Structural basis of fatty acid substrate binding to cyclooxygenase-2

Article abstract of DOI:10.1074/jbc.M110.119867

The cyclooxygenases (COX-1 and COX-2) are membrane-associated heme-containing homodimers that generate prostaglandin H₂ from arachidonic acid (AA). Although AA is the preferred substrate, other fatty acids are oxygenated by these enzymes with varying efficiencies. We determined the crystal structures of AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) bound to Co³⁺-protoporphyrin IX-reconstituted murine COX-2 to 2.1, 2.4, and 2.65 A , respectively. AA, EPA, and docosahexaenoic acid bind in different conformations in each monomer constituting the homodimer in their respective structures such that one monomer exhibits nonproductive binding and the other productive binding of the substrate in the cyclooxygenase channel. The interactions identified between protein and substrate when bound to COX-1 are conserved in our COX-2 structures, with the only notable difference being the lack of interaction of the carboxylate of AA and EPA with the side chain of Arg-120. Leu-531 exhibits a different side chain conformation when the nonproductive and productive binding modes of AA are compared. Unlike COX-1, mutating this residue to Ala, Phe, Pro, or Thr did not result in a significant loss of activity or substrate binding affinity. Determination of the L531F:AA crystal structure resulted inAA binding in the same global conformation in each monomer. We speculate that the mobility of the Leu-531 side chain increases the volume available at the opening of the cyclooxygenase channel and contributes to the observed ability of COX-2 to oxygenate a broad spectrum of fatty acid and fatty ester substrates.

Full text of DOI:10.1074/jbc.M110.119867

Protein Structure and Folding:
Structural Basis of Fatty Acid Substrate
Binding to Cyclooxygenase-2
Alex J. Vecchio, Danielle M. Simmons and
Michael G. Malkowski
J. Biol. Chem. 2010, 285:22152-22163.
doi: 10.1074/jbc.M110.119867 originally published online May 12, 2010
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 29, pp. 22152–22163, July 16, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Structural Basis of Fatty Acid Substrate Binding to
□
S
Cyclooxygenase-2*
Received for publication, March 3, 2010, and in revised form, April 27, 2010 Published, JBC Papers in Press, May 12, 2010, DOI 10.1074/jbc.M110.119867
Alex J. Vecchio‡§, Danielle M. Simmons‡§, and Michael G. Malkowski‡§1
From the ‡Hauptman-Woodward Medical Research Institute and §Department of Structural Biology, State University of New York,
Buffalo, New York 14203
catalyze the first committed step in prostaglandin (PG)2 bio-
synthesis (reviewed in Refs. 1, 2). The product, PGH2, is pro-
duced as a result of two sequential reactions that are performed
in separate but functionally linked active sites. In the first reac-
tion, arachidonic acid (AA; 20:4 ␻-6) bound in the cyclooxyge-
nase channel undergoes a bis-oxygenation to form the interme-
diate PGG2. The released intermediate is then bound in the
peroxidase active site, where the 15-hydroperoxide group of
PGG2 is reduced to form PGH2 in the second reaction. Both
COX-1 and COX-2 require a preliminary catalytic turnover at
the peroxidase active site to generate an oxy-ferryl porphyrin
radical that is subsequently transferred to Tyr-385 for the ini-
tiation of cyclooxygenase catalysis.
The cyclooxygenases (COX-1 and COX-2) are membrane-as-
sociated heme-containing homodimers that generate prostag-
landin H2 from arachidonic acid (AA). Although AA is the pre-
ferred substrate, other fatty acids are oxygenated by these
enzymes with varying efficiencies. We determined the crystal
structures of AA, eicosapentaenoic acid (EPA), and docosa-
hexaenoic acid (DHA) bound to Co3؉-protoporphyrin IX-re-
˚
constituted murine COX-2 to 2.1, 2.4, and 2.65 A, respectively.
AA, EPA, and docosahexaenoic acid bind in different conforma-
tions in each monomer constituting the homodimer in their
respective structures such that one monomer exhibits nonpro-
ductive binding and the other productive binding of the sub-
strate in the cyclooxygenase channel. The interactions identi-
fied between protein and substrate when bound to COX-1 are
conserved in our COX-2 structures, with the only notable differ-
ence being the lack of interaction of the carboxylate of AA and
EPA with the side chain of Arg-120. Leu-531 exhibits a different
side chain conformation when the nonproductive and produc-
tive binding modes of AA are compared. Unlike COX-1, mutat-
ing this residue to Ala, Phe, Pro, or Thr did not result in a sig-
nificant loss of activity or substrate binding affinity.
Determination of the L531F:AA crystal structure resulted in AA
binding in the same global conformation in each monomer. We
speculate that the mobility of the Leu-531 side chain increases
the volume available at the opening of the cyclooxygenase chan-
nel and contributes to the observed ability of COX-2 to oxygen-
ate a broad spectrum of fatty acid and fatty ester substrates.
Understanding the similarities and differences associated
with the structure and function of COX-1 and COX-2 and the
rationale for the existence of two isoforms has been the focus of
much recent research (3–5). COX-1 and COX-2, which are
encoded by separate genes (6), display ϳ60% sequence identity
within the same species and greater than 85% sequence identity
among orthologs from different species (7). Accordingly, the
crystal structures of COX-1 and COX-2 are virtually superim-
posable, and the catalytic mechanism is conserved between iso-
forms (1, 7). The major difference between COX-1 and COX-2
is in their observed expression patterns (8). In most cases,
COX-1 is expressed constitutively, producing PGs that mediate
“housekeeping” functions, whereas COX-2 is expressed in
response to growth factors, tumor promoters, or cytokines (8).
PGs produced via COX-2 are intimately involved in a number
of pathologies, including inflammation, pain, and colon cancer
(9). Other differences between isoforms include the following:
(a) the lower concentration of reducing cosubstrate required by
COX-2 for activation compared with COX-1, allowing for the
utilization of AA when it is a small constituent in a fatty acid
pool in vivo (4, 5, 10); and (b) the ability of aspirin-acetylated
COX-2 to oxygenate AA, which shifts the reaction specificity
such that (15R)-hydroxyeicosatetraenoic acid ((15R)-HETE) is
formed instead of PGH2 (11, 12).
The cyclooxygenase enzymes (COX-1 and COX-2) are mem-
brane-associated heme-containing bifunctional enzymes that
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grant R01 GM077176 (to M. G. M.) from the NIGMS. This work was also
supported by an Arthritis Investigator award (to M. G. M.) from the Arthritis
Foundation as part of the Segal Osteoarthritis Initiative. This work was
based upon research conducted at the Cornell High Energy Synchrotron
Source (CHESS), which is supported by the National Science Foundation
Award DMR-0225180, using the Macromolecular Diffraction at CHESS
(MacCHESS) facility, which is supported by National Institutes of Health
Award RR-01646 from NCRR.
Although AA is the preferred substrate for COX-1 and
COX-2, other fatty acids can be oxygenated by both isoforms
with varying efficiencies (13–15). A prime example is the 30%
The on-line version of this article (available at http://www.jbc.org) contains efficiency (compared with AA) by which COX-2 can oxygenate
□S
supplemental Tables S1–S11, Figs. S1 and S2, and additional references.
the fatty acid eicosapentaenoic acid (EPA) (20:5 ␻-3) as
The atomic coordinates and structure factors (codes 3HS5, 3HS6, 3HS7, and
3KRK) have been deposited in the Protein Data Bank, Research Collaboratory
for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
(http://www.rcsb.org/).
2 The abbreviations used are: PG, prostaglandin; AA, arachidonic acid; (15R)-
HETE, (15R)-hydroxyeicosatetraenoic acid; EPA, eicosapentaenoic acid;
DHA, docosahexaenoicacid;ov, ovine;mu, murine;␤OG, ␤-octylglucoside;
r.m.s.d., root mean square deviation.
1 To whom correspondence should be addressed: Hauptman-Woodward
Medical Research Institute, 700 Ellicott St., Buffalo, NY 14203. Tel.: 716-898-
8624; Fax: 716-898-8660; E-mail: malkowski@hwi.buffalo.edu.
