3OLT

X-ray crystal structure of arachidonic acid bound to the cyclooxygenase channel of R513H murine COX-2

PDB DOI: https://doi.org/10.2210/pdb3OLT/pdb

Classification: OXIDOREDUCTASE
Organism(s): Mus musculus
Expression System: Spodoptera frugiperda
Mutation(s): Yes

Deposited: 2010-08-26 Released: 2011-04-13
Deposition Author(s): Vecchio, A.J., Malkowski, M.G.

Experimental Data Snapshot

Method: X-RAY DIFFRACTION
Resolution: 2.45 ?
R-Value Free: 0.219
R-Value Work: 0.163
R-Value Observed: 0.166

Starting Model: experimental
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Ligand Structure Quality Assessment

This is version 2.1 of the entry. See complete history.

Literature

The structural basis of endocannabinoid oxygenation by cyclooxygenase-2.

Vecchio, A.J., Malkowski, M.G.

(2011) J Biol Chem 286: 20736-20745

PubMed: 21489986 Search on PubMedSearch on PubMed Central
DOI: https://doi.org/10.1074/jbc.M111.230367
Primary Citation of Related Structures:
3MDL, 3OLT, 3OLU

PubMed Abstract:
The cyclooxygenases (COX-1 and COX-2) oxygenate arachidonic acid (AA) in the committed step of prostaglandin biogenesis. Substitutions of I434V, H513R, and I523V constitute the only differences in residues lining the cyclooxygenase channel between COX-1 and COX-2. These changes create a hydrophobic pocket in COX-2, with Arg-513 located at the base of the pocket, which has been exploited in the design of COX-2-selective inhibitors. Previous studies have shown that COX-2, but not COX-1, can oxygenate endocannabinoid substrates, including 2-arachidonoyl glycerol (2-AG). To investigate the isoform-specific structural basis of endocannabinoid binding to COX-2, we determined the crystal structure of the 2-AG isomer 1-arachidonoyl glycerol (1-AG) in complex with wild type and R513H murine (mu) COX-2 to 2.2 and 2.35 ?, respectively, and R513H muCOX-2 in complex with AA to 2.45 ? resolution. The 2,3-dihydroxypropyl moiety of 1-AG binds near the opening of the cyclooxygenase channel in the space vacated by the movement of the Leu-531 side chain, validating our previous hypothesis implicating the flexibility of the Leu-531 side chain as a determinant for the ability of COX-2 to oxygenate endocannabinoid substrates. Functional analyses carried out to compliment our structural findings indicated that Y355F and R513H muCOX-2 constructs had no effect on the oxygenation of 1-AG and 2-AG, whereas substitutions that resulted in a shortened side chain for Leu-531 had only modest effects. Both AA and 1-AG bind to R513H muCOX-2 in conformations similar to those observed in the co-crystal structures of these substrates with wild type enzyme.

Organizational Affiliation

Department of Structural Biology, Hauptman-Woodward Medical Research Institute,The State University of New York, Buffalo, New York 14203, USA.

Macromolecule Content

Total Structure Weight: 141.53 kDa
Atom Count: 9,793
Modelled Residue Count: 1,102
Deposited Residue Count: 1,184
Unique protein chains: 1

Macromolecules

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(by identity cutoff) | 3D Structure

Entity ID: 1
Molecule	Chains	Sequence Length	Organism	Details	Image
Prostaglandin G/H synthase 2	A, B	592	Mus musculus	Mutation(s): 1 Gene Names: Ptgs2, Cox-2, Cox2, Pghs-b, Tis10 EC: 1.14.99.1
UniProt
Find proteins for Q05769 (Mus musculus) Explore Q05769 Go to UniProtKB: Q05769
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt Group	Q05769
Glycosylation
Glycosylation Sites: 3	Glycosylation Focus chain A Focus chain B	Go to GlyGen: Q05769-1
Sequence Annotations Expand
Reference Sequence

Oligosaccharides

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Entity ID: 2
Molecule	Chains	Length	2D Diagram	Glycosylation	3D Interactions
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose	C, E	2		N-Glycosylation	Interactions Focus chain C Focus chain E Interactions & Density Focus chain C Focus chain E
Glycosylation Resources
GlyTouCan: G42666HT GlyCosmos: G42666HT GlyGen: G42666HT

Entity ID: 3
Molecule	Chains	Length	2D Diagram	Glycosylation	3D Interactions
alpha-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose	D, F	3		N-Glycosylation	Interactions Focus chain D Focus chain F Interactions & Density Focus chain D Focus chain F
Glycosylation Resources
GlyTouCan: G62182OO GlyCosmos: G62182OO GlyGen: G62182OO

