properties to promote their survival and propagation. Furthermore, these viral-derived structural
insights shed light on the mechanisms of ligand
signal-tuning and constitutive activity of mammalian GPCRs as a whole. The tunability of US28, and
perhaps other viral GPCRs, suggests that chemokine ligand-engineering strategies to elicit differential and biased signaling from GPCRs may be a
productive way to create new agonistic and inhibitory ligands.
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We thank B. Kobilka and members of the Kobilka lab for advice and
discussions; S. Kim, N. Latorraca, and A. Sanborn for assistance
with MD simulations and analysis; J. Spangler for helpful discussions;
E. Özkan for assistance with data collection; and H. Axelrod for advice
on refinement strategies. We also acknowledge beamline resources
and staff of Advanced Photon Source GM/CA beamlines 23-ID-B and
23-ID-D and Stanford Synchrotron Radiation Lightsource beamline
12-2. The data in this paper are tabulated in the main manuscript and
in the supplementary materials. Structure factors and coordinates
have been deposited in the Protein Data Bank with identification (ID)
numbers 4XT1 and 4XT3. We acknowledge support from the Cancer
Research Institute (J.S. B.), Howard Hughes Medical Institute (K.C.G.),
the Keck Foundation Medical Scholars Program (K.C.G.), NIH RO1
GM097015 (K.C.G.), a Terman Faculty Fellowship (R.O.D.), Swiss
National Science Foundation (A.A.), NIH Pioneer award (H.L.P.), and
Ludwig Foundation for Cancer Research (A.A.).
Materials and Methods
Figs. S1 to S10
Tables S1 and S2
17 December 2014; accepted 23 January 2015
Crystal structure of the chemokine
receptor CXCR4 in complex with a
Ling Qin,1 Irina Kufareva,1*† Lauren G. Holden,1 Chong Wang,2 Yi Zheng,1
Chunxia Zhao,1 Gustavo Fenalti,2 Huixian Wu,2 Gye Won Han,3,4 Vadim Cherezov,3
Ruben Abagyan,1 Raymond C. Stevens,3,4† Tracy M. Handel1†
Chemokines and their receptors control cell migration during development, immune system
responses, and in numerous diseases, including inflammation and cancer. The structural basis of
receptor:chemokine recognition has been a long-standing unanswered question due to the
challenges of structure determination for membrane protein complexes. Here, we report the
crystal structure of the chemokine receptor CXCR4 in complex with the viral chemokine antagonist
vMIP-II at 3.1 angstrom resolution. The structure revealed a 1:1 stoichiometry and a more extensive
binding interface than anticipated from the paradigmatic two-site model. The structure helped
rationalize a large body of mutagenesis data and together with modeling provided insights into
CXCR4 interactions with its endogenous ligand CXCL12, its ability to recognize diverse ligands,
and the specificity of CC and CXC receptors for their respective chemokines.
The chemokine receptor CXCR4 controls cell migration during immune surveillance and evelopment of the cardiovascular, hema- topoietic, and central nervous systems (1–3). Like many other chemokine receptors (CKRs),
CXCR4 contributes to inflammatory diseases and
cancer (4, 5). It also functions as one of two coreceptors that facilitate entry of HIV into host immune cells (6). Despite the importance of CXCR4
and CKRs in general, structural insights into CKR:
chemokine recognition have been limited to nuclear
magnetic resonance studies of chemokines with
peptides derived from CKR N termini (7–9). This is
partly due to the challenges of structure determination for full-length membrane proteins and
Here, we present the structure of CXCR4 in complex with vMIP-II, a CC chemokine encoded by
Kaposi’s sarcoma–associated herpesvirus. vMIP-II functions as a broad-spectrum antagonist of
many human CKRs (10) and helps the virus to escape
the host immune response (11). We chose vMIP-II
for structural studies because it is a high-affinity
antagonist of CXCR4 [median inhibitory concentration, 6 to 15 nM (10, 12)] and, as a ligand for both
CC and CXC chemokine receptors, was expected to
provide insight into ligand recognition specificity.
Design of an irreversible
Despite high affinity in membranes, the CXCR4:
vMIP-II complex was insufficiently stable in de-
tergent to justify crystallization trials. We there-
fore employed a strategy that uses disulfide trapping
to generate an irreversible complex (13, 14). Co-
expression of pairs of single cysteine mutants of
CXCR4 and vMIP-II was expected to result in
spontaneous formation of a disulfide bond if the
disulfide was compatible with the native geometry
of the CKR:chemokine complex. Guided by three-
dimensional models of CXCR4:chemokine com-
plexes (14), 37 cysteine mutant pairs were designed,
and, for each pair, the abundance of disulfide-
trapped complexes was evaluated (15). These pairs
included seven N-terminal cysteine mutants of
vMIP-II that were systematically coexpressed
with two CXCR4 cysteine mutants, D972.63C or
D187ECL2C [superscript denotes the Ballesteros-
Weinstein index (16, 17) for helical domain res-
idues; ECL is extracellular loop]. Of all mutant
pairs analyzed, CXCR4(D187C) coexpressed with
vMIP-II(W5C) formed the highest percentage
of trapped complex (Fig. 1A). It also showed an
unfolding temperature of 63°C (Fig. 1B), which
is 4° to 14°C higher than other mutant combina-
tions, and excellent monodispersity when ana-
lyzed by size-exclusion chromatography (fig. S1).
By comparison, the mutant pair with the second
highest melting temperature, CXCR4(D187C):
vMIP-II(H6C) (59°C), was produced in signif-
icantly lower yield and showed lower monodis-
persity, despite the adjacent position of the
vMIP-II cysteine (fig. S1). CXCR4(D97C) formed
little or no covalent complex with any of the
seven vMIP-II mutants tested (Fig. 1, A and B).
The observed sensitivity of several biophysical
properties of the complex to precise cysteine
placement suggests specificity of the disulfide-
trapping approach and supports compatibility
of the D187C: W5C disulfide bond with the na-
tive complex geometry. We therefore selected
CXCR4(D187C):vMIP-II(W5C) for crystalliza-
tion in lipidic cubic phase (LCP) (18) and de-
termined the structure at 3.1 Å resolution. Data
1University of California, San Diego, Skaggs School of
Pharmacy and Pharmaceutical Sciences, La Jolla, CA 92093,
USA. 2Department of Integrative Structural and
Computational Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
3Department of Chemistry, Bridge Institute. 4Department of
Biological Sciences, Bridge Institute, University of Southern
California, Los Angeles, CA 90089, USA.
*These authors contributed equally to this work. †Corresponding
author. E-mail: firstname.lastname@example.org (T.M.H.); email@example.com
(R.C.S.); firstname.lastname@example.org (I.K.)