22152 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 29•JULY 16, 2010

Fatty Acid Substrate Binding in COX-2
opposed to the less than 5% efficiency by which COX-1 oxygen- zyme for crystallization trials. The apoenzyme was reconsti-
tuted with Co3ϩ-protoporphyrin IX and fatty acid substrate to
generate the appropriate enzyme-substrate complexes for x-ray
crystallographic analyses. Crystal structures of muCOX-2 in
complex with AA, EPA, and docosahexaenoic acid (DHA) (22:6
␻-3) were elucidated to resolutions of 2.1, 2.4, and 2.65 Å,
respectively. The structures were utilized to detail and func-
tionally characterize the nuances involved in the binding of
fatty acid substrates in the cyclooxygenase channel of COX-2.
ates this substrate (14, 16). COX-2 can also oxygenate a more
extensive array of substrates compared with COX-1, presum-
ably because of an ϳ20% larger active site created by a highly
conserved I523V substitution between isoforms (17). Marnett
and co-workers (18–20) have established that COX-2, but not
COX-1, oxygenates the endocannabinoids 2-arachidonylglyc-
erol, arachidonylethanolamide, and N-arachidonylglycine, uti-
lizing the same general catalytic mechanism derived for AA.
These substrates are larger than AA due to the additional
chemical constituent attached to the ␦-end of the fatty acid.
Crystal structures of ovine (ov) COX-1 have been elucidated
in the presence of AA and other fatty acid substrates to moder-
ate resolutions (ϳ3.0 Å) (16, 21–23). The fatty acids in these
structures are all oriented in the cyclooxygenase channel such
that the carboxylate of the substrate interacts with the guani-
dinium group of Arg-120 and the ␻-end of the substrate binds
in a hydrophobic groove above Ser-530, resulting in the place-
ment of C-13 below Tyr-385 (7). The ovCOX-1:fatty acid crys-
tal structures provided experimental verification at the molec-
ular level of fundamental components of the cyclooxygenase
reaction previously identified via biochemical studies, includ-
ing the following: the required interaction between Arg-120
and the carboxylate of the fatty acid substrate (22, 24); the posi-
EXPERIMENTAL PROCEDURES
Materials—AA ((5Z,8Z,11Z,14Z)-eicosatetraenoic acid),
EPA ((5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid), and DHA
((4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoic acid) were pur-
chased from Cayman Chemical (Ann Arbor, MI). Decyl malto-
side and ␤-octylglucoside (␤OG) were purchased from
Anatrace (Maumee, OH). Co3ϩ-protoporphyrin IX was pur-
chased from Porphyrin Products (Logan, UT). The
QuikChange mutagenesis kit was purchased from Stratagene
(La Jolla, CA), and the Bac-to-Bac baculovirus expression kit
and associated reagents, including Spodoptera frugiperda 21
(Sf21) insect cells, fetal bovine serum, fungizone, penicillin/
streptomycin, and sf-900 II serum-free media were purchased
tioning of the 13-pro-S hydrogen below Tyr-385 for subsequent from Invitrogen. Hi-Trap HP chelating and HiPrep Sephacryl
abstraction (2, 22); and the placement of the ␻-end of the fatty
acid substrate within the hydrophobic groove, where the termi-
nal carbon lies adjacent to Gly-533 (22, 25). Furthermore, anal-
ysis of the spatial arrangement of AA within the cyclooxygenase
channel of the ovCOX-1:AA crystal structure allowed for the
proposal of a valid sequence of events for the generation of
PGG2 and recycling of the tyrosyl radical during cyclooxygen-
ase catalysis (22). AA bound in its productive conformation in
ovCOX-1 has been utilized as a model to wed structure with
functional studies carried out on both COX-1 and COX-2 that
have investigated the catalytic process and molecular determi-
nants governing substrate binding, specificity, and product for-
mation (16, 19–21, 23, 26–30).
S200-HR chromatography columns were purchased from GE
Healthcare. Oligonucleotides used for site-directed mutagene-
sis were purchased from Integrated DNA Technologies (Cor-
alville, IA).
Preparation of His6 N580A Murine COX-2 and Leu-531
Mutants—Native muCOX-2 was cloned into the NotI and
BamHI sites of the vector pFastbac1. The pFastbac1 construct
was then used as a template to insert six histidine residues
behind the signal sequence and between Asn-19 and Pro-20 of
muCOX-2 using the QuikChange mutagenesis kit as described
in Ref. 32 and the following primer (note: forward primer listed
only; inserted amino acids are boldface and underlined): 5Ј-
CTCAGCCAGGCAGCAATTCATCACCATCACCATCAC-
CCTTGCTGTTCCAATCCA-3Ј. As muCOX-2 is variably
glycosylated at Asn-580, we further generated an N580A mutant
construct to remove this N-glycosylation site utilizing the
QuikChange mutagenesis kit and the His6 muCOX-2 construct
in pFastbac1 generated above as the template, with the follow-
ing primer (forward primer only; site of mutation boldface and
underlined): 5Ј-ACAGCCACCATCGCTGCAAGTGCCTCC-
3Ј. Finally, Leu-531 mutants were generated using the
QuikChange mutagenesis kit and the His6 N580A muCOX-2
construct as a template, utilizing the following primers (for-
ward primer only; site of mutation bolded and underlined):
L531A, 5Ј-GGAGCACCATTCTCCGCGAAAGGACTTAT-
GGG-3Ј; L531P, 5Ј-GGAGCACCATTCTCCCCGAAAGGA-
CTTATGGG-3Ј; L531T, 5Ј-GGAGCACCATTCTCCACGA-
AAGGACTTATGGG-3Ј; L531F, 5Ј-GGAGCACCATTCTC-
CTTCAAAGGACTTATGGG-3Ј. Proper insertion of the His6
tag, N580A, and Leu-531 mutations were confirmed by DNA
sequencing, which was performed at the Roswell Park Cancer
Attempts to study the structure of AA bound in the cyclooxy-
genase channel of COX-2 have yielded interesting but some-
what ambiguous results. Kiefer et al. (31) reported the structure
of AA bound to the apoenzyme form of murine (mu) COX-2 in
an attempt to prevent oxygenation of the substrate during crys-
tallization. The electron density observed in the cyclooxygen-
ase channel of the resulting structure suggested a mixture of AA
and PGG2. The authors concluded that contamination of the
protein preparation with heme-containing COX-2 led to the
oxygenation of AA during the crystallization process. In
another attempt, a peroxidase-inactive mutant (H207A) of
muCOX-2 was used to form a complex with AA. Analysis of the
electron density within the cyclooxygenase channel of this
structure showed that AA had indeed bound, but in a nonpro-
ductive conformation in which the carboxylate of AA hydro-
gen-bonded to Tyr-385 and Ser-530 at the apex of the channel
instead of near the opening. We report here studies designed to
further investigate the structural basis of fatty acid substrate
binding to COX-2. We engineered a His6-tagged N580A
muCOX-2 construct to produce milligram quantities of apoen- Institute DNA Sequencing Laboratory.
JULY 16, 2010•VOLUME 285•NUMBER 29 JOURNAL OF BIOLOGICAL CHEMISTRY 22153

Fatty Acid Substrate Binding in COX-2
Generation of Recombinant Baculovirus and Expression of trode. Each standard assay mixture contained 3 ml of 100 mM
Murine COX-2—Positive baculovirus was generated for His6 Tris, pH 8.0, 1 mM phenol, 5 ␮M hematin, and 1–200 ␮M sub-
N580A muCOX-2 and mutant constructs via the transforma- strate. Reactions were initiated by the addition of up to 20 ␮g of
tion of max efficiency DH10Bac cells with the appropriate con- protein in a volume of 20 ␮l. Km and Vmax values were deter-
struct in pFastbac1, followed by transfection of Sf21 insect cells mined by measuring oxygen uptake using 1–200 ␮M fatty acid
with the isolated bacmid DNA. The initial p1 virus stock was substrate and fitting the data to the Michaelis-Menten equation
amplified in 50 ml of Sf21 insect cells using a multiplicity of using GraphPad Prism 5.0 for Windows (GraphPad Software,
infection (MOI) of 0.1 plaque-forming unit/ml. The resulting San Diego). Peroxidase activity was measured spectrophoto-
p2 virus was harvested 48 h post-infection, plaque-assayed, and metrically at 25 °C. The reaction mixture in the cuvette con-
used to generate large quantities of p3 virus for subsequent tained 100 mM Tris, pH 8.0, 100 ␮M N,N,NЈ,NЈ-tetramethylphe-
large scale expressions. For expression, Sf21 insect cells were nylenediamine, 1.7 ␮M hematin, and up to 100 ␮g of protein.
grown at 27 °C in sf-900 II serum-free media supplemented Reactions were initiated by adding 100 ␮l of 300 ␮M H2O2, and
with 1.5% (v/v) fetal bovine serum and the appropriate antibi- the oxidation of N,N,NЈ,NЈ-tetramethylphenylenediamine was
otics and additives and maintained in suspension in an 8-liter monitored at 610 nm over time.
