Article https://doi.org/10.

1038/s41467-024-52045-7

Structural basis for CCR6 modulation by

allosteric antagonists

Received: 22 March 2024 David Jonathan Wasilko1, Brian S. Gerstenberger 2, Kathleen A. Farley 1,
Wei Li3, Jennifer Alley3, Mark E. Schnute 2, Ray J. Unwalla2, Jorge Victorino1,
Accepted: 23 August 2024
Kimberly K. Crouse3, Ru Ding3, Parag V. Sahasrabudhe 1, Fabien Vincent 1,
Richard K. Frisbie1, Alpay Dermenci4, Andrew Flick 4, Chulho Choi 4,
Gary Chinigo4, James J. Mousseau4, John I. Trujillo4, Philippe Nuhant2,
Check for updates Prolay Mondal4, Vincent Lombardo4, Daniel Lamb5,6, Barbara J. Hogan 5,6,
Gurdeep Singh Minhas5, Elena Segala5, Christine Oswald5, Ian W. Windsor 1,
Seungil Han 1, Mathieu Rappas5,6, Robert M. Cooke5, Matthew F. Calabrese 1,
1234567890():,;
1234567890():,;

Gabriel Berstein3, Atli Thorarensen2 & Huixian Wu 1

The CC chemokine receptor 6 (CCR6) is a potential target for chronic

inflammatory diseases. Previously, we reported an active CCR6 structure in
complex with its cognate chemokine CCL20, revealing the molecular basis of
CCR6 activation. Here, we present two inactive CCR6 structures in ternary
complexes with different allosteric antagonists, CCR6/SQA1/OXM1 and CCR6/
SQA1/OXM2. The oxomorpholine analogues, OXM1 and OXM2 are highly
selective CCR6 antagonists which bind to an extracellular pocket and disrupt
the receptor activation network. An energetically favoured U-shaped con-
formation in solution that resembles the bound form is observed for the active
analogues. SQA1 is a squaramide derivative with close-in analogues reported as
antagonists of chemokine receptors including CCR6. SQA1 binds to an intra-
cellular pocket which overlaps with the G protein site, stabilizing a closed
pocket that is a hallmark of inactive GPCRs. Minimal communication between
the two allosteric pockets is observed, in contrast to the prevalent allosteric
cooperativity model of GPCRs. This work highlights the versatility of GPCR
antagonism by small molecules, complementing previous knowledge of CCR6
activation, and sheds light on drug discovery targeting CCR6.

The chemokine receptors (CKRs) are a group of class A G protein- CX3C, and C receptors, where C refers to the conserved N-terminal
coupled receptors (GPCRs) that respond to a family of cytokines cysteines and X represents the amino acids between the two
known as chemokines1. More than twenty distinct CKRs have been N-terminal cysteines of the chemokine1. Upon activation by chemo-
discovered in humans, including 18 conventional CKRs that are typi- kines, CKRs mediate a wide range of physiological processes including
cally Gi-coupled and 5 atypical CKRs which are structurally related to embryonic development, immune cell proliferation, activation, dif-
the conventional receptors but are non-signalling2. CKRs can also be ferentiation, and cell death. Consequently, CKRs play a significant role
classified by their primary type of chemokine ligands as CC, CXC, in the development and homoeostasis of the immune system, and are

1
Discovery Sciences, Medicine Design, Pfizer Inc., Groton, CT, USA. 2Medicine Design, Pfizer Inc., Cambridge, MA, USA. 3Inflammation and Immunology
Research, Pfizer Inc., Cambridge, MA, USA. 4Medicine Design, Pfizer Inc., Groton, CT, USA. 5Sosei Heptares, Steinmetz Building, Granta Park, Great Abington,
Cambridge, UK. 6Present address: Nxera Pharma UK Limited, Steinmetz Building, Granta Park, Great Abington, Cambridge, UK.
e-mail: huixian.wu@pfizer.com

