction of the TEV protease cleavage site and purification of the CterClACLY. Two
regions in subunit A of ClACLY were chosen to introduce the TEV protease cleavage site. The
site that worked best was introduced with site-directed mutagenesis, changing residue 345 from
3
5
K to F and inserting Y before it, to match the consensus sequence EXXYXQˆS/G with
EVMYFQˆG, where ˆ represents the cleavage site. Primers are given in Table S1. Sequences
TM
were confirmed and the plasmid was used to transform E. coli strain BL21 STAR (DE3).
ClACLY with the TEV protease cleavage site was produced and purified by affinity
chromatography and stored as an ammonium sulfate precipitate. The precipitate was collected by
centrifugation and dissolved in cleavage buffer (50 mM TrisHCl pH 8.00, 0.5 mM EDTA, 1 mM
DTT). TEV protease was added in a 1:100 w/w ratio and the sample was left for 24 h at 4°C.
After digestion, the sample was diluted with cation exchange buffer (20 mM MES pH 6.3, 15%
glycerol, 10 mM 2-ME) and loaded onto a HiTrap SP HP column (GE Healthcare). The protein
eluted with a salt gradient (20 mM MES, 15% glycerol, 10 mM 2-ME, 1 M KCl, pH 6.3). Eluted
protein was diluted with anion exchange buffer (20 mM TrisHCl, 15% glycerol, 10 mM 2-ME,
pH 8.0), loaded onto a HiTrap Q HP column and eluted with a salt gradient (20 mM Tris, 15%
glycerol, 10 mM 2-ME, 1 M KCl, pH 8.0). Eluted protein was concentrated and exchanged to 20
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mM potassium phosphate pH 7.0, 10 mM 2-ME before storage at -80°C. The protein
concentration was measured spectrophotometrically using its predicted absorbance 0.1% at 280
nm, i.e. 1.141.
Crystal structure of the C-terminal portion of ClACLY cleaved by TEV protease. Screening
used the PEG/Ion Screen (Hampton Research) and hanging drops with 0.5 µL protein solution (5
mg/mL protein, 5 mM acetyl-CoA and 20 mM potassium phosphate pH 7.0) and 0.5 µL well
solution. Optimized well solution contained 20% w/v PEG3350, 200 mM sodium acetate, 100
mM MES pH 6.3. Seeds for streak seeding were grown with a well solution containing 17% w/v
PEG3350 and 200 mM sodium acetate pH 5.2. A hair was touched to these crystals, then
streaked across a drop containing 10% w/v PEG3350, 100 mM sodium acetate, 50 mM MES pH
6
.2, 2.5 mg/mL protein, 2.5 mM acetyl-CoA and 10 mM potassium phosphate equilibrated for
one hour over well solutions containing 20% w/v PEG3350, 200 mM sodium acetate, 100 mM
MES pH 6.2. Crystals grew to 25 µM × 25 µM × 100 µM, were cryo-protected using 20% v/v
glycerol, 20% w/v PEG 3350, 200 mM sodium acetate, 100 mM MES pH 6.3, vitrified in
nitrogen at 100 K, stored under liquid nitrogen and shipped to the synchrotron.
Two high resolution data sets were collected, one at the CMCF-ID beamline at the Canadian
Light Source on a Pilatus3 S 6M detector and the second at IMCA-CAT beamline 17-ID at the
Advanced Photon Source on a Pilatus 6M detector. Data were processed using XDS or DIALS
3
6–41
42
43
via XIA2.
ARCIMBOLDO Shredder was run on a HTCondor Grid of 130 nodes to
4
4
determine initial phases using a truncated model of Acetobacter aceti CS (PDB ID:2H12 ). It
4
5
performed eLLG-guided location of template-derived fragments with PHASER. Additional
degrees of freedom modified the fragments during molecular replacement through gyre
4
6
refinement against the rotation function and gimble refinement after placement. Best scored
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4
7
phase sets were subject to density modification and autotracing with SHELXE leading to a
main chain trace comprising 672 residues and characterized by a CC of 31%. The model and
4
8
electron density maps were visualized using COOT and the model was refined using
4
9,50
phenix.refine.
This first model was used to solve the second structure by molecular
5
1
replacement using PHASER. TLS refinement in PHENIX improved both models.
Acknowledgments Research was supported by a Discovery Grant from NSERC (MEF),
Canada Graduate Scholarship - Master’s Program (NS), Queen Elizabeth II Scholarship (VHN).