spinner flask equipped with an air-sparging line for proper aer-
Crystallization and Data Collection—For crystallization,
ation. The cells were infected with p3 virus stock at a multiplic- purified apo-His6 N580A muCOX-2 (or the L531F mutant) was
ity of infection of 1.0 once the cell density reached ϳ2.0 ϫ 106 reconstituted with a 2-fold molar excess of Co3ϩ-protoporphy-
cells/ml in the spinner flask and harvested via centrifugation rin IX, followed by dialysis overnight at 4 °C against 20 mM Tris,
72–96 h post-infection in conjunction with a drop in cell via- pH 8.0, 100 mM NaCl, 0.6% (w/v) ␤OG. Just prior to setup, a
bility to below 80%. Cell pellet was harvested via centrifugation, 10-fold molar excess of substrate was added to the reconsti-
pooled, and stored at Ϫ80 °C.
tuted protein. Initial crystallization screening was carried out
Purification of Murine COX-2—The cell pellet from 2 liters of on the muCOX-2:AA complex using the 1536 microbatch-un-
culture was resuspended in 50 mM Tris, pH 8.0, 300 mM NaCl, der-oil screen offered by the High Throughput Crystallization
10 mM imidazole, and 1 mM 2-mercaptoethanol (2 ml/g cell Laboratory, at The Hauptman-Woodward Medical Research
pellet) and subsequently ruptured using a microfluidizer Institute (34). One subsequent crystallization lead was further
(Microfluidics, Newton, MA). Decyl maltoside was added to the optimized to generate crystals of the COX-2:fatty acid com-
cell lysate to a final concentration of 0.7% (w/v), and the protein plexes utilized in this study. Specifically, crystals were grown in
was solubilized for 1 h with stirring at 4 °C. The mixture was sitting-drops by combining 3 ␮l of protein solution with 3 ␮l of
then centrifuged at 40,000 rpm at 4 °C for 1 h to remove insol- 23–34% polyacrylic acid 5100, 100 mM HEPES, pH 7.5, 20 mM
uble debris. The clarified supernatant was loaded on a 5-ml MgCl2, and 0.6% (w/v) ␤OG and equilibrating over reservoir
HiTrap chelating HP column and washed with buffer A (50 mM solutions containing 23–34% polyacrylic acid 5100, 100 mM
Tris, pH 8.0, 300 mM NaCl, 20 mM imidazole, 1 mM 2-mercap- HEPES, pH 7.5, and 20 mM MgCl2. Trays were incubated at
toethanol, and 0.5% (w/v) decyl maltoside). An intermediate 23 °C, and plate-like crystals were formed in 3 days up until
wash step consisting of 10 column volumes was carried out approximately 4 weeks. Crystals were cryopreserved via trans-
using buffer B (buffer A containing a final concentration of 60 fer to a solution consisting of 30% polyacrylic acid 5100, 100 mM
mM imidazole), followed by elution with buffer C (buffer A con- HEPES, pH 7.5, 20 mM MgCl2, 0.6% (w/v) ␤OG, and 10% eth-
taining 200 mM imidazole) over 5 column volumes. Function- ylene glycol. Following incubation in cryoprotectant for
ally active fractions were subsequently pooled and dialyzed 10–120 min, the crystals were looped and flash-cooled directly
overnight at 4 °C against 50 mM Tris, pH 8.0, 300 mM NaCl, and into the nitrogen gas stream prior to diffraction analysis. Data
0.53% (w/v) ␤OG to initiate detergent exchange for crystalliza- were collected on beamline A1 at the Cornell High Energy Syn-
tion. The sample was concentrated to ϳ2 ml using a Millipore chrotron Source (Ithaca, NY) at a wavelength of 0.9789 Å using
Ultrafree-15 spin concentrator with a 50-kDa nominal molec- an Area Detector Systems CCD Quantum-210 Detector. Data-
ular mass cutoff. The concentrated protein sample was then sets were integrated and scaled using MOSFLM and SCALA,
trypsin-digested for 20–90 min at 25 °C with a 30:1 ratio COX- respectively, in the CCP4 suite of programs (35). For the L531F
2:trypsin (33). The reaction was terminated via the addition of 2 mutant COX-2:AA structure, the dataset was assembled from
mM phenylmethylsulfonyl fluoride. Trypsin digestion has no diffraction measurements from two crystals. The data from
deleterious affect on COX-2 function, as digested protein con- each crystal was processed individually using MOSFLM and
tained cyclooxygenase and peroxidase activity comparable with then scaled together using SORTMTZ and SCALA. Details of
the undigested sample. The protein was then subjected to size- the data collection statistics are summarized in Table 1.
exclusion chromatography utilizing a HiPrep 16/60 Sephacryl
Structure Solution and Refinement—Although crystal struc-
S-300 HR column equilibrated in 25 mM Tris, pH 8.0, 150 mM tures of both COX-1 and COX-2 have been elucidated, exten-
NaCl, and 0.53% (w/v) ␤OG. The peak fractions of the purified sive care was taken to remove model bias during the structure
His6 N580A muCOX-2 constructs were pooled and concen- solution and refinement process given the crystallographic
trated to 3 mg/ml for crystallization trials.
observations reported previously for AA in complex with apo-
Cyclooxygenase and Peroxidase Assays—For measurement of and H207A muCOX-2 (31). As such, difference Fourier maps
cyclooxygenase activity, the initial rate of O2 uptake was mea- were not utilized to generate initial phases for the enzyme-sub-
sured at 37 °C using a model 5300 biological oxygen monitor strate complexes. Instead, a modified version of monomer A of
(YSI Inc., Yellow Springs, OH), equipped with an oxygen elec- Protein Data Bank entry 1CVU (31) was created in which all
22154 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 29•JULY 16, 2010

Fatty Acid Substrate Binding in COX-2
TABLE 1
Data collection and refinement statistics
Crystallographic
parameter
COX-2:AA
L531F:AA
COX-2:EPA
COX-2:DHA
Space group
I222
I222
I222
I222
No. in asymmetric unit
2
2
2
2
Unit cell length
a
119.98 Å
132.55 Å
180.52 Å
90°
120.79 Å
133.04 Å
180.65 Å
90°
121.85 Å
132.34 Å
180.29 Å
90°
119.29 Å
131.71 Å
179.24 Å
90°
b
c
␣ ϭ ␤ ϭ ␥
Wavelength
0.9789 Å
20.0-2.10 Å
2.21-2.10 Å
8.4 (58.2)
0.9777 Å
20.0-2.40 Å
2.53-2.40 Å
17.4 (32.2)
0.9789 Å
20.0-2.40 Å
2.53-2.40 Å
9.6 (55.9)
0.9789 Å
20.0-2.65 Å
2.79-2.65 Å
12.4 (48.9)
Resolution
Highest resolution shella
b
Rmerge
b
RPIM
4.1 (29.5)
423,435 (56,778)
83,526 (12,044)
12.9 (2.4)
7.2 (23.6)
320,496 (18,069)
55,133 (7387)
8.0 (2.1)
5.6 (33.7)
223,307 (30,395)
56,982 (8181)
11.0 (2.4)
5.7 (22.6)
244,651 (34,902)
41,205 (5946)
10.9 (3.4)
Total observations
Total uniquec
I/␴(I)
Completeness
Multiplicity
99.6% (99.3%)
5.1 (4.7)
96.8% (90.2%)
5.8 (2.4)
99.8% (99.6%)
3.9 (3.7)
99.8% (100%)
5.9 (5.9)
Wilson B factor
28.5 Å2
38.5 Å2
40.7 Å2
50.4 Å2
No. of atoms in refinement
10,230
0.169 (0.245)
0.210 (0.293)
33.6 Å2
9920
9766
9565
Rwork
0.179 (0.226)
0.228 (0.298)
29.1 Å2
0.176 (0.248)
0.223 (0.325)
34.8 Å2
0.181 (0.263)
0.234 (0.347)
38.2 Å2
d
Rfree
Average B factor, protein
Average B factor, solvent
26.5 Å2
22.0 Å2
23.5 Å2
14.0 Å2
Average B factor, fatty acid
Monomer A
43.7 Å2
52.6 Å2
0.284 Å
0.008 Å
1.121°
49.2 Å2
51.8 Å2
0.292 Å
0.008 Å
1.490°
44.5 Å2
45.6 Å2
0.296 Å
0.008 Å
1.104°
64.7 Å2
64.3 Å2
0.348 Å
0.006 Å
1.021°
Monomer B
Mean positional errore
r.m.s.d. in bond length
r.m.s.d. in bond angle
a The values in parentheses represent the values in the outermost resolution shell.
b Rmerge and RPIM are as defined in Ref. 57.
c Data represent reflections with F Ͼ 0 ␴F, which were used in the refinement.
d 5.0% of the total reflections were used to generate the test set.