Nature Communications | (2024)15:7574 1

Article https://doi.org/10.1038/s41467-024-52045-7

important drug targets for cancer, infectious diseases, inflammation, which contains seven thermostabilizing alanine-substitutions20,21 in the
and autoimmune disorders3,4. transmembrane domain and a point mutation L1343.41W (superscripts
The CC chemokine receptor 6 (CCR6)5 is a member of the family indicate Ballesteros–Weinstein numbering for GPCRs22) to increase
expressed on a variety of lymphocytes including T cells, particularly the receptor expression23. Binding of the SQA and OXM analogues to the
T helper 17 (Th17) cells6, B cells, and dendritic cells. CCR6 is activated by thermostabilized CCR6 construct was confirmed with comparable
its specific ligand CCL207, which stimulates the migration of CCR6+ potency to the wild-type (WT) receptor (Supplementary Fig. 1). Addi-
lymphocytes to sites of inflammation. The expression of both CCL20 tive stabilization between the SQA and OXM analogues was also
and CCR6 is upregulated in the epithelial tissues of patients with chronic observed with the stabilized construct (Fig. 1d). For structural studies,
inflammatory conditions such as psoriasis8 and inflammatory bowel twenty-seven residues at both N and C-termini of CCR6 were truncated
disease (IBD)9. Therefore, inhibiting CCL20-mediated CCR6 signaling is a to improve protein homogeneity. Furthermore, the residues between
promising strategy for anti-inflammatory drug discovery10. Indeed, a L241 and K251 of intracellular loop 3 (ICL3) were replaced by a ther-
monoclonal antibody targeting CCL20 has resulted in effective allevia- mostabilized apocytochrome b562 (BRIL)24. Two amino acid linkers
tion of inflammation in preclinical models11. In addition, CCR6 knockout were grafted from the A2A adenosine receptor25 to stabilize continuous
mice are protected from induced psoriasis-like inflammation12. Accord- alpha-helices as the junctions between TM5 (CCR6) and helix 1 (BRIL),
ingly, a small molecule antagonist of CCR6 could be useful as an oral as well as TM6 (CCR6) and helix 4 (BRIL). The engineered CCR6-BRIL
treatment for autoimmune disorders. chimeric construct (Supplementary Fig. 2) was co-purified with ana-
Previously, we reported an active CCR6 structure in complex with logues from both SQA and OXM series. An anti-BRIL Fab26 with an anti-
CCL20 and G protein13. This structure revealed a shallow CCL20 pocket Fab nanobody (Nb)27 was employed to complex with the BRIL in the
formed mainly by extracellular loops and the N-terminus of CCR6, leav- chimeric CCR6 construct. The BRIL/Fab/Nb module was introduced as
ing the orthosteric pocket within the seven-transmembrane (7TM) a fiducial marker in the single-particle cryo-EM analysis to facilitate
domain largely unoccupied. The CCL20-CCR6 recognition mode as well particle alignment28. This approach led to the successful determination
as the large extracellular pocket of this protein receptor pose significant of two inactive CCR6 structures bound by ligands from the SQA and
challenges to the development of small molecule antagonists targeting OXM series.
the orthosteric site. Alternatively, modulating CKRs via allosteric ligands, The complex structures of CCR6/SQA1/OXM1 and CCR6/SQA1/
notable for its versatility and diversity of mechanisms-of-action (MoA), OXM2 were determined at overall resolutions of 3.3 Å and 3.0 Å,
has been an attractive and promising approach in CKR drug discovery14–18. respectively (Supplementary Figs. 3, 4, Supplementary Table 1). Cryo-
In this work, we present two inactive CCR6 structures in ternary EM reconstructions of both complexes reveal robust density, allowing
complexes with different allosteric antagonists from the squaramide the unambiguous fitting of BRIL, Fab, Nb, and the 7TM of CCR6
(SQA) and the oxomorpholine (OXM) series. The structures are (Fig. 2a, b). Most extracellular loops (ECLs) and intracellular loops
determined using single-particle cryogenic electron microscopy (cryo- (ICLs) are unresolved or ambiguous in the reconstruction except for
EM) facilitated by an engineered fiducial marker. The SQA and OXM the bottom of ECL2, which adopts a β-hairpin structure like that
analogues occupy two different pockets, binding of which can occur reported in the active CCR6 and other CKR structures13. The dis-
independently and simultaneously on CCR6. Small molecule NMR ordered loops in the final reconstructions are not unexpected based
suggests an energetically favoured U-shaped conformation in solution on the known intrinsic flexibility of these regions in GPCRs. In both
is critical for the activity of OXMs. We show different allosteric structures, the densities of the SQA and OXM analogues are robustly
mechanisms and no cooperativity between SQA and OXM analogues to resolved, with each series binding to a distinct but well-defined pocket
antagonize CCR6, highlighting the diversity of GPCR allosteric mod- within the 7TM bundle, allowing confident modelling of ligand binding
ulation by small molecules. for subsequent structural analysis (Fig. 2, Supplementary Figs. 3, 4).

Results The OXM analogues bind to an extracellular allosteric pocket

Additive stabilization of CCR6 by SQA and OXM U-shaped density is resolved in an extracellular pocket for both
SQA1 (Fig. 1a) is a SQA derivative that was initially discovered as a reconstructions and can be fitted by the two different OXM analo-
CXCR2 inhibitor and later found to also inhibit CCR6 activity19. gues, OXM1 and OXM2 (Fig. 2a, b). Binding of the OXM analogues to
Conversely, the OXM analogues OXM1 and OXM2 (Fig. 1a) were dis- CCR6 is mainly mediated by hydrophobic interactions with side
covered in a ligand-based virtual screen using SQA1 as the seed and chains from TMs 3, 4, 5, 6, and ECL2 (Figs. 2c, 3a–d). In particular,
subsequent structure-activity relationship (SAR) studies. All three the p-chlorophenyl group of OXM1 and the p-trifluoromethylphenyl
small molecules demonstrate robust inhibition of CCL20-mediated moiety of OXM2 are sandwiched between TMs 5 and 6 with their
chemotaxis in primary human T cells expressing endogenous CCR6 para-substitutions pointing towards the hydrophobic membrane,
(Fig. 1b). Significant CCR6 stabilization was observed in a thermal while making hydrophobic interactions with F1293.36, L2195.43,
shift assay (Fig. 1c) upon incubation with the small molecules, con- F2235.47, I2686.49, M2726.53, and L2756.56. On the other end of the bent
firming direct target engagement for the SQA and OXM molecules. molecules are hydrophobic substitutions on the amide nitrogen of
Surprisingly, the stabilization of CCR6 by analogues from the two the oxomorpholine core, i.e., an indane moiety in OXM1 and a
series is additive, as a significantly larger thermal shift was observed bicyclo[1.1.1]pentane in OXM2. These two groups bind in a hydro-
when analogues from both series were added to the receptor. Such phobic pocket capped by the C-terminal tip of ECL2 that connects to
additive stabilization was not dependent on the order of ligand TM5. Due to the compact size of this subpocket, the larger indane
addition between SQA1 and OXM analogues but was not observed group of OXM1 bends down towards the p-chlorophenyl group,
when CCR6 was co-incubated with the two different OXM molecules. forming an intramolecular T-shaped π stacking interaction which
These results suggest that despite the discovery of the OXM series likely further stabilizes the observed U-shaped conformation of
via a SQA-based virtual screen, the two series bind to different OXM1. Other than the hydrophobic interactions, several H-bonds
pockets on CCR6 and can bind independently and simultaneously to are mediated by the two amide groups of the OXM analogues with
the receptor. polar side chains such as N1303.37, K1223.29, T1824.60, and N2716.52.
Remarkably, a majority of the OXM binding pocket side chains are
Construct engineering for cryo-EM studies CCR6-specific (Supplementary Fig. 5). Consequently, OXM1 was
To reveal how the SQA and OXM analogues bind to CCR6, we con- found to be highly selective for CCR6 and is not active against other
ducted cryo-EM studies using an engineered CCR6 construct Nα7.1 CKRs (Supplementary Table 2).