Small Molecules

Ligands 5 Unique
ID	Chains	Name / Formula / InChI Key	2D Diagram	3D Interactions
COH Query on COH Download Ideal Coordinates CCD File SDF format, chain O [auth A] SDF format, chain Z [auth B] MOL2 format, chain O [auth A] MOL2 format, chain Z [auth B] mmCIF format, chain O [auth A] mmCIF format, chain Z [auth B]	O [auth A], Z [auth B]	PROTOPORPHYRIN IX CONTAINING CO C₃₄ H₃₂ Co N₄ O₄ AQTFKGDWFRRIHR-RGGAHWMASA-L		Interactions Focus chain O [auth A] Focus chain Z [auth B] Interactions & Density Focus chain O [auth A] Focus chain Z [auth B]
ACD Query on ACD Download Ideal Coordinates CCD File SDF format, chain N [auth A] SDF format, chain Y [auth B] MOL2 format, chain N [auth A] MOL2 format, chain Y [auth B] mmCIF format, chain N [auth A] mmCIF format, chain Y [auth B]	N [auth A], Y [auth B]	ARACHIDONIC ACID C₂₀ H₃₂ O₂ YZXBAPSDXZZRGB-DOFZRALJSA-N		Interactions Focus chain N [auth A] Focus chain Y [auth B] Interactions & Density Focus chain N [auth A] Focus chain Y [auth B]
BOG Query on BOG Download Ideal Coordinates CCD File SDF format, chain Q [auth A] MOL2 format, chain Q [auth A] mmCIF format, chain Q [auth A]	Q [auth A]	octyl beta-D-glucopyranoside C₁₄ H₂₈ O₆ HEGSGKPQLMEBJL-RKQHYHRCSA-N		Interactions Focus chain Q [auth A] Interactions & Density Focus chain Q [auth A]
NAG Query on NAG Download Ideal Coordinates CCD File SDF format, chain AA [auth B] SDF format, chain P [auth A] MOL2 format, chain AA [auth B] MOL2 format, chain P [auth A] mmCIF format, chain AA [auth B] mmCIF format, chain P [auth A]	AA [auth B], P [auth A]	2-acetamido-2-deoxy-beta-D-glucopyranose C₈ H₁₅ N O₆ OVRNDRQMDRJTHS-FMDGEEDCSA-N		Interactions Focus chain AA [auth B] Focus chain P [auth A] Interactions & Density Focus chain AA [auth B] Focus chain P [auth A]
EDO Query on EDO Download Ideal Coordinates CCD File SDF format, chain G [auth A] SDF format, chain H [auth A] SDF format, chain I [auth A] SDF format, chain J [auth A] SDF format, chain K [auth A] SDF format, chain L [auth A] SDF format, chain M [auth A] SDF format, chain R [auth B] SDF format, chain S [auth B] SDF format, chain T [auth B] SDF format, chain U [auth B] SDF format, chain V [auth B] SDF format, chain W [auth B] SDF format, chain X [auth B] MOL2 format, chain G [auth A] MOL2 format, chain H [auth A] MOL2 format, chain I [auth A] MOL2 format, chain J [auth A] MOL2 format, chain K [auth A] MOL2 format, chain L [auth A] MOL2 format, chain M [auth A] MOL2 format, chain R [auth B] MOL2 format, chain S [auth B] MOL2 format, chain T [auth B] MOL2 format, chain U [auth B] MOL2 format, chain V [auth B] MOL2 format, chain W [auth B] MOL2 format, chain X [auth B] mmCIF format, chain G [auth A] mmCIF format, chain H [auth A] mmCIF format, chain I [auth A] mmCIF format, chain J [auth A] mmCIF format, chain K [auth A] mmCIF format, chain L [auth A] mmCIF format, chain M [auth A] mmCIF format, chain R [auth B] mmCIF format, chain S [auth B] mmCIF format, chain T [auth B] mmCIF format, chain U [auth B] mmCIF format, chain V [auth B] mmCIF format, chain W [auth B] mmCIF format, chain X [auth B]	G [auth A] H [auth A] I [auth A] J [auth A] K [auth A] G [auth A], H [auth A], I [auth A], J [auth A], K [auth A], L [auth A], M [auth A], R [auth B], S [auth B], T [auth B], U [auth B], V [auth B], W [auth B], X [auth B]	1,2-ETHANEDIOL C₂ H₆ O₂ LYCAIKOWRPUZTN-UHFFFAOYSA-N		Interactions Focus chain G [auth A] Focus chain H [auth A] Focus chain I [auth A] Focus chain J [auth A] Focus chain K [auth A] Focus chain L [auth A] Focus chain M [auth A] Focus chain R [auth B] Focus chain S [auth B] Focus chain T [auth B] Focus chain U [auth B] Focus chain V [auth B] Focus chain W [auth B] Focus chain X [auth B] Interactions & Density Focus chain G [auth A] Focus chain H [auth A] Focus chain I [auth A] Focus chain J [auth A] Focus chain K [auth A] Focus chain L [auth A] Focus chain M [auth A] Focus chain R [auth B] Focus chain S [auth B] Focus chain T [auth B] Focus chain U [auth B] Focus chain V [auth B] Focus chain W [auth B] Focus chain X [auth B]

Binding Affinity Annotations
ID	Source	Binding Affinity
ACD	BindingDB: 3OLT	Kd: 8070 (nM) from 1 assay(s)

Experimental Data & Validation

Experimental Data

Method: X-RAY DIFFRACTION
Resolution: 2.45 ?
R-Value Free: 0.219
R-Value Work: 0.163
R-Value Observed: 0.166
Space Group: I 2 2 2

Unit Cell:

Length ( ? )	Angle ( ? )
a = 121.148	α = 90
b = 132.048	β = 90
c = 180.758	γ = 90

Software Package:

Software Name	Purpose
Adxv	data processing
PHASER	phasing
REFMAC	refinement
MOSFLM	data reduction
SCALA	data scaling

Structure Validation

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Ligand Structure Quality Assessment

Entry History

Deposition Data

Released Date: 2011-04-13

Deposition Author(s):

Vecchio, A.J.

Malkowski, M.G.

Revision History (Full details and data files)

Version 1.0: 2011-04-13
Type: Initial release
Version 1.1: 2011-07-13
Changes: Version format compliance
Version 1.2: 2017-11-08
Changes: Refinement description
Version 2.0: 2020-07-29
Type: Remediation
Reason: Carbohydrate remediation
Changes: Advisory, Atomic model, Data collection, Database references, Derived calculations, Structure summary
Version 2.1: 2023-09-06
Changes: Data collection, Database references, Refinement description, Structure summary