e Coordinate error was calculated by Luzatti plot.
ligands and waters as well as residues 33–144, 320–325, 344– MAC5 refinement were carried out, followed by the calculation
391, and 500–553 were deleted. The regions deleted encom- of 2Fo Ϫ Fc and Fo Ϫ Fc electron density maps. In the areas
pass the N and C termini, as well as the membrane binding where the carbon positions differ between the substrates, we
domain and walls of the cyclooxygenase channel. The remain- would expect to see the appropriate “positive” and “negative”
ing structure (ϳ60% of the total residues) was then utilized as electron density peaks in the calculated Fo Ϫ Fc electron density
the search model for molecular replacement calculations on maps. A similar method has been used to verify the changes
each dataset using the program PHASER (36). The subsequent observed between fatty acid substrates bound in the cyclooxy-
phases were then used as input to ARP/wARP (37) utilizing the genase channel of ovCOX-1 (16, 23). Inspection of the resulting
“automated model building starting from experimental phases” electron density maps clearly shows positive difference density
option, which further minimizes the introduction of model bias in the hydrophobic groove above Ser-530 in monomer B, when
(38). ARP/wARP was able to build the majority of missing por- AA and EPA are modeled in their nonproductive conforma-
tions of the protein model. Iterative cycles of manual model tions. Similarly, negative difference density is observed for the
building and refinement, using the programs COOT (39) and misplacement of the ␻-end of each substrate in the hydropho-
REFMAC5 (40), were then carried out to fit the remaining res- bic groove above Ser-530 in monomer A when AA and EPA are
idues and to add waters, substrate, sugar moieties, and other modeled in their productive conformations. Moreover, nega-
ligand molecules. The coordinates and stereochemical dictio- tive difference density is localized around the side chain of Leu-
naries for AA, EPA, and DHA were downloaded from the PRO- 531 in each monomer of the muCOX-2:AA crystal structure as
DRG server (41). TLS refinement (42), utilizing the TLSMD this side chain is forced to reposition to accommodate the dif-
web server (43, 44), was carried out on each structure during the ferent modeled conformations of AA.
final rounds of REFMAC5 refinement. Final refinement statis-
tics are summarized in Table 1.
Structural Analysis—van der Waals and hydrogen bond
interactions were calculated using the program COOT. The
To verify that the observed differences between the confor- upper limit on distance for consideration as a van der Waals
mations of AA and EPA in each monomer were significant, we contact is 4.0 Å. Superposition of coordinates between struc-
performed the following exercise in both the muCOX-2:AA tures was done using the program LSQAB within the CCP4
and muCOX-2:EPA crystal structures. The substrate model suite of programs and the coordinates for all C␣ atoms unless
built in the nonproductive conformation in monomer A was otherwise stated. The root mean square deviation reported for
replaced with the substrate model built in the productive con- the side chain of Arg-120 was derived from the measured dis-
formation from monomer B and vice versa. Cycles of REF- tances between the positions of the C␣ and all side chain atoms
JULY 16, 2010•VOLUME 285•NUMBER 29
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Fatty Acid Substrate Binding in COX-2
TABLE 2
Crystallographic Structure Determination of Native and
Mutant COX-2:Fatty Acid Complexes—To generate stable
complexes of the fatty acid substrates within the cyclooxygen-
ase channel, we reconstituted purified apo-His6 N580A
muCOX-2 with Co3ϩ-protoporphyrin IX to create a native-like
enzyme that lacks both peroxidase and cyclooxygenase activity
and hence does not form PG product when incubated with
substrates (49). This method has been used successfully to trap
AA and other fatty acid substrates within the cyclooxygenase
channel of ovCOX-1 for structural studies (16, 21–23). We sub-
sequently determined the crystal structures of His6 N580A
muCOX-2 in complex with the fatty acid substrates AA, EPA,
and DHA using synchrotron radiation. (Accordingly, we use
the naming conventions of muCOX-2:AA, muCOX-2:EPA,
and muCOX-2:DHA, when describing the co-crystal
structures.)
Fatty acid substrate oxygenation by His6 N580A muCOX-2
Oxygen consumption was measured using an oxygen electrode with the fatty acids
AA, EPA, and DHA as described under “Experimental Procedures.” kcat and Km
values calculated for the utilization of AA and EPA were from duplicate measure-
ments, and those for DHA were from triplicate measurements. We define the rela-
tive rate as the rate at which His6 N580A muCOX-2 utilizes EPA and DHA as
substrates normalized to the utilization of AA, which is set to 100%.
Substrate
kcat
s
Km
␮M
k
cat/Km Relative rate
Ϫ1
%
AA (20:4 ␻-6)
EPA (20:5 ␻-3)
27.0 Ϯ 0.40 5.14 Ϯ 0.29
5.24
0.92
0.09
100
32.3
12.8
8.70 Ϯ 0.21 9.45 Ϯ 0.73
DHA (22:6 ␻-3) 3.45 Ϯ 0.15 36.8 Ϯ 4.25
of this residue (eight total atoms) in the structures compared.
Simulated annealing omit maps were calculated using CNS
(45). Model validation was carried out using MOLPROBITY (46).
Figures were created in CCP4MG (47). The coordinates and
structure factors for Co3ϩ-protoporphyrin IX-reconstituted
muCOX-2 in complex with AA, EPA, DHA, as well as for the
L531F muCOX-2:AA complex have been deposited in the Protein
Data Bank (codes 3HS5, 3HS6, 3HS7, and 3KRK, respectively).
Each of the muCOX-2:fatty acid structures contains two
monomers in the crystallographic asymmetric unit (termed
“monomer A” and “monomer B” for comparison purposes) that
form the canonical biological dimer. Interpretable electron
density was observed for Co3ϩ-protoporphyrin IX and the car-
bohydrate moieties linked to Asn-68, Asn-144, and Asn-410 in
each monomer (residue numbering based on ovCOX-1 num-
bering scheme), and a single ␤OG molecule was observed at a
crystallographic special position for each dimer (data not
shown). No electron density was observed for C-terminal resi-
dues beyond Gln-584 in monomer A and Val-583 in monomer
B in any of the muCOX-2:fatty acid complexes. The domain
architecture, which includes the N-terminal epidermal growth
factor domain, the membrane-binding domain, and the cata-
lytic domain, is well resolved for each monomer in all three
structures. There are no significant structural differences
between monomers within each muCOX-2:fatty acid structure
(A versus B) or between monomers of the different muCOX-2:
fatty acid structures (A versus A, B versus B, and A versus B)
when compared, as calculated root mean square deviations
(r.m.s.d.) between monomers are within the mean positional
error of these structures (supplemental Table S1).
RESULTS
To characterize the binding of fatty acid substrates within the
cyclooxygenase channel of COX-2, we generated a construct of
muCOX-2 that was optimized for structural biology studies. A
His6 tag was engineered into muCOX-2 two residues beyond
the signal sequence at the N terminus of the enzyme to facilitate
purification of the enzyme (32). In addition, we mutated Asn-
580, one of four known N-glycosylation sites in COX-2 (48) to
alanine. Roughly 50% of the COX-2 molecules are N-glycosy-
lated at Asn-580 (48). Subsequent replacement of this residue
with alanine results in the removal of N-glycosylation hetero-
geneity. The resulting His6 N580A muCOX-2 construct was
utilized in an insect cell-based expression system to overpro-
duce recombinant enzyme, and a two-step purification proto-
col, consisting of immobilized metal affinity and size-exclusion
chromatography steps, was implemented to generate milligram
quantities of the protein. The purified His6 N580A muCOX-2
enzyme was functionally characterized by spectrophotometric
measurement of the peroxidase activity and determination of
the oxygen consumption kinetics using an oxygen electrode,
with AA, EPA, and DHA utilized as fatty acid substrates. The
enzyme retains complete peroxidase activity with respect to
untagged, natively glycosylated wild-type muCOX-2 (data not
shown). Moreover, calculated kcat and Km values are nearly
identical to those reported previously for huCOX-2 and
muCOX-2 when AA and EPA are utilized as substrates (Table
2) (14, 15). DHA is a poor substrate for huCOX-2, exhibiting a
specific activity of ϳ10% relative to the oxygenation of AA (15).