Nature Communications | (2024)15:7574 2

Article https://doi.org/10.1038/s41467-024-52045-7

a b
Inhibition of CCL20-Mediated T Cell Chemotaxis

120 120 120

SQA1 100 100 100

% Inhibition
% Inhibition

% Inhibition
80 80 80

60 60 60

40 40 40

20 20 20
OXM1 0 0 0
-10 -9 -8 -7 -6 -5 -4 -10 -9 -8 -7 -6 -5 -4 -10 -9 -8 -7 -6 -5 -4
log [SQA1], M log [OXM1], M log [OXM2], M

OXM2

c d
WT CCR6 Thermostabilized CCR6
p < 0.0001 p < 0.0001
12 p < 0.0001 12 p = 0.0004
p < 0.0001
p < 0.0001
10 10 p < 0.0001
p = 0.0013
ΔTM (°C)

ΔTM (°C)
8 8 p = 0.1741
p = 0.3736
p = 0.5476 p = 0.4536
6 6

4 4

2 2
OXM1 + - + - + - OXM1 + - + - + -
OXM2 - + + - - + OXM2 - + + - - +
SQA1 - - - + + + SQA1 - - - + + +

Fig. 1 | SQA and OXM analogues inhibit CCL20-mediated activation of CCR6 and independent experiments. c,d Thermal shifts of c, WT and d, thermostabilized
display additive binding stabilization to the receptor. a Chemical structures of CCR6 Nα7.1 from Apo to liganded conditions as indicated. Data are presented as
SQA1, OXM1 and OXM2. b Inhibition of CCL20-mediated CCR6+ human T-cell mean ± s.d. from n = 4 (WT) and n = 6 (Nα7.1) independent experiments. p value
chemotaxis demonstrated by SQA1 (pIC50 = 6.7 ± 0.8), OXM1 (pIC50 = 6.8 ± 0.2), and presented in c and d is two-sided from unpaired t test, p < 0.05 was considered
OXM2 (pIC50 = 6.78 ± 0.11). Data are presented as mean ± s.d. from n = 3 significant. Source data are provided as a Source Data file.

Conformational restraints are required for OXM activity We rationalize the conformational restraints conferred by the
The observed bent pose of OXM1 in the ligand-bound structure cyclopropyl group as well as the T-shaped π stacking interactions
prompted us to study the solution conformation of this molecule between the indane and the p-chlorophenyl groups are critical factors
using Nuclear Magnetic Resonance (NMR) experiments to under- in stabilizing the observed U-shaped conformation of OXM1 in both
stand the energy landscape of this conformation. Rotating-frame bound and free forms. To test this hypothesis, we synthesized and
nuclear Overhauser Enhancement signals (ROEs) were collected analysed two additional OXM analogues, OXM3 which bears a gem-
using established methods29. Robust ROEs were obtained between dimethyl replacement at the cyclopropyl position, and OXM4 that
the indane C(sp3)-H (H-18) and the aromatic hydrogens from the p- lacks the para-Cl substitution on the phenyl ring (Fig. 3e). As expected,
chlorophenyl ring (Hs-3,4,6,7) (Supplementary Fig. 6), indicating extended conformations in solution are observed for OXM3 and
intramolecular interactions between the indane and the p-chlor- OXM4, and no long-range ROEs or nuclear Overhauser enhancement
ophenyl moieties are also maintained in solution. Additionally, we signals (NOEs) were observed (Fig. 3e, Supplementary Figs. 14–27,
performed proton and carbon assignments for OXM1 (Supplemen- Supplementary Table 3). Consequently, neither OXM3 nor OXM4 are
tary Figs. 7–13, Supplementary Table 3) in a compressed dimethyl active on CCR6. Finally, we also characterized the solution con-
sulfoxide (DMSO) gel30,31 which were used to calculate the residual formation of OXM2 used in the cryo-EM studies. Despite the absence
dipolar coupling (RDC) values of the proton-carbon and proton- of the indane moiety, a curved conformation that largely resembles the
nitrogen J-couplings. The RDC data was fit to a conformational bound conformation was revealed in its solution state (Fig. 3e, Sup-
ensemble that consisted of low energy structures generated by plementary Figs. 28–34, Supplementary Table 3). The curved con-
molecular dynamics simulations in a solvent-dependent manner (see formation of OXM2 in solution was further confirmed by long-range
methods). This exercise output several U-shaped conformations of NOEs observed between the bicyclo[1.1.1]pentane (Hs-21,24,25) and the
OXM1 as the best fits. Ninety-two percent of the low energy con- aromatic hydrogens from the p-trifluoromethylphenyl ring (Hs-
formers greatly resemble the bound pose observed in the cryo-EM 16,17,19,20) (Supplementary Fig. 35). In sum, the NMR studies support
structure (Fig. 3e), suggesting a low energy conformation for the the hypothesis that a low-energy curved conformation in the free state
bound state. is critical for OXM analogues to be active on CCR6.

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Article https://doi.org/10.1038/s41467-024-52045-7

a b

c d
EC OXM1 OXM2 IC SQA1
III III VII
IV IV
V VI VIII
V V
II
II
III

VI VI II I
VII I
VII I IV

e f
SQA1 OXM1 SQA1 OXM2

Fig. 2 | Overall structures of CCR6 in complex with OXM and SQA analogues. salmon; Nb, yellow; CCR6, aquamarine in a and deep teal in b. c Extracellular (EC)
a,b Cryo-EM map (left) and model (right) of CCR6 complex with anti-BRIL Fab and view of CCR6 bound to OXM analogues, OXM1 (left) and OXM2 (right).
anti-Fab Nb bound to a OXM1 (orange carbon spheres) and SQA1 (yellow carbon d Intracellular (IC) view of CCR6 bound to the SQA analogue, SQA1. Both c and
spheres), or b OXM2 (slate carbon spheres) and SQA1 (yellow carbon spheres). d use the same colour code as a and b. e SQA1, OXM1, and f SQA1, OXM2 fits into the
Protein is coloured by subunit as follows: Fab heavy chain, magenta; Fab light chain, cryo-EM density maps of the two structures from two viewing angles.