IU was supported by grants BIO2015-64216-P and MDM2014-0435, AM is supported by a
fellowship BES-2017-080368 (Spanish Ministry of Science and Innovations). Beamline 08ID-1
at the Canadian Light Source is supported by the Canada Foundation for Innovation, Natural
Sciences and Engineering Research Council of Canada (NSERC), University of Saskatchewan,
Government of Saskatchewan, Western Economic Diversification Canada, National Research
Council Canada, and Canadian Institutes of Health Research. Use of resources at the Industrial
Macromolecular Crystallography Association Collaborative Access Team (IMCA-CAT)
beamline 17-ID was supported by the companies of the Industrial Macromolecular
Crystallography Association through a contract with Hauptman-Woodward Medical Research
Institute. The Advanced Photon Source is a U.S. Department of Energy (DOE) Office of Science
User Facility operated for the DOE Office of Science by Argonne National Laboratory under
Contract No. DE-AC02-06CH11357.
Competing Interests The authors have no competing interests.
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Table 1. Kinetic parameters for ClACLY and the H491A mutant.
-
1
app
m
app
m
app
m
k
cat (s )
K
for ATP (mM)
K
for CoA (μM)
K
for citrate (mM)
2
2
2
(
R value)
(R value)
14.3 ± 1.6
(0.92)
(R value)
Wild-type
0.022 ± 0.001
0.29 ± 0.04
0.93)
0.28 ± 0.04
0.90)
1.6 ± 0.2
(0.96)
(
H491A mutant 0.008 ± 0.001
17.6 ± 2.4
(0.92)
1.6 ± 0.4
(0.80)
(
Table 2. Thermodynamic data for binding of CoA.
ClACLY
H491A&D543A double mutant
2.00 ± 0.01
Binding Sites
2.14 ± 0.21
5
-1
5
-1
Association Constant (K
b
)
(2.03 ± 0.39) x 10 M
(6.27 ± 0.71) x 10 M
Dissociation Constant (K
d
)
4.93 ± 0.91 µM
1.59 ± 0.18 µM
-1
-1
Binding Enthalpy (∆H)
-6.78 ± 0.09 kcal mol
-4.50 ± 0.08 kcal mol
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FIGURE LEGENDS
Figure 1. Domains of succinyl-CoA synthetases and ATP-citrate lyases. Members of the
acyl-CoA synthetase (NDP-forming) superfamily have five domains in common. In most
succinyl-CoA synthetases, domains 1 and 2 form the α-subunit, while domains 3, 4 and 5 form
the β-subunit. ATP-citrate lyases also have a domain showing sequence similarity to citrate
synthase (CS). Human ATP-citrate lyase is a homotetramer where all domains are in a single
polypeptide chain, but C. limicola ATP-citrate lyase is a heterooctamer with two different
polypeptide chains. Sites I, II and III are the three catalytic sites. Site I is at the interface of
domains 1, 2 and 5, site II is within the ATP-grasp fold formed by domains 3 and 4 and site III is
within the CS domain.
Figure 2. Crystal structure of the CterClACLY. The protein is a tetramer that possesses 222
symmetry. Both views are along two-fold axes, with the model rotated 90° about the vertical axis
between the views. The four polypeptide chains are colored differently in the ribbon diagrams,
and the termini that are visible are labelled N or C. The two 3’-phosphoadenosine portions of
acetyl-CoA and the side chains of the active site residues that were mutated are drawn as stick
5
2
models. This figure and Figures 3 and 4 were drawn using PYMOL.
Figure 3. Binding of the 3’-phosphoadenosine end of acetyl-CoA to the C-terminal portion
of ClACLY. A. The portion of acetyl-CoA that could be modelled into electron density near
chain A and the surrounding residues are drawn as stick models, colored according to atom type.
o c
Electron density from the 2F -F map is displayed in grey, contoured at 1 rmsd. B. The
3
’-phosphoadenosine end of acetyl-CoA binds in a similar location to the acetyl-CoA analogue,
2
2
trifluoroacetonyl-CoA, in chicken CS (PDB ID: 6CSC ). The crystal structures were superposed
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based on main chain atoms of residues 485 to 495 of chain A of ClACLY and residues 314 to
3
6
24 of chain A of citrate synthase. Colors are similar to those in part A, but the carbon atoms in
CSC are cyan. For reference, citrate is shown bound to CS. Only one of the two conformations
of trifluoroacetonyl-CoA is drawn. In both A and B, hydrogen bonds are represented by dashed
lines. The adenine portion of acetyl-CoA has only one hydrogen bond with the protein and
interacts primarily via van der Waals interactions.
Figure 4. Substitution of histidine in CS with phenylalanine in ACLY. A conserved
phenylalanine residue in ACLY (A) replaces the active site histidine residue in CS (B). In
chicken CS, the imidazole donates a hydrogen bond to citrate. The phenylalanine has no
hydrogen bond donor in its side chain, but the peptide bond between F450 and G451 is flipped
relative to the peptide bond between H274 and G275. This allows the amide nitrogen to serve as
the hydrogen bond donor. The crystal structures were superposed based on Cα atoms of residues
3
98 to 446 of chain A of ClACLY and residues 221 to 269 of chain A of chicken citrate synthase
2
2
(
PDB ID: 6CSC ).
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.