When His6 N580A muCOX-2 was tested with DHA, we
observed a similar relative activity to that reported for
huCOX-2, with a Km value ϳ7-fold higher than that of AA (Table
2). Taken together, the His6 and N580A modifications that were
introduced into the native muCOX-2 construct to facilitate the
steps associated with generating the required quantities of protein
Interpretable electron density was observed for fatty acid
substrate in the cyclooxygenase channel of each monomer of
the muCOX-2 dimer for the structures determined. However,
there are significant conformational differences observed for
AA within the cyclooxygenase channels in the muCOX-2:AA
crystal structure and EPA in the muCOX-2:EPA crystal struc-
ture when their corresponding monomers (monomer A and
monomer B) are examined. Specifically, AA is bound in mono-
mer A such that the carboxylate of the fatty acid substrate inter-
acts with Tyr-385 and Ser-530 at the apex of the channel, sim-
ilar to that observed by Kiefer et al. (31) and generally referred
to as a nonproductive binding mode for AA (Fig. 1A). In stark
contrast, AA is bound in monomer B such that the carboxylate
lies near Arg-120 and Tyr-355 at the opening of the cyclooxy-
genase channel, similar to the productive binding mode of AA
observed in the ovCOX-1:AA crystal structure (Fig. 1B) (22).
Similarly, EPA binds in a nonproductive conformation in mon-
omer A (supplemental Fig. S1) and a productive conformation
for x-ray crystallographic studies do not influence or alter the abil- in monomer B (Fig. 2A) in the muCOX-2:EPA crystal structure.
ity of the enzyme to oxygenate fatty acid substrates.
Interestingly, DHA adopts similar global substrate conforma-
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Fatty Acid Substrate Binding in COX-2
AA and EPA make a total of 54 and 56 contacts, respectively,
with 19 residues that line the cyclooxygenase active site in
the muCOX-2:AA and muCOX-2:EPA crystal structures
(supplemental Table S2 and S3). The 19 residues participating
in these interactions are essentially conserved with respect to
those observed to form contacts with fatty acid substrates
bound in the cyclooxygenase channel of ovCOX-1 (16, 22, 23).
The major difference between the active site residue contacts
made by AA and EPA in the muCOX-2:AA and muCOX-2:EPA
crystal structures compared with those made between sub-
strate and active site residues in ovCOX-1 is the lack of an
interaction between the carboxylate of these two substrates and
the side chain of Arg-120. This interaction is a required molec-
ular determinant for the binding of fatty acid substrates to
COX-1 (24), but not COX-2, and our structural data confirm
previous mutagenesis studies to this effect (50). Indeed, only
one hydrophilic contact is made by AA and EPA with cyclooxy-
genase active site residues in these structures, a hydrogen bond
between the carboxylate oxygen O1 of each substrate and the
phenolic hydroxyl of Tyr-355, compared with three in struc-
tures of fatty acids bound to ovCOX-1 (16, 22, 23). The lack of a
required interaction between the carboxylate of AA and EPA
and side chain of Arg-120 coupled with the larger volume of the
COX-2 cyclooxygenase channel also provides these substrates
with greater conformational freedom within the active site of
COX-2. This is borne out in r.m.s.d. values of 1.04 and 0.92 Å
calculated for the productive conformations of AA and EPA in
the muCOX-2:AA and muCOX-2:EPA structures, respectively,
compared with the conformation exhibited by AA in the
ovCOX-1:AA structure (supplemental Table S4 and Table S5).
COX-1 is essentially inactivated when EPA is utilized as a
substrate due to EPA binding in a strained conformation within
the cyclooxygenase channel, which results in the misalignment
of C-13 below Tyr-385 (14, 16). The conformation of EPA
within the cyclooxygenase channel of monomer B of the
muCOX-2:EPA crystal structure is significantly different from
that observed in the ovCOX-1:EPA crystal structure, with a
calculated r.m.s.d. of 2.28 Å between the 22 atoms of the sub-
strate (supplemental Table S6). The observed conformation of
EPA in monomer B is more closely related to that observed for
AA in both the ovCOX-1:AA and muCOX-2:AA (monomer B)
crystal structures (Fig. 3, B and C). In this orientation, C-13 of
EPA is properly aligned below Tyr-385, consistent with kinetic
data showing that COX-2 can utilize EPA as a substrate (Table
2) (14). The decreased efficiency observed for the oxygenation
of EPA by COX-2 compared with AA may be explained by the
presence of an additional double bond at the ␻-end of the sub-
strate. The increased rigidity likely constrains the conforma-
tional flexibility at the ␻-end, which in turn limits its maneu-
verability within the cyclooxygenase channel during catalysis.
Indeed, there is an ϳ2-fold increase in the Km value for AA and
EPA binding to muCOX-2 (Table 2).
FIGURE 1. AA bound in the cyclooxygenase channel of the muCOX-2:AA
complex. A, stereo view of AA bound in the “nonproductive” conformation in
the cyclooxygenase channel of monomer A. Alternate positions are observed
for the C8–C9 double bond of AA bound in the nonproductive conformation.
B, stereo view of AA bound in the “productive” conformation in the cyclooxy-
genase channel of monomer B. The positions of the hydrogen atoms have
been modeled onto C-13 of AA. The 13-pro-S hydrogen lies 2.9 5Å below the
phenolic oxygen of Tyr-385. The Fo Ϫ Fc-simulated annealing omit map den-
sity, contoured at 4␴, is shown with the final refined models of AA (dark blue).
tions in both monomers in the muCOX-2:DHA crystal struc-
ture, with the carboxylate group located near Arg-120 and Tyr-
355 (Fig. 2, B and C). The observed binding mode of substrate
within these structures is described in detail below.
AA and EPA Bound in a Productive Conformation within the
Cyclooxygenase Channel of COX-2—The conformations of AA
and EPA observed in monomer B of the muCOX-2:AA and
muCOX2:EPA crystal structures exhibit the “L-shaped” config-
uration observed for AA and other fatty acid substrates bound
in the cyclooxygenase channel of ovCOX-1 (Fig. 3, A and C) (16,
22, 23). In both cases, the carboxylate of the substrate is posi-
tioned at the channel opening near the side chains of Arg-120
and Tyr-355, and the ␻-end is encased by residues Phe-205,
Phe-209, Val-228, Val-344, Phe-381, and Leu-534, which form a
hydrophobic groove above Ser-530. The central substrate car-
bons of AA and EPA weave around the side chain of Ser-530,
properly placing C-13 of each substrate 2.95 and 2.96 Å, respec-
tively, below the phenolic oxygen of Tyr-385 for abstraction of
the 13-pro-S hydrogen. As such, the observed conformations of
AA and EPA in monomer B of these structures can be consid-
ered as productive substrate conformations given that they
exhibit the proper positioning and stereochemical alignment
necessary for initiation of the cyclooxygenase reaction (22).
The spatial positions of the side chains of the cyclooxygenase
active site residues in monomer B of the muCOX-2:AA and
muCOX-2:EPA crystal structures remain mostly unchanged
compared with their positions in the ovCOX-1:AA and
ovCOX-1:EPA crystal structures. The only notable exceptions
are differences exhibited by the side chains of Arg-120, Ser-530,
JULY 16, 2010•VOLUME 285•NUMBER 29
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Fatty Acid Substrate Binding in COX-2
action between the carboxylate of
the substrate and the side chain of
Arg-120 in COX-2 results in both
the N␩1 and N␩2 atoms of Arg-120
engaging the O⑀1 and O⑀2 atoms of
Glu-524 in a dual ionic interaction
that is reinforced by a hydrogen
bond, which contributes to the sta-
bilization of both side chains and
the closing of the cyclooxygenase
channel entrance upon substrate
binding. Finally, we observe an
alternate rotamer conformation for
the side chain of Leu-531 in mono-
mer B of the muCOX-2:EPA crystal
structure. The implications of the
mobility of this side chain are dis-
cussed below.
