collection and refinement statistics are shown
in table S1.
Overall complex geometry
In complex with vMIP-II, CXCR4 possesses the
typical seven transmembrane (TM) helical topology. Whereas previous dimeric structures of
CXCR4 suggested that chemokines might bind
receptors in a 2:1 CKR:chemokine stoichiometry (19, 20), the present structure demonstrates
that the stoichiometry is 1:1, in agreement with
a recent study (14). The chemokine interacts
via its globular core with the receptor N terminus
[chemokine recognition site 1 (CRS1) (21)] and
via its N terminus with the receptor TM pocket
(CRS2) (Fig. 1C). Clear electron density is observed
for the entire chemokine N terminus, including the CXCR4(D187C):vMIP-II(W5C) disulfide
bond, which adopts a favorable geometry (Fig.
1D). Residues 1 to 22 of the receptor are not visible in the density, consistent with the moderate
stability of the CRS1 interaction between CXCR4
and vMIP-II, as suggested by disulfide-trapping
experiments (fig. S2) and previous mutagenesis
Molecular interactions between
CXCR4 and vMIP-II
The CXCR4:vMIP-II interaction is mediated by
an extensive (1330 Å2) contiguous interface, with
every residue in the chemokine N terminus and
N loop (1-LGASCHRPDKCCLGYQ-16) contacting
the receptor (Fig. 2 and table S2). Although parts
of the interface can be classified as CRS1 or CRS2,
the absence of a distinct boundary prompted in-
troduction of an intermediate region, CRS1.5 (Fig.
2, A and B). The CRS1 interaction involves CXCR4
N-terminal residues 23-SMKEP-27 packing against
the chemokine N loop (residues 13-LGYQ-16) and
its third b strand (b3, residues 49-QVC-51) (Fig. 2,
C and D, and table S2). This interaction continues
toward CRS1.5, where receptor residues 27-PCFRE-
31 bind to chemokine residues 8-PDKCC-12 (Fig.
2, C and D) and form an antiparallel b sheet. In
CRS2, the chemokine N terminus makes hydrogen bonds to receptor residues D972.63, D2626.58,
and E2887.39 and numerous van der Waals packing interactions (Fig. 2, C and D, and table S2).
Most of the interacting CXCR4 residues are
known determinants of either vMIP-II binding (table S3) or CXCL12 binding and activation (22–26). The dominant role of the vMIP-II
N terminus is supported by the fact that an isolated vMIP-II(1-21) peptide binds CXCR4 with
appreciable affinity [190 nM (12) versus 6 to 15 nM
for wild-type (WT) vMIP-II (10, 12)], which is dramatically reduced by mutations L1A, R7A, and
K10A (27) (table S3). Notably, a W5A mutation
has only a moderate effect (27). Disulfide-trapping
studies also support the role of the chemokine N
loop (fig. S2).
Comparison of CXCR4:vMIP-II with
The conformation of the observed part of the
receptor N terminus differs significantly from
previous small-molecule and peptide-bound struc-
tures (19) in that it adopts an orientation almost
perpendicular to the membrane to form a b-sheet
interaction in CRS1.5 with chemokine residues
C11 and C12 (Fig. 3, A and B). To accommodate
this change as well as binding of the chemokine
N terminus in the TM pocket, the extracellular
half of helix I is laterally shifted outward by ~2.4 Å,
forming an extra a-helical turn and bending at
the top (Fig. 3A). ECL2 forms a b hairpin as in
other CXCR4 structures but is more closed onto
the binding pocket (Fig. 3A), bringing D181 and
D182 of CXCR4 in close proximity with K10 of
vMIP-II (Fig. 2, C and D).
The binding pocket of CXCR4 is open and
negatively charged (Fig. 3C) and can be separated into a major and minor subpocket (28).
Similar to the small-molecule antagonist, IT1t,
the chemokine N terminus makes the majority of
contacts in the minor subpocket and makes polar interactions with D972.63 and E2887.39 (Fig. 3,
C and D). By contrast, the spatial overlap between the vMIP-II N terminus and CVX15 is
moderate, with common recognition determinants
including D187ECL2 and D2626.58 (Fig. 3, C and E).
The limited overlap between CVX15 and the
chemokine N terminus may enable the design of
modulators that simultaneously occupy the minor and major subpockets; in fact, a series of
CXCR4 ligands obtained by grafting the N terminus of CXCL12 onto a peptide analog of CVX15
(29) may bind CXCR4 in this manner.
As in five earlier structures (19), CXCR4 forms
a dimer in the vMIP-II–bound form (Fig. 4A).
The preservation of similar dimerization patterns
in all CXCR4 structures (Fig. 4B) suggests possible physiological relevance and is consistent
with numerous reports of CXCR4 homo- and
heterodimerization in cells (30). The structure
III VII VI
Fig. 1. Design and crystallization of a disulfide-trapped CXCR4:vMIP-II
complex. (A) Nonreducing SDS–polyacrylamide gel electrophoresis and
Western blot of CXCR4(D97C) (left) and CXCR4(D187C) (right) coexpressed
with cysteine mutants of vMIP-II (residues 1 to 7). Uncomplexed CXCR4 and
disulfide-trapped complexes have molecular weights of ~45 and 55 kD, respec-
tively. Band identities were confirmed by Western blot using antibodies against
the FLAG and hemagglutinin (HA) tags at the N and C termini of CXCR4 and
vMIP-II, respectively (second and third rows). The 55-kD band was labeled by
antibodies to FLAG and HA (second to fourth rows); the band at 45 kD was only
labeled by the antibody to FLAG (second and fourth rows). (B) Thermal
stabilities of the complexes measured by a CPM assay (40) are shown as
mean T SEM measurements performed in triplicate. (C) Overall structure of
the CXCR4:vMIP-II complex (gray:magenta ribbon and transparent mesh).
(D) Zoomed view of the vMIP-II N terminus in the CXCR4 pocket showing the
CXCR4(D187C):vMIP-II(W5C) disulfide bond. The 2mFo – DFc electron
density map around the N terminus is contoured at 1.0 s and colored blue.