SQA1 binds to an intracellular pocket 8.47 position provides a pocket that allows the squaramide core to
In contrast to the OXM analogues, an intracellular binding site is bind. Indeed, a CCR6 mutant bearing G3208.47V completely abolished
revealed for the SQA molecule SQA1 (Figs. 2d, 4). The binding poses of SQA1 binding (Fig. 4c, d). Extensive polar interactions are observed
SQA1 from both structures are nearly identical. Therefore, the CCR6/ between the squaramide-picolinamide core of SQA1 and CCR6,
SQA1/OXM1 complex that led to a higher-resolution reconstruction including the salt bridges between D822.40 and the squaramide NHs, as
was used for analysis hereafter. Side chains interacting with SQA1 are well as H-bonds mediated by side chains of S792.37, T812.39, R1433.50, and
from TMs 1, 2, 3, 6, 7, and helix 8 (Fig. 4a, b). CCR6 has a glycine residue K3228.49 with the 3’-hydroxyl-picolinamide moiety. Consequently, SQA1
G3208.47 as the TM7-helix 8 linker. The absence of a side chain at the binding was abolished on mutants bearing single point mutations such

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Article https://doi.org/10.1038/s41467-024-52045-7

a b
P199ECL2
N186ECL2
K1223.29
T1824.60 III

L2155.39 Y1253.32
VI VII

IV L2756.56
N1303.37
V
N2716.52
M2726.53
L2195.43 F1293.36

F2235.47
c d
P199ECL2
N186ECL2

III
T1824.60
K1223.29
Y1253.32
L2155.39 VII
L2756.56
IV VI
N1303.37
N2716.52
V
L2195.43 M2726.53
F1293.36
F2235.47 I2686.49
e

Bound structure Conformer 1 = 51%

Conformer 1 = 61% Bound structure
Conformer 2 = 40% Conformer 1 = 100%
Conformer 2 = 31% Conformer 1 = 100%
Conformer 3 = 7%
Conformer 3 = 8%

OXM1 OXM2 OXM3 OXM4

IC50 158 nM IC50 166 nM IC50 > 10 PM IC50 > 10 PM

as D822.40N, S792.37A, T812.39L, R1433.50A, or K3228.49A (Fig. 4c, d). On the A co-crystal structure of CXCR2 in complex with SQA1 was
other hand, binding of the 1-(4-isopropyl furan-2-yl)propyl moiety is reported previously32. Comparison of the bound structures of SQA1
largely mediated by hydrophobic interactions with side chains from on CCR6 and CXCR2 reveal a highly similar mode adopted by the
TMs 1, 2, 7, and helix 8. Particularly, the 1-propyl binds in a hydrophobic molecule in binding to a mostly conserved pocket shared by the two
pocket formed by the side chains of L852.43, Y3167.53, and V671.53. CCR6 receptors (Fig. 4e, Supplementary Fig. 36). As observed in CCR6,
mutants bearing mutations in this pocket such as L852.43A or Y3167.53A extensive polar interactions are also seen in the CXCR2 structure,
no longer respond to SQA1 (Fig. 4c, d). mediated by the squaramide-picolinamide core with conserved side

Nature Communications | (2024)15:7574 5

Article https://doi.org/10.1038/s41467-024-52045-7

Fig. 3 | Structures of OXM analogues bound to CCR6 and comparison to solu- according to degrees of exposure, scaled by size. Light-blue halos around residues
tion conformations. a Binding pocket of OXM1 (orange carbon sticks). b Protein- indicate the degree of interaction with ligand, scaled by size. The dotted contour
ligand interaction diagram for OXM1 prepared with Molecular Operating Environ- around the ligand reflects steric room for methyl substitution. e Chemical struc-
ment (MOE)52. c Binding pocket of OXM2 (slate carbon sticks). d Protein-ligand tures of different OXM analogues and their solution conformations were deter-
interaction diagram for OXM2 prepared with MOE52. In a and c, CCR6 protein is mined by NMR using residual dipolar couplings (RDC)30. Three most populated
shown in aquamarine and deep teal ribbons, respectively. Side chains interacting conformations are illustrated by dark green, light green, and orange carbon sticks,
with the small molecules are shown as white carbon sticks. Hydrogen bonds are respectively. The bound conformations of OXM1 and OXM2 are shown as purple
highlighted by yellow dashed lines. In b and d, OXM pocket residues are presented carbon sticks in the overlays for comparison. The IC50 value of each OXM analogue
as follows: polar residues in pink, hydrophobic residues in green, acidic residues determined in the CCL20-mediated human CCR6+ T-cell chemotaxis assay is shown
with a red contour ring, basic residues with a blue contour ring. Green dotted under the corresponding chemical structure. Solution conformer library files of the
arrows indicate hydrogen and halogen bonds mediated by side chains that con- OXM analogues are provided as Source Data files.
tribute to ligand binding. Ligand atoms exposed to environment are shaded in blue