DHA Bound in the Cyclooxygen-
ase Channel of COX-2—Despite
being two carbons longer and pos-
sessing two additional unsaturated
double bonds with respect to AA,
DHA binds in an L-shaped confor-
mation in both monomers of the
muCOX-2:DHA crystal structure,
similar to that observed for 18 and
20 carbon fatty acid substrates
bound to ovCOX-1 (16, 22, 23) and
muCOX-2 (Fig. 2, B and C). The
substrate is constrained within the
known boundaries constituting
the cyclooxygenase channel, such
that the carboxylate of DHA inter-
acts with Arg-120 and Tyr-355 at
the base of the channel and the
␻-end abuts the side chain of
Ile-377 near Gly-533 in the hydro-
phobic groove above Ser-530 (7, 16,
FIGURE 2. EPA and DHA bound in the cyclooxygenase channel of the muCOX-2:EPA and muCOX-2:DHA
complexes. A, stereo view of EPA bound in the productive conformation in the cyclooxygenase channel of
monomer B of the muCOX-2:EPA crystal structure. The positions of the hydrogen atoms have been modeled
onto C-13 of EPA. The 13-pro-S hydrogen lies 2.96 Å below the phenolic oxygen of Tyr-385. The observed
conformation of DHA in monomer A is shown in B, and monomer B in the muCOX-2:DHA crystal structure is
shown in C. DHA binds in each cyclooxygenase channel such that the carboxylate interacts with the side chains
of Arg-120 and Tyr-355 at the opening of the channel. The Fo Ϫ Fc-simulated annealing omit map density,
contoured at 4␴, is shown with the final refined models of EPA (cyan) and DHA (yellow).
and Leu-531 (Fig. 3, A–C). The side chain of Ser-530 exhibits a 22, 23). To facilitate binding, carbons C1–C9 of DHA take on a
dual conformation in the muCOX-2:AA crystal structure. The compacted and coiled orientation, similar to that observed for
mobility and flexibility of this side chain likely facilitates access EPA binding to the cyclooxygenase channel of ovCOX-1 (Fig.
of the ␻-end of the substrate to the hydrophobic groove and, 3D) (16). The distinct orientation of these carbons is necessary
although not essential for catalysis, may contribute to the to accommodate the rigidity associated with the C4–C5 and
proper alignment of C-13 below Tyr-385 (7). Shifts in the posi- C7–C8 unsaturated bonds. The constrained conformation of
tion of the side chain of Ser-530 have been observed previously DHA places carbon-15 ϳ3.0 Å below the phenolic oxygen of
in the ovCOX-1:DHLA crystal structure (23). A reduction in Tyr-385. The oxygenation of DHA by COX-2 is inefficient
the mobility of the side chain of Ser-530 upon aspirin treatment (Table 2) and results in the generation of monohydroxy fatty
could be a factor in the generation of (15R)-HETE by aspirin- acid products (15, 51). Hence, it is clear from the observed con-
acetylated COX-2 (11, 27). The conformation of the side chain formation of DHA that the inherent rigidity and compacted
of Arg-120 is different in the muCOX-2:AA and muCOX-2: nature of binding of this substrate in the cyclooxygenase chan-
EPA crystal structures, compared with its positioning in the nel would favor the production of monohydroxy fatty acids and
ovCOX-1:AA and ovCOX-1:EPA crystal structures (r.m.s.d. preclude the formation of a cyclic endoperoxide product. The
between side chain atoms of 1.27 and 1.61 Å, respectively; Fig. 3, residues lining the cyclooxygenase channel that contact DHA
A and B). The N⑀, N␩1, and N␩2 side chain atoms of Arg-120 are also conserved with respect to those making contacts with
forms a bifurcated interaction with both the carboxylate of the AA and EPA when bound to muCOX-2 (supplemental
substrate and the O⑀2 atom of Glu-524 in the cyclooxygenase Tables S7 and S8). Not surprisingly, the constrained orientation
channel of ovCOX-1 (16, 22, 23). In contrast, the lack of inter- of DHA results in this fatty acid making ϳ23% more contacts with
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Fatty Acid Substrate Binding in COX-2
carboxylate of the fatty acid is stabi-
lized by two hydrogen bonds, one
each to the phenolic oxygen of Tyr-
385 and the hydroxyl group of the
side chain of Ser-530 and two water
molecules occupy the empty hydro-
phobic groove above Ser-530. The
carbon atoms of both AA and EPA
are directed toward the opening of
the cyclooxygenase channel. The
␻-end of each substrate occupies
the space above Arg-120 and abuts
the side chain of Leu-531, whose
side chain is now forced into a
different rotameric conformation
compared with its location in mon-
omer B of the muCOX-2:AA and
muCOX-2:DHA crystal structures
and the crystal structures of
ovCOX-1 complexed with fatty acid
substrates (16, 22, 23). An alternate
conformation of the nonproductive
binding mode is also observed for
AA, as the C8–C9 unsaturated
bond can be modeled in two dif-
ferent positions (Fig. 1A). The
observed nonproductive confor-
mations of both AA and EPA are
very similar to that seen for AA in
the H207A apo muCOX-2 crystal
structure (supplemental Fig. S2)
(31).
Mobility of Leu-531 Influences
Substrate Binding to COX-2—To
investigate the importance of the
FIGURE 3. Comparison of the binding modes of fatty acid substrates in the cyclooxygenase channel. The
difference in the observed side chain
position of Leu-531 in our muCOX-
2:fatty acid crystal structures, we
constructed site-directed mutants
superposition of fatty acid substrates within the cyclooxygenase channel are shown. A, superposition of the
productive conformation of AA in monomer B of muCOX-2:AA (dark blue) and ovCOX-1:AA (Protein Data Bank
code 1DIY; pink). Carbon-13 of AA is labeled accordingly. B, superposition of the productive conformation of
EPA in monomer B of muCOX-2:EPA (cyan) with the nonoptimally aligned conformation observed in the crystal
structure of ovCOX-1:EPA (Protein Data Bank code 1IGX; purple). The position of C-13 for EPA in each structure
is labeled with a cyan and purple asterisk. The positions of cyclooxygenase active site residues in A and B are of Leu-531 and determined the abil-
shown in green for COX-2 and yellow for COX-1. C, superposition of the productive conformation of AA in
monomer B of muCOX-2:AA (dark blue) with the productive conformation of EPA in monomer B of muCOX-2:
EPA (cyan). The location of C-13 is labeled accordingly, and the position of the side chain of Leu-531 in the
muCOX-2:EPA structure is shown in pink. D, superposition of the productive conformation of AA in monomer
B of muCOX-2:AA (dark blue) with the conformation of DHA observed in monomer B of muCOX-2:DHA (yellow).
The locations of C-13 of AA and carbon-15 of DHA are denoted by a red asterisk. The positions of the side chains
of Arg-120 and Ser-121 in monomer B of muCOX-2:DHA are shown in pink.
ity of these mutants to oxygenate
AA. Specifically, Leu-531 was sub-
stituted with Ala, Phe, Pro, and Thr
utilizing the optimized His6 N580A
muCOX-2 construct as a template,
followed by expression and purifica-
residues lining the cyclooxygenase channel, including two salt tion as described above. All four mutant constructs were puri-
links between the carboxylate oxygen and Arg-120 and a hydrogen fied to levels equivalent to wild-type His6 N580A muCOX-2
bond between the carboxylate and phenolic oxygen of Tyr-355. utilized in crystallization trials and also exhibited wild-type lev-
The conformations observed for DHA are virtually identical els of peroxidase activity when measured spectrophotometri-
within each monomer of the muCOX-2:DHA crystal structure, cally (data not shown). Interestingly, none of the four Leu-531
with an r.m.s.d. of 0.71 Å between the 24 atoms of the substrate substitutions resulted in significant loss of cyclooxygenase
(supplemental Table S9), which would be expected for a rigid sub- activity (Table 3). There is less than a 2-fold reduction in rela-
strate bound in the cyclooxygenase channel.
tive Vmax compared with wild-type muCOX-2 when AA is uti-
Nonproductive Conformations of AA and EPA—AA and EPA lized as the substrate. Moreover, the L531A and L531P con-
exhibit the nonproductive binding conformation in monomer structs exhibit lower Km values for AA than wild-type enzyme
A of the muCOX-2:AA and muCOX-2:EPA crystal structures (Table 3). In stark contrast, substitution of Leu-531 to Ala in
(Fig. 1A and supplemental Fig. S1). In this conformation, the ovCOX-1 results in a mutant construct with greater than
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Fatty Acid Substrate Binding in COX-2
TABLE 3
Oxygenation of AA by Leu-531 mutants of His6 N580A muCOX-2
Oxygen consumption was measured using an oxygen electrode and AA as the fatty
acid substrate as described under “Experimental Procedures.” The oxygenation of
AA by Leu-531 mutant constructs is normalized to the oxygenation of AA by wild-
type His6 N580A muCOX-2 and reported as a relative Vmax. Relative Vmax values for
L531T and L531F were measured at an AA concentration of 25 ␮M in duplicate. The
relative Vmax and Km values reported for L531A and L531P represent results from
triplicate measurements. ND, not determined.
Construct
Relative Vmax
Km
%
␮M
Wild-type
L531A
L531P
100
58
64
56
89
5.14 Ϯ 0.29
3.65 Ϯ 0.32
2.95 Ϯ 0.27
ND
L531T
L531F
ND
21-fold reduction and 27-fold increase in relative specific activ-
ity and Km values, respectively, compared with wild-type
ovCOX-1 (26). Substitutions of Leu-531 with Asn, Asp, Ile, and
Val in ovCOX-1 also result in 5–14-fold reductions in relative
specific activity (26, 52). However, as is the case for the COX-2
substitutions, Km values for these ovCOX-1 substitutions
remain unchanged or are slightly lower compared with wild-
type enzyme (26, 52). Hence, Leu-531 substitutions are more
tolerated in muCOX-2, whereas a reduction in size or conserv-
ative replacement of this side chain in ovCOX-1 significantly
affects the proper alignment of substrate for oxygenation (26).
