National Laboratory of Biomacromolecules, National Center of Protein Science–Beijing, CAS Center for Excellence in Biomacromolecules, Institute of
Biophysics, Chinese Academy of Sciences, Beijing, China.
CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai, China.
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
University of Chinese Academy of Sciences, Beijing, China.
College of Life Science and Technology, Collaborative Innovation Center for
Genetics and Development, and Key Laboratory of Molecular Biophysics of the Ministry of Education, Huazhong University of Science and Technology,
Department of Pharmacology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China.
Department of Chemistry,
Bridge Institute, University of Southern California, Los Angeles, CA, USA.
Drug Discovery and Design Center, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.
iHuman Institute, ShanghaiTech University, Shanghai, China.
School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
CAS Center for Excellence in Biomacromolecules, Chinese Academy
of Sciences, Beijing, China.
These authors contributed equally: Can Cao, Qiuxiang Tan. *e-mail: email@example.com; firstname.lastname@example.org
AFR, a member of the G-protein-coupled receptor (GPCR)
superfamily, is expressed on the surfaces of a variety of cells
and is involved in numerous pathophysiologi-
cal activities related to inflammation and immune responses as
well as cardiovascular, reproductive, respiratory and nervous-
. After binding to its endogenous agonist, PAF
(1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), PAFR is cou
pled to G
protein and consequently activates various downstream
. Many compounds with high structural diver-
sity have been characterized as PAFR antagonists and inverse ago-
nists, and show differential effects on receptor conformation and
. The highly potent and selective PAFR ligands SR 27417
(N-(2-dimethylamino ethyl)-N-(3-pyridinyl methyl)[4-(2,4,6-tri
isopropylphenyl)thiazol-2-yl]amine) and ABT-491 (4-ethynyl-N,N
inhibit PAF signaling both in vitro and in vivo, and have been sug
gested as drug candidates for the treatment of diseases including
asthma and cardiovascular diseases
. To reveal PAFR’s modes of
ligand binding and to better understand the signaling mechanism of
PAFR, we solved crystal structures of human PAFR in complex with
SR 27417 and ABT-491 (Fig. 1 and Table 1).
Structural determination of PAFR–SR 27417 and PAFR–ABT-
491 complexes. To obtain crystals of PAFR, we replaced residues
P217–N223 in the third intracellular loop (ICL3) of the receptor
with residues 2–148 of a modified flavodoxin (P2A Y98W)
Desulfovibrio vulgaris to generate a PAFR-flavodoxin fusion con
struct, and we made a PAFR–minimal T4 lysozyme (mT4L) fusion
construct by inserting the mT4L
between residues V218 and A224
in ICL3. To improve protein homogeneity, we truncated 26 amino
acids (residues C317–N342) at the C terminus of the receptor in
both constructs. Additionally, we introduced four point muta
A and D289
indicates residue numbering in Ballesteros–Weinstein nomencla
), into the PAFR gene to improve protein yield, homogeneity
and thermal stability (Supplementary Fig. 1). Another mutation,
N169D in the second extracellular loop (ECL2), was introduced
to remove a heterogeneous glycosylation site. Our ligand-binding
assay showed that the flavodoxin and mT4L fusion proteins and
the five mutations had little effect on the binding affinity of PAFR
toward PAF, SR 27417 and ABT-491 (Supplementary Fig. 2 and
Supplementary Table 1).
The modified PAFR constructs were cloned into a pFastBac 1
vector for expression in Spodoptera frugiperda (Sf9) insect cells,
thus generating PAFR-flavodoxin and PAFR-mT4L fusion proteins,
which were copurified with SR 27417 and ABT-491, respectively.
Crystallization trials of the four PAFR fusion protein–ligand com
plexes were set up by reconstituting the proteins in lipidic cubic
phase (LCP) with cholesterol supplementation. Crystals of PAFR–
flavodoxin in complex with SR 27417 and of PAFR-mT4L in com
Structural basis for signal recognition and
transduction by platelet-activating-factor receptor
, Qiuxiang Tan
, Chanjuan Xu
, Lingli He
, Linlin Yang
, Ye Zhou
, Yiwei Zhou
, Minmin Lu
, Cuiying Yi
, Gye Won Han
, Xianping Wang
, Xuemei Li
, Huaiyu Yang
, Hualiang Jiang
, Yongfang Zhao
, Jianfeng Liu
, Raymond C. Stevens
, Xuejun C. Zhang
* and Beili Wu
Platelet-activating-factor receptor (PAFR) responds to platelet-activating factor (PAF), a phospholipid mediator of cell-to-cell
communication that exhibits diverse physiological effects. PAFR is considered an important drug target for treating asthma,
inflammation and cardiovascular diseases. Here we report crystal structures of human PAFR in complex with the antagonist
SR 27417 and the inverse agonist ABT-491 at 2.8-Å and 2.9-Å resolution, respectively. The structures, supported by molecular
docking of PAF, provide insights into the signal-recognition mechanisms of PAFR. The PAFR–SR 27417 structure reveals an
unusual conformation showing that the intracellular tips of helices II and IV shift outward by 13 Å and 4 Å, respectively, and
helix VIII adopts an inward conformation. The PAFR structures, combined with single-molecule FRET and cell-based functional
assays, suggest that the conformational change in the helical bundle is ligand dependent and plays a critical role in PAFR activa-
tion, thus greatly extending knowledge about signaling by G-protein-coupled receptors.
NATURE STRUCTURAL & MOLECULAR BIOLOGY | VOL 25 | JUNE 2018 | 488–495 | www.nature.com/nsmb
© 2018 Nature America Inc., part of Springer Nature. All rights reserved.