inward toward the center of the TM bundle, similar to the position seen in the active-state structure of the b2-adrenergic receptor (b2AR) and
contrasting with the corresponding arginine in
the inactive CCR5 structure that projects away (Fig.
5C) (2, 21). The DRY motif can stabilize the inactive conformation of some GPCRs by participating in a salt bridge between Arg3.50 and an acidic
residue at position 6.30, in what is known as the
“ionic lock” (20, 22). US28 lacks an acidic residue
at position 6.30, so absence of this contact could
be one factor contributing to stabilization of the
active state in the absence of ligand.
Further structural evidence for the active state
of US28 is demonstrated by the intracellular
half of TM7, which is shifted toward the center
of the TM bundle (Fig. 5A). This is seen in the
active-state b2AR but not the inactive CCR5 struc-
ture (2, 21). The inward movement of TM7 re-
sults in Tyr2917.53 of the NPXXY motif shifting 7 Å
toward the center of the TM bundle, which is
close enough to TM5 and TM3 to form hydrogen
bonds with Tyr2085.58 and Ile1223.43 through a
water molecule (Fig. 5B). Tyr2085.58, in turn, forms
a hydrogen bond with Arg1293.50 of the DRY motif
(Fig. 5C). This completes a hydrogen bond net-
work connecting TM3, TM5, and TM7 that has
been seen in previously solved active-state struc-
tures (20, 21).
CX3CL1 has been shown to exhibit both agonist and inverse agonist activities in US28 signaling assays. This apparent discrepancy has been
explained by CX3CL1 being a “camouflaged agonist” that signals but exhibits diminished agonist
activity due to ligand-induced internalization and
degradation (23). These structures support an
interpretation that CX3CL1 is an agonist, not an
inverse agonist, because it does not induce an inactive state of the receptor. CX3CL1 binding may
either stabilize the ligand-independent active
state of US28 or alter the active conformation to
induce a slightly different signaling outcome from
the unliganded state.
Structural basis for constitutive activity
and ligand action
Constitutive activity is a common property of viral
GPCRs that enhances viral pathogenesis (18) and
is also seen to varying degrees in some mam-
malian GPCRs (24, 25). Although structures of
certain constitutively active rhodopsin mutants
are available (26, 27), the mechanistic basis
through which viral GPCRs have gained this evo-
lutionarily advantageous constitutive activity has
To address this question, we performed atomic-level molecular dynamics (MD) simulations of
US28, both with and without bound CX3CL1 (see
supplementary materials and methods). Atomic-level simulations have provided mechanistic insight into important functional properties of other
GPCRs (28, 29). Because the crystal structures of
US28 with and without the nanobody exhibit
essentially identical conformations of the TM helices, we initiated our simulations from the 2.9 Å
structure but omitted the nanobody.
Using integrated analysis of sequence, structure, and simulations of US28, we uncovered
molecular features of US28 that may lead to its
constitutive activity. In particular, we found that
US28 has evolved a distinctive structure environment around Asp1283.49, near the cytoplasmic
end of TM3, that probably results in a destabilization of the receptor’s inactive state (Fig. 6).
Asp3.49 is part of the conserved DRY motif, which
SCIENCE sciencemag.org 6 MARCH 2015 • VOL 347 ISSUE 6226 1115
Fig. 3. Interaction of the CX3CL1 N terminus with the US28 ligand binding pocket (site 2). (A) Side chain contacts between CX3CL1 site 2 region
(blue) and US28 (orange). (B) Two-dimensional plot of side-chain contacts between the CX3CL1 N-terminal hook and US28.
Fig. 4. Comparison of US28-CX3CL1 with chemokine receptor small-molecule and peptide complexes. (A) Overall superposition of the US28
(orange), CCR5 (green; PDB ID: 4MBS), and CXCR4 (purple; PDB ID: 3ODU)
TM helices from the side (left) and as viewed from extracellular space (right).
(B) Surface cutaway side views comparing ligand binding modes for US28-
CX3CL1 (orange-blue), CCR5-maraviroc (green-red; PDB ID: 4MBS), and
CXCR4-CVX15 (purple-yellow; PDB ID: 3OE0).