To investigate the structural consequences of removing the
flexibility associated with the Leu-531 side chain on AA binding
in the cyclooxygenase channel of COX-2, we determined the
crystal structure of Co3ϩ-protoporphyrin IX-reconstituted
L531F muCOX-2 in complex with AA (denoted L531F:AA). As
with the other muCOX-2:fatty acid complexes, the domain
architecture, Co3ϩ-protoporphyrin IX, and carbohydrate moi-
eties are well resolved in the crystal structure, and there are no
significant structural differences between monomers (data not
shown). In contrast to what we observe in the muCOX-2:AA
and muCOX-2:EPA crystal structures, the L531F mutation
causes AA to bind in the same global conformation in both
monomers of the L531F:AA crystal structure, with the carbox-
ylate of AA located near the entrance of the cyclooxygenase
channel in each monomer (Fig. 4, A and B). The ␻-end of the
substrate is bound in the hydrophobic groove above Ser-530,
FIGURE 4. AA bound in the cyclooxygenase channel of the L531F:AA com-
plex. Stereo view of AA bound in the cyclooxygenase channel of monomer B
(A) and monomer A (B) of the L531F:AA crystal structure. The L531F mutation
causes AA to bind with its carboxylate near Arg-120 and Tyr-355 at the open-
ing of the channel in each monomer of the biological dimer. The Fo Ϫ Fc-
and the contacts made between AA and the residues lining the simulated annealing omit map density, contoured at 4␴, is shown, and the
final refined models of AA (magenta) and hydrogen atoms have been mod-
cyclooxygenase channel are conserved with respect to those
eledontoC-13ofAA. ThesuperpositionoftheproductiveconformationofAA
in monomer B of muCOX-2:AA (dark blue) with the observed conformation of
AA in monomer A (C) and monomer B (D) of L531F:AA (magenta) is shown.
The location of C-13 of AA in each structure is denoted with a dark blue and
magenta asterisk accordingly. The positions of the side chains of Arg-120,
Glu-524, Ser-530, and Phe-531 in the L531F:AA structure are colored in pink.
identified when fatty acid substrates bind productively in the
cyclooxygenase channel (supplemental Tables S10 and S11)
(16, 22, 23).
The side chain of Leu-531 does not interact with fatty acid
substrate when it is bound in a productive conformation to
muCOX-2 or when it is bound to ovCOX-1 (16, 22, 23). Instead, boxylate of AA at the opening of the cyclooxygenase channel.
it forms hydrophobic interactions with the side chain of Arg- The carboxylate is perturbed with respect to its position in
120, suggesting a stabilizing role for this side chain in the main- monomer B of the muCOX-2:AA crystal structure, forming
tenance of high affinity binding of substrate to ovCOX-1 (26). two interactions with the N⑀ and N␩1 atoms of Arg-120 in
In the L531F:AA crystal structure, the side chain of Phe-531 lies addition to its interaction with the side chain atoms of Glu-524
above helix D of the membrane-binding domain in the vicinity and analogous to that observed for AA and other fatty acid
of Arg-120, where it exhibits a single rotamer conformation substrates bound to ovCOX-1 (16, 22, 23).
(Fig. 4, A and B). Substitution of Leu for Phe makes the side
The similar L-shaped conformations of AA observed in mon-
chain long enough to make an additional hydrophobic contact omer A and monomer B of the L531F:AA crystal structure
with C-1 of AA. This in turn influences the binding of the car- shows that the L531F substitution results in a decrease in the
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Fatty Acid Substrate Binding in COX-2
volume of the cyclooxygenase channel available for AA binding conformation of AA bound to ovCOX-1 (22) is also valid for AA
(Fig. 4, C and D). This loss of volume combined with the rigidity and EPA, given their similar overall L-shaped conformations
of the Phe-531 side chain impedes AA from binding in a non- and the optimal positioning of C-13 beneath the phenolic oxy-
productive conformation in monomer A. Moreover, further gen of Tyr-385 for hydrogen abstraction. Moreover, the
inspection of AA binding in each monomer of the L531F:AA observed constrained conformation of DHA provides for an
crystal structure indicates that AA does in fact bind differently explanation at the molecular level as to why monohydroxy acids
in each monomer despite their similar global orientations in the are formed upon oxygenation of this substrate by COX-2,
cyclooxygenase channel. As observed for AA and EPA in the rather than a cyclic endoperoxide product.
muCOX-2:AA and muCOX-2:EPA crystal structures, AA is
When the binding of AA and EPA in COX-1 and COX-2 are
bound productively in monomer B, with C-13 located ϳ3.2 Å compared, the first notable difference in cyclooxygenase active
below Tyr-385 for hydrogen abstraction (Fig. 4, A and D). site residues is related to the flexibility of Ser-530, which exhib-
Accordingly, the observed longer distance between C-13 and its alternate conformations for its side chain in the muCOX-
the phenolic oxygen of Tyr-385 compared with that observed in 2:AA crystal structure. The observed movement of the Ser-530
the muCOX-2:AA crystal structure likely explains the slight side chain may provide for easier access of the ␻-end of the
decrease in relative specific activity for this mutant (Table 3). substrate into the hydrophobic groove and also aid in the
However, AA binds in a nonoptimal conformation in monomer proper positioning of C-13 for catalysis (7). Furthermore, it is
A, as C-13 is displaced with respect to its position in monomer possible that a reduction of the flexibility of the Ser-530 side
B such that the modeled 13-pro-S hydrogen points away from chain plays a role in the generation of (15R)-HETE upon aspirin
Tyr-385 in a manner similar to that observed for EPA binding to acetylation of COX-2.
ovCOX-1 (Fig. 4, B and C) (16). Hence, our functional analysis
The second notable difference is the lack of interaction
of Leu-531 mutants in COX-2 coupled with the L531F:AA crys- between the carboxylate of AA and EPA and the side chain of
tal structure suggests that the side chain of Leu-531 does not Arg-120 in the muCOX-2 crystal structures. An ionic interac-
play a significant role in high affinity binding of substrate to tion between the carboxylate and Arg-120 is required for fatty
COX-2 and only a minor role in aligning C-13 optimally below acid binding to COX-1 but not COX-2, and the muCOX-2:AA
Tyr-385 for hydrogen abstraction. Rather, the mobility and muCOX-2:EPA crystal structures confirm previous
observed for the side chain of Leu-531 results in an increase in mutagenesis results to this effect. Rieke et al. (50) postulated
active site volume at the opening of the cyclooxygenase channel that the lack of a required interaction between the carboxylate
that influences the binding of certain fatty acid substrates.
of AA and the side chain of Arg-120 in COX-2 suggested that
the hydrophobic residues lining the cyclooxygenase channel
contributed more significantly to fatty acid substrate binding to
DISCUSSION
In light of the difficulties and nuances associated with earlier COX-2. Indeed, carbons C13–C20 of AA and EPA make 40 and
attempts to elucidate the structure of AA bound to muCOX-2, 38 of the 54 and 56 observed contacts (74 and 68%), respec-
we applied techniques developed to produce ovCOX-1:fatty tively, with residues lining the hydrophobic groove above Ser-
acid substrate complexes for crystallographic studies in an 530 within the cyclooxygenase channel in monomer B of the
attempt to determine the crystal structures of fatty acid sub- muCOX-2:AA and muCOX-2:EPA crystal structures, com-
strates bound to muCOX-2. All of the structures reported here pared with 26 of the 52 contacts (50%) made by the equivalent
crystallized with I222 space group symmetry, with a biological carbons of AA in the ovCOX-1:AA crystal structure. Hence, the
dimer in the asymmetric unit of the crystal. Thus, there is no lack of interaction between the carboxylate and Arg-120 is now
crystallographic symmetry relating one monomer to the other compensated for by an increased number of hydrophobic con-
(as is the case with ovCOX-1:fatty acid crystal structures), and tacts at the ␻-end of the substrate, ultimately providing AA and
the biological dimer observed in these muCOX-2:fatty acid EPA with the ability to achieve greater conformational flexibil-
structures can be considered to contain two independent ity for the carboxylate end of the substrate in the cyclooxygen-
monomers and hence two independent cyclooxygenase active ase channel of COX-2. This is supported by the greater than 0.9
sites. As such, significant efforts were taken to eliminate the Å r.m.s.d. calculated for these substrates compared with the
introduction of model bias during the building and refinement position of AA bound in the cyclooxygenase channel of
of fatty acid substrates within the cyclooxygenase channels so ovCOX-1 and in agreement with molecular dynamics simula-
that potential differences between substrate conformations or tions carried out to explore the equilibrium behavior of AA
active site residues in each monomer could be identified.