chains shared by the two receptors. The most striking difference in all utilizing a highly similar molecular mechanism to regulate their
the ligand binding pose is for the 1-(4-isopropyl furan-2-yl)propyl corresponding receptors.
moiety (Fig. 4f). In CXCR2, this moiety is pushed about 1 Å downward Located in the extracellular half of the 7TM, the OXM pocket is
towards the intracellular side compared to the bound position in about 9 Å away from the CCL20 binding site (Fig. 5g). Binding of the
CCR6. Movement of the 1-(4-isopropyl furan-2-yl)propyl moiety is OXM analogues, however, reshapes the orthosteric pocket of CCR6 to
likely caused by the difference of side chains at the 1.57 position, a conformation that is vastly different from that observed in the
which is F711.57 in CCR6 and is replaced by I731.57 in CXCR2. The ali- CCL20-bound form (Fig. 5b, c). Particularly, by interacting with N186
phatic isoleucine side chain reaches deeper into the 1-propyl binding from ECL2 and the C-terminal tip of the loop, OXM1 or OXM2 binding
pocket, resulting in a smaller subpocket in CXCR2 that pushes the brings the β-hairpin of ECL2 4 Å closer to the centre of the EC pocket, a
1-propyl group down. Furthermore, to avoid clashing into the iso- conformation that would clash with the N-terminus of CCL20 resolved
leucine side chain, the 4-isopropyl furan of SQA1 in CXCR2 under- in the active structure. Moreover, sandwiched in between TMs 5 and 6,
goes a rotation away from TM1 compared to the bound structure the para-substituted phenyl groups of OXM1 and OXM2 wedge TM5
in CCR6. 8 Å away from the centre of the 7TM bundle, which makes room to
accommodate the 4–5 Å inward movement of the entire TM6 asso-
SQA and OXM antagonize CCR6 via different allosteric ciated with receptor inactivation. ECL3 provides an important docking
mechanisms site for the N-loop and the 30s-loop of CCL20 during receptor acti-
The bound CCR6 structures are resolved in inactive conformations vation. The large movement of TM6 observed in the OXM analogue-
(Fig. 5a). A comparison of the SQA- and OXM-bound CCR6 structures bound structures results in an EC tip conformation of the helix that is
to the previous CCL20-bound structure reveals that the binding sites now no longer compatible with CCL20 docking. Altogether, OXM
of the OXM and SQA analogues do not overlap with the CCL20 binding binding stabilizes a distinct receptor conformation of the EC region
site (Fig. 5b), indicating both OXM and SQA molecules are allosteric from that utilized by CCL20, thereby allosterically inhibiting CCL20-
antagonists of CCR6. Compared to the active structure, a significant mediated CCR6 activation.
rearrangement of the CCR6 7TM bundle is observed in the inactive Positive cooperativity between orthosteric agonists and G protein
structures, which includes a 4–5 Å inward movement of the entire TM6 is a hallmark of GPCR activation. Cooperative stabilization of inactive
towards the centre of the TM bundle and a 4.6-Å outward movement at CCR2 between orthosteric and allosteric antagonists has also been
the IC tip of TM7 (Fig. 5c, d). These changes result in a closed IC pocket reported17. However, crosstalk between different allosteric ligands has
that is not compatible with G protein coupling. Furthermore, the not been well characterized to date. To understand the communica-
conserved functional motifs such as P2265.50-M1333.40-F2636.44, D1423.49- tion between the extra- and intracellular allosteric pockets discovered
R1433.50-Y1443.51, and N3127.49-P3137.50-x-x-Y3167.53 are now resolved in on CCR6, we examined binding of [3H]SQA1 to WT CCR6 with and
conformations resembling those observed in most other inactive class without OXM1. To our surprise, the presence of OXM1 did not enhance
A GPCR structures (Fig. 5e–g)33. In the inactive structures of CCR718 and the binding of [3H]SQA1 (Supplementary Fig. 37a, b), in contrast to the
CCR916 reported previously, a conserved N6.52-Y3.32-Q6.48 H-bond net- report that the presence of an orthosteric CCR2 antagonist increased
work was observed as a hallmark of the inactive state13. In the inactive binding of the intracellular antagonist, CCR2-RA17. We also studied the
CCR6, a similar H-bond network mediated by Y1253.32, Q2676.48, and impact of SQA1 on the binding of OXM1 using SPR with a stabilized
N2716.52 is also observed (Fig. 5h). Notably, both Y1253.32 and N2716.52 are CCR6. The small differences in association and dissociation rate con-
residues located in the OXM binding pocket, and the side chains of stants, ka and kd, and the equilibrium affinity constant, KD, in the
these residues mediate specific protein-ligand interactions via hydro- absence and presence of SQA1 are within experimental error (Sup-
phobic packing and a H-bond interaction with the oxomorpholine plementary Fig. 37c, d, Supplementary Table 4). Taken together, these
core, respectively. Therefore, binding of OXM analogues likely further binding data suggest minimal communication occurred between the
stabilizes the Y1253.32-Q2676.48-N2716.52 H-bond network that is impor- two allosteric pockets occupied by the OXM and SQA molecules, dif-
tant for the inactive state of CCR6. fering from the cooperativity observed between orthosteric agonists
Bound to an inactive IC pocket, SQA1 conformationally and and intracellular signalling transducers.
sterically hinders the coupling of the G protein (Fig. 5b). Furthermore,
SQA1 engages the side chains of R1433.50 and Y3167.53, which are part of Discussion
the D3.49R3.50Y3.51 and the N7.49P7.50xxY7.53 motifs involved in G protein Despite the significant advancement of cryo-EM and its tremendous
binding and CCR6 activation. In the SQA1 co-structures, R1433.50 and success in active GPCR structure determination35, limited examples
Y3167.53 are found in inactive conformations (Fig. 5f, g). Therefore, have been reported on the application of cryo-EM to small molecule-
SQA1 binding spatially competes with G protein binding and prevents bound inactive GPCR structure determination. The challenges are
conformational changes of the TMs and the critical activation motifs, twofold: the small size of the receptors alone, and the highly dynamic
which altogether inhibits CCL20-mediated CCR6 activation. In addi- nature of this protein family. Here we present a successful example of
tion to SQA1, a few other intracellular antagonists have been reported the application of cryo-EM in tackling inactive GPCR structure deter-
for other CKRs, such as CXCR1/215,34, CCR217, CCR718, and CCR916, likely mination for small molecule drug discovery. A BRIL fusion was

Nature Communications | (2024)15:7574 6

Article https://doi.org/10.1038/s41467-024-52045-7

a b
G3208.47 Y3167.53
F3238.50
V2556.36
VI VIII
A2526.33 V671.53
L852.43
T701.56
I2566.37
II Y3268.53
R1433.50 I

III K3228.49 Y741.60

F711.57
T812.39 S792.37 D822.40
c d
CPM

39

43

47
37

50

53

49
04
2.

2.

8.
2.

3.

7.

8.
2.

e f
Y7.53 L2.43
VII VII
VIII
II

F8.50
III II I D2.40

VIII
CCR6 T701.56/V721.56 F711.57/I731.57
CXCR2 (CCR6/CXCR2) I (CCR6/CXCR2)
Fig. 4 | SQA1 binds to an intracellular pocket. a Structure of SQA1 (yellow carbon shown by black circles and columns. CPM (counts per minute) of [3H]-SQA1 mea-
sticks) bound to CCR6 (aquamarine ribbon) from the higher-resolution CCR6/ sured at 1,000 nM ligand concentration from c normalized to WT are shown as
SQA1/OXM1 cryo-EM structure. Side chains interacting with SQA1 are shown by mean ± s.d. from n = 3 independent experiments (blue circle and columns).
white carbon sticks. Hydrogen bonds are highlighted by yellow dashed lines. e Overlay of CCR6 and CXCR2 (PDB ID 6LFL) inactive structures bound by SQA1.
b Protein-ligand interaction diagram for SQA1 prepared with MOE52. Definitions of f Major differences of the binding pockets in CCR6 and CXCR2 SQA1-bound
the schematic representation are same as that in Fig. 3b, d. c Saturation binding structures. Side chains of pocket residues are shown in sticks. In e and f, CXCR2 and
curves of [3H]-SQA1 on CCR6 WT and mutants as indicated. Binding of [3H]-SQA1 on SQA1 from 6LFL are shown by dark grey ribbon and dark grey sticks.
WT provided a KD of 250 ± 22 nM. Data shown as mean ± s.d. are from n = 3 inde- CCR6 structure is shown by the same cartoon representations as in a. Source data
pendent experiments. d Expression are confirmed for all mutants transiently are provided as a Source Data file.
expressed in HEK293 cells. Normalized surface expression of mutants to WT are

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a Active Inactive b

EC

IC

c IV d
III VI VII
V V VIII

EC VI IC
II III
I
II

VII I IV

e f g h
III N3127.49 P3137.50 IV II
P2265.50 D1423.49 Y1253.32
M1333.40 II
VI III
I
III VII
Y3167.53
V
V Y1443.51 VII
VI N2716.52 Q2676.48
V R1433.50 VIII
F2636.44 VII VI

Fig. 5 | Different mechanisms of allosteric antagonism of CCR6 by OXM same colour codes as in a. Blue lines depict pockets revealed by the structures.
and SQA. a Overall structures of the active CCR6 (magenta, left, PDB ID 6WWZ) in c–d, Structural overlays of the active (magenta) and inactive (aquamarine)
complex with its cognate chemokine ligand CCL20 (yellow) and a heterotrimeric G CCR6 structures from c EC and d IC views. e–h, Conformational changes of con-
protein coloured by subunit (Gαo, green, Gβ, cyan, and Gγ, light grey), and the served functional motifs, e P2265.50-M1333.40-F2636.44, f D1423.49-R1433.50-Y1443.51,
inactive CCR6 (aquamarine, right) in complex with SQA1 (yellow carbon spheres) g N3127.49-P3137.50-x-x-Y3167.53, and h Y1253.32-Q2676.48-N2716.52 revealed by structural
and OXM1 (orange carbon spheres). The light grey box highlights the location of overlay of active (magenta) and inactive (aquamarine) CCR6. In c–h significant TM
cell membranes. b Overlays of CCL20, OXM1, and SQA1 bound structures onto the movements and side chain conformational changes from active to inactive states
active (left) and the inactive (right) CCR6 structures. Structures are presented in the are highlighted by black arrows.

introduced to replace the ICL3 with a carefully designed junction to our work are also likely present in other CKRs. Therefore, the lessons
achieve optimal rigidity. The BRIL/Fab/Nb module is proven to be an described herein are useful for structure-based drug discovery of small
effective fiducial marker that allows robust particle alignment in cryo- molecules targeting CCR6 and other related CKRs.
EM28. Thermostabilizing mutations together with a ternary complex
strategy of incorporating two different allosteric antagonists led to a Methods
highly stabilized CCR6. As a result, two high-resolution structures in CCR6 thermostabilization and characterization
complex with analogues from the SQA and OXM series were deter- HEK293T cells (ATCC, Cat. No. CRL-3216) used for transient transfec-
mined in this study, providing the only known examples of inactive tion for thermostabilization experiments were cultured in DMEM +
CCR6 structures bound by small molecules. The structures reveal that GlutaMAX™-I with 4.5 g/L D-Glucose and Pyruvate (Gibco, Cat. No.
SQA and OXM utilize different allosteric mechanisms to inhibit CCR6 31966-021), supplemented with 10% (v/v) of foetal calf serum (FCS).
activation. Importantly, the allosteric pockets of CCR6 discovered in Transient transfections were carried out using GeneJuice (Merck) in