The conformation of AA, EPA, and DHA observed in mon-
bound to COX-1 and COX-2 (29).
The side chain of Leu-531 must move to accommodate the
omer B of their respective crystal structures provides the first ␻-end of the substrate when AA is bound nonproductively in
insights at the molecular level for the productive binding of COX-2. Movement of this side chain is the only notable differ-
these substrates in the cyclooxygenase channel of COX-2. The ence when the positions of equivalent residues are compared
observed productive conformations of AA and EPA are in with the active sites of ovCOX-1:AA and monomer B of
accord with early models put forth for AA binding in the muCOX-2:AA. In contrast to ovCOX-1, Leu-531 substitutions
cyclooxygenase channel of COX-2 that placed the carboxylate in muCOX-2 did not result in a significant loss of cyclooxyge-
near the opening of the channel near Arg-120 and the ␻-end in nase activity or binding affinity when AA was utilized as the
a hydrophobic groove above Ser-530 (25, 50). The sequence of substrate. The side chain of Leu-531 does not interact with fatty
events put forth for the cyclooxygenase reaction based on the acid substrates bound productively to ovCOX-1 (16, 22, 23) or
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Fatty Acid Substrate Binding in COX-2
muCOX-2. In both ovCOX-1 and monomer B of the muCOX- higher affinity, which can then act to allosterically modulate
2:AA structure, the side chain of Leu-531 forms interactions cyclooxygenase catalysis in the partner monomer, although the
with the side chain of Arg-120. This interaction has been pos- residues and mechanism involved in mediating the cross-talk
tulated to provide stabilization of Arg-120 for high affinity between monomers have not been identified (15).
binding of substrate to ovCOX-1 (26). When AA is bound in a
The crystal structures presented here agree with aspects of
nonproductive conformation, the ␻-end of the substrate inter- the model proposed for half-of-sites activity for cyclooxygen-
acts with the side chains of both Arg-120 and Leu-531, effec- ase. Specifically, we see fatty acid substrate bound in the
tively replacing the direct interaction between the two side cyclooxygenase channel of both monomers of the canonical
chains, which occurs when substrate is bound productively. It is cyclooxygenase homodimer. Moreover, only one substrate is
unclear from a structural perspective whether or not the bound in an optimal conformation for subsequent cyclooxyge-
observed flexibility of Leu-531 in the muCOX-2:AA crystal nase catalysis. In all of our COX-2:fatty acid crystal structures,
structure is also a feature of the COX-1 homodimer, given that the substrate in monomer A is bound in either a nonproductive
the known crystal structures of ovCOX-1 in complex with fatty conformation (muCOX-2:AA and muCOX-2:EPA) or in a non-
acid substrates all crystallized with a single monomer in the optimal or perturbed conformation that results in the misalign-
asymmetric unit. However, the significant decrease in specific ment of C-13 (C-15 of DHA) for catalysis (muCOX-2:DHA and
activity observed for conservative substitutions of Leu-531 in L531F:AA), whereas substrate is always bound productively in
ovCOX-1 coupled with the strict requirement for the carboxy- monomer B. It remains unclear as to whether or not the non-
late of the fatty acid substrate to interact with the side chain of productive binding mode observed for AA and EPA in mono-
Arg-120 for high affinity binding suggests that Leu-531 would mer A of their respective crystal structures represents an “allo-
not exhibit the same side chain flexibility in the cyclooxygenase steric” conformation that contributes to the signaling at the
channel of COX-1.
dimer interface (15). It is interesting to note that the global
Structural elucidation of the L531F:AA crystal structure conformational differences observed for AA and EPA in mon-
revealed AA bound in the same global conformation in each omer A of the muCOX-2:AA and the muCOX-2:EPA crystal
monomer, with the side chain of Phe-531 exhibiting a single structures versus the binding of DHA and AA in monomer A of
rotamer conformation. The L531F substitution introduces the COX-2:DHA and L531F:AA crystal structures correlate
rigidity at this site that decreases the volume of the cyclooxyge- with the ability of these substrates to attenuate Cu2ϩ/o-phe-
nase channel available for substrate binding, which in turn nanthroline-induced cross-linking studies carried out using
impedes the binding of AA in the nonproductive conformation. P127C/S541C ⌬C huPGHS-2 (15). Based on these results, the
The substitution also introduces a hydrophobic contact authors suggest that there are differences in the way that select
between this side chain and carbon-1 of AA that influences the substrates interact within the cyclooxygenase channel of
carboxylate of AA to form the requisite ionic and hydrogen COX-2 (15).
bond interactions with the side chain of Arg-120, analogous to
The different Leu-531 side chain conformations are the most
that observed for substrate binding to ovCOX-1. Thus, our significant changes observed when the areas in and around the
results indicate that the flexibility of the side chain of Leu-531 is cyclooxygenase channel of monomer A and monomer B of
important for increasing the volume available for substrate these crystal structures are compared. It is too early to speculate
binding at the opening of the cyclooxygenase channel of as to whether or not the movement of this side chain is directly
COX-2. As such, we postulate that the observed increase in or indirectly involved in mediating the cross-talk between
active site volume achieved by this movement is a significant monomers. We also do not observe any significant changes in
contributor to the observed ability of COX-2 to bind a broad residues at the dimer interface between monomers, which sug-
spectrum of fatty acid substrates and esters compared with gests that a large backbone movement is not the mechanism by
COX-1 (18, 20, 53).
which the monomers relay the allosteric signal. It should be
Why do we observe significantly different conformations of noted, however, that large conformational backbone changes
AA and EPA in each monomer of the biological dimer in the are not the exclusive means by which proteins transmit allo-
muCOX-2:AA and muCOX-2:EPA crystal structures? It has steric signals. Indeed, there are emerging ideas and new ways of
recently been demonstrated that the cyclooxygenase enzymes thinking about allostery and cross-talk that do not involve a
oxygenate fatty acid substrates via a half-of-sites reactivity significant movement or change in shape (55, 56), and it is clear
mechanism (54). Accordingly, it has been postulated that at any that additional structural, functional, and biophysical analyses
given time only one monomer of the COX-2 homodimer is will be required to shed light on the issue with respect to COX-1
functional, and it alone accounts for the cyclooxygenase activity and COX-2.
of the enzyme. Yuan et al. (54) initially speculated that only one
In summary, the crystal structures of Co3ϩ-protoporphyrin
substrate molecule bound in the cyclooxygenase channel of one IX-reconstituted muCOX-2 in complex with AA, EPA, and
monomer, which then could cause the partner monomer to DHA presented here provide the first molecular insight into the
undergo a conformational change that inhibits substrate from productive binding of these substrates in the cyclooxygenase
binding to this monomer. Subsequent analyses from the same channel of COX-2. Moreover, our structure-function analyses
laboratory have since demonstrated that substrate indeed binds contribute to the verification of previous biochemical studies
concurrently to each monomer of the dimer. As such, the cur- undertaken to characterize the role that Arg-120 and Leu-531
rent model of half-of-sites activity for cyclooxygenase involves play in fatty acid substrate binding to COX-1 and COX-2.
one monomer of the biological dimer binding substrate with a Finally, we show that the mobility of the side chain of Leu-531
22162 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 29•JULY 16, 2010

Fatty Acid Substrate Binding in COX-2
26. Thuresson, E. D., Lakkides, K. M., Rieke, C. J., Sun, Y., Wingerd, B. A.,
Micielli, R., Mulichak, A. M., Malkowski, M. G., Garavito, R. M., and
Smith, W. L. (2001) J. Biol. Chem. 276, 10347–10357
influences the binding of AA and EPA within the cyclooxygen-
ase channel of COX-2 and suggest that this inherent flexibility
contributes to increase the volume available at the opening of
the cyclooxygenase channel so that COX-2 can oxygenate a
wide ranging array of fatty acid substrates and esters compared
with COX-1. The structural elucidation of COX-2 complexed
with these fatty acid-based COX-2-specific substrates will be
required to validate our hypothesis.
27. Schneider, C., Boeglin, W. E., Prusakiewicz, J. J., Rowlinson, S. W., Mar-
nett, L. J., Samel, N., and Brash, A. R. (2002) J. Biol. Chem. 277, 478–485
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Acknowledgments—We thank Drs. Alan Brash and Claus Schneider
in the Department of Pharmacology, Vanderbilt University, for sup-
plying the cDNA for native murine COX-2.
31. Kiefer, J. R., Pawlitz, J. L., Moreland, K. T., Stegeman, R. A., Hood, W. F.,
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