Nature Communications | (2024)15:7574 8

Article https://doi.org/10.1038/s41467-024-52045-7

accordance with manufacturer’s protocols. Full-length WT CCR6 used 10/300 (Cytiva) in buffer containing 50 mM HEPES pH 7.5, 150 mM
as a template for thermostabilization was cloned into pcDNA3.1-based NaCl, 0.003% LMNG, 0.0003% CHS, 50 μM of OXM1 or OXM2 and
plasmid vector to allow transient expression with C-terminal GFP-TEV- 50 μM of SQA1. The SEC peak fraction was collected and con-
myc-His×10 tags. Thermostability of transiently expressed CCR6 con- centrated to 1 mg/ml for cryo-EM grid preparation.
structs in HEK293T cells was assayed by solubilized [3H]-SQA1 binding.
Cells were solubilized in detergent (1% (w/v) n-Dodecyl-β-D-Mal- Cryo-EM sample preparation and data acquisition
topyranoside (DDM, Anatrace), n-Decyl-β-D-Maltopyranoside (DM, For both samples, cryo-EM grids were prepared with a Vitrobot Mark IV
Anatrace), or 2% (w/v) n-Nonyl-β-D-Glucopyranoside (NG, Anatrace) (Thermo Scientific) by applying 3 µl of sample to freshly glow-
with cholesteryl hemisuccinate (CHS, Sigma) added at 0.012% (w/v) as discharged Quantifoil Au R1.2/1.3 200-mesh grids and plunge freez-
appropriate) prior to incubation with radioligand and heating, and ing into liquid ethane. The grids were prepared using a blot force of +3,
unbound radioligand was subsequently removed using microscale a blot time of 2 s, and an absorption time of 20 s. The sample chamber
immobilized metal affinity chromatography (IMAC). Melting tem- was kept at 4 °C and 100% humidity.
perature (Tm) was defined as the temperature at which 50% of maximal The CCR6/SQA1/OXM1 dataset was collected using EPU v3.2
radioligand binding was measured. Stabilizing mutants were identified (Thermo Scientific) on a Titan Krios operating at 300 KeV and equip-
and combined using [3H]-SQA1 binding20,21. The thermostabilized ped with a Selectris energy filter (Thermo Scientific) at a slit width of
construct CCR6_Nα7.1 used in the cryo-EM study contains the follow- 10 eV and a Falcon 4 direct electron detector (Thermo Scientific) at a
ing seven mutations: L471.33A, L862.44A, V962.54A, S1403.47A, R159A, magnification of 215,000× for a magnified pixel size of 0.575 Å per
F2365.60A, G2957.32A (superscripts indicate Ballesteros–Weinstein pixel. Data were collected using a defocus range of −0.8 to −2.4 μm and
numbering for GPCRs22). The thermostabilized construct CCR6_Nβ10.2 a dose of 7.93 electrons per pixel per s for a total exposure time of
used in the crosstalk SPR study contains F731.59A, V2766.57A, and 1.76 s divided into 60 subframes for a total accumulated dose of 40
A2796.60L in addition to the seven mutations of Nα7.1. electrons per Å2.
The affinity of [3H]-SQA1 binding to CCR6 constructs was deter- The CCR6/SQA1/OXM2 dataset was collected using EPU
mined by saturation binding. Increasing concentrations of [3H]-SQA1 v2.12.1.2782REL (Thermo Scientific) on a Titan Krios G3i operating at
were incubated for 2 h at 25 °C with aliquots of HEK293T cells tran- 300 KeV and equipped with a Bioquantum imaging filter at a slit width
siently expressing CCR6 (or mock-transfected control) resuspended in of 20 eV and a K3 direct electron detector (Gatan) at a magnification of
50 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2 and protease inhibi- 130,000× for a magnified pixel size of 0.654 Å per pixel. Data were
tors. For the crosstalk study, the cells were prebound by 10 μM of collected using a defocus range of −0.4 to −1.8 μm and a dose of 18.27
OXM1 for 1 h before addition of [3H]-SQA1. The cell suspension with electrons per pixel per s for a total exposure time of 1.06 divided into
ligand mix was then chilled to 4 °C prior to receptor solubilization in 1% 45 subframes for a total accumulated dose of 45 electrons per Å2.
DDM and 0.012% CHS, and unbound radioligand was separated using
microscale IMAC. Non-specific binding to mock-transfected controls Cryo-EM data processing
was subtracted from binding to CCR6-containing samples prior to Data processing was carried out using cryoSPARC v3.3.1 (Structura
calculation of Kd in GraphPad Prism v9.5.1, using one site specific curve Biotechnology). For the CCR6/SQA1/OXM1 dataset, 12,852 movies
fitting. were imported into cryoSPARC and subjected to patch motion cor-
rection and patch CTF estimation. Exposures with CTF fits better than
Expression, membrane preparation, and protein purification 5 Å were selected (12,776 movies) and blob picking was used to select
The thermostabilized CCR6_Nα7.1-BRIL chimeric construct was and extract 5,332,464 particles. These particles were subjected to
cloned into a modified pFastBac1 vector (Invitrogen) with a hae- multiple rounds of 2D classification. Ab initio model generation was
magglutinin (HA) signal sequence followed by a cleavable FLAG- carried out with 476,361 particles and 3 classes. The best model was
10×His tags. Protein expression was done in Spodoptera frugiperda used as a template for heterogeneous refinement starting with
(Sf9) insect cells (ATCC) using the Bac-to-Bac Baculovirus Expression 2,168,721 particles using one good class model and 3 junk models.
System (Invitrogen). The insect cells were infected at a density of When most of the particles were sorted to the good class (487,232
2–3 × 106 cells/ml with high titre viral stock multiplicity of infection particles), the particles were re-extracted using the full box size of 520
(MOI) of 5.0. At 48 h post infection, cells were collected. Cell pellet pix. These particles (482,484 particles) were subjected to nonuniform
was resuspended by dounce homogenization in buffer containing refinement, resulting in a map of 3.3 Å.
10 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, 20 mM KCl, For the CCR6/SQA1/OXM2 dataset, 15,650 movies were subjected
nuclease (Pierce) and EDTA-free cOmpleteTM protease inhibitor to patch motion correction and patch CTF estimation. Exposures with
cocktail (Roche). Cell debris were removed by centrifugation at CTF fits better than 5 Å were selected (14,790 movies) and blob picking
1000×g for 20 min. The supernatant was then subjected to ultra- was used to select and extract 10,786,639 particles. These particles
centrifugation at 158,420×g for 30 min and the membrane pellet was were subjected to multiple rounds of 2D classification. Ab initio model
resuspended by dounce homogenization in 50 mM HEPES pH 7.5, generation for three classes was carried out with 129,004 particles
150 mM NaCl, 10% glycerol, and EDTA-free cOmpleteTM protease selected from 2D classification. The best model was used as a template
inhibitor cocktail (Roche). The resuspension was solubilized at 4 °C for heterogeneous refinement starting with 1,127,335 particles using
for 4 h with 2% (w/v) Lauryl Maltose Neopentyl Glycol (LMNG, Ana- one good class model and 3 junk models. After three rounds of het-
trace) and 0.2% (w/v) CHS in the presence of 2 mg/ml iodoacetamide erogeneous refinement, most of the particles sorted to the good class
and 50 μM of the compounds. The sample was clarified by ultra- (371,301 particles), the particles were re-extracted using the full box
centrifugation at 340,252×g for 30 min at 4 °C, and the supernatant size of 520 pix (368,745 particles). These particles were subjected to
was batch bound to TALON cobalt-affinity resin (Takara) overnight at nonuniform refinement, resulting in a map of 3.0 Å.
4 °C. The resin was washed with successively lower concentrations of
detergent and eluted in buffer containing 50 mM HEPES pH 7.5, Model building and refinement
500 mM NaCl, 0.003% LMNG, 0.0003% CHS, 300 mM imidazole, For each of the CCR6 structures presented, an atomic model predicted
50 μM OXM1 or OXM2, and 50 μM SQA1. The elution fractions were by AlphaFold236 (thermostabilized CCR6_Nα7.1ICL3BRIL fusion con-
concentrated and incubated with anti-BRIL Fab and Fab-Nb in a molar struct sequence) was rigid-body fit into the map density. Starting
ratio of 1:1.2:1.5 on ice for 1 h. The complex was then subjected to models of the anti-BRIL Fab and the hinge-binding nanobody were
size-exclusion chromatography (SEC) using a Superose 6 GL Increase obtained from PDB entry 6WW2. Following the docking of each model

Nature Communications | (2024)15:7574 9

Article https://doi.org/10.1038/s41467-024-52045-7

and ligands into the EM density map, iterative manual adjustment and were measured relative to the irradiated peak which was arbitrarily
real-space refinement was carried out using Coot v0.9.8.137. Subse- assigned a value of –100 and used a mixing time of 0.3 s for NOEs and
quently, global refinement and minimization in real space was per- 0.2 s for ROEs and an interpulse delay of 2 s. Calibration for this
formed using phenix.real_space_refine in Phenix v1.2038. The final experiment used a ROE/NOE intensity for the adjacent aromatic pro-
models were visually inspected for fit to the map, and Molprobity ton at a distance of 2.8 Å.
v4.439 was used for geometry validation. All structure figures were
generated by using PyMOL v2.5.440, UCSF Chimera v1.1641, and UCSF RDC NMR solution conformations
ChimeraX v1.442. The proton and carbon assignments for OXM1, OXM2, OXM3, and
OXM4 were completed using a combination of 1D proton, 2D COSY, 2D
Intrinsic fluorescence-based thermal shift assay HSQC, 2D HMBC and 1D carbon experiments using standard Bruker
Purified CCR6 ICL3-BRIL fusion protein (9 μM) was incubated with pulse sequences and a concentration of 7 mg/200 μl at 300 K. The
10 μM of the specified compounds in buffer containing 50 mM HEPES assignments are shown in Supplementary Figs. 7–11, 14–18, 21–25,
pH 7.5, 500 mM NaCl, and 0.003% LMNG/0.0003% CHS. After a 5 min 28–32. All NMR experiments were collected on a Bruker AVANCE III
incubation at 25 °C, the samples were loaded into capillary tubes for spectrometer operating at a 1H-Larmor frequency of 600.1 MHz and at
Tycho NT.6 instrument (NanoTemper). Tryptophan fluorescence at 150.90 MHz for 13C that was equipped with a 5 mm TCI helium cryop-
emission wavelengths of 350 nm and 330 nm were measured as the robe using Topspin 3.2. At a minimum, a 2 K × 128 data matrix was
sample temperature was ramped from 35 °C to 95 °C according to the acquired for each 2D spectrum using a minimum of 64 scans with a
preprogrammed instrument protocol. The ratio of the emission spectral width of 7200 Hz in the f2 dimension. The 2D data sets were
fluorescence was used to determine the Ti for the melting curves zero-filled to at least 512 data points. All spectra were assigned using
reported by the instrument software. MestreNova software version 11.0. The chemical shifts are referenced
to residual solvent (deuterated DMSO). Both 1JCH and 1JNH’s48 were
CPM-based thermal shift assay collected for the four OXM analogues using compressed DMSO-d6
CPM-based thermal shift assay was conducted for CCR6 WT by a dif- swollen poly-HEMA gels in New Era Enterprises compression devices.
ferential scanning fluorimetry assay adapted from a previous For the proton-carbon and proton-nitrogen J-couplings, two J-scaled
publication43 using UNit (Unchained Labs). In brief, 5 μM of protein was Bird (JSB) HC-HSQC spectra were collected for each compound (Sup-
incubated for 5 min at 25 °C with the presence of 10 μM of testing plementary Figs. 12, 13, 19, 20, 26, 27, 33, 34). The first JSB HC-HSQC
compound or 1% DMSO as control in buffer containing 50 mM HEPES was collected on the compound in DMSO-d6 alone (isotropic media)
pH 7.5, 500 mM NaCl, 0.05% DDM, and 2 μM of the thiol-specific while the second JSB HC-HSQC was collected on the compound in a
fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]- poly-HEMA gel (anisotropic media). The residual dipolar coupling
maleimide (CPM) dye. After incubation, the samples were heated from (RDC) values (1D) for proton-carbon and proton-nitrogen J-couplings
20 °C to 80 °C at a temperature ramping rate of 2 °C/min, during which were determined using the established formula: 1D = 1T – 1J30. Overall,
the CPM fluorescence (excitation 384 nm, emission 470 nm) was RDCs (1JCH and 1JNH) in a range of −8.9 to +13.3 Hz were measured for
recorded. Melting temperature (Tm) is defined as the temperature at each compound in DMSO-d6 as shown in Supplementary Table 3. The
which 50% of the protein was denatured based on Boltzmann sig- RDC data was fit to each member of a conformational ensemble that
moidal fitting using GraphPad Prism v9.5.1. consisted of 80–160 low energy structures that were generated in a
solvent dependent manner based on molecular dynamic simulations.
OXM discovery by SQA-based virtual screen Two files were generated for each compound: a linear conformer file
A field-based 3D similarity search as implemented in the Blaze module and a U- shaped conformer file. The RDC restraints were fit to each file
within the Cresset software (v2.0)44–46 was performed using the small independently and the best results (lowest Q) are reported. The RDC
molecule x-ray conformation47, of an SQA analogue as a query. Briefly, data was fit using Mestre MSpin/Stereofitter software version 2.3.2
the method uses electrostatics and shape of the query ligand to rapidly using the input files provided as a Source Data file49. With this software,
search large chemical collections for molecules with similar properties. the Cornilescu Q-factor (Q)50 was used to judge the goodness of the fit
The basis of electrostatic potential calculations is the polarizable XED for each conformation. The conformations with the best fit had
force field which allows an accurate description of the charges around Q = 0.005 for OXM1, Q = 0.097 for OXM2, Q = 0.001 for OXM3, and
atoms distribution. Each molecule is described as a Field Point using Q = 0.035 for OXM4. In addition, the experimental RDC’s correlated
the local extrema of four molecular fields: positive electrostatic, well with the back-calculated values as shown in Supplementary
negative electrostatic, surface (van der Waals interactions), and Table 3.
hydrophobic. Unlike traditional electrostatic field methods that place
single point partial charges at atom centres, this field redefines charges Conformer generation for RDC fitting experiments
towards a multipole electron distribution around the atom. The All structures were first subject to geometry optimization in Macro-
molecular fields developed from this representation are condensed to model (v10.5)51 using the OPLS3e forcefield and water as solvent, with
their local field points and used to rapidly search and score scaffolds default parameters and convergence criteria. A conformational search
with similar electronic properties for molecules in our internal data- was performed on each structure using the mixed MCMM/low-mode
base. The OXM analogue OXM1 with a similarity score of 0.74 was search as implemented in the Macromodel program with 1000 steps
identified from this approach. per rotatable bond and an energy window of 21 kJ mol−1 for retention of
conformers. The conformer files (OXM1, OXM2, OXM3, and OXM4) are
NMR experiments using ROE techniques provided in the Source Data file for each compound.
Rotating-frame nuclear Overhauser enhancements (ROEs) or nuclear
Overhauser effect (NOE) experiments were collected on a Bruker OXM binding measurement by SPR
AVANCE III spectrometer operating at a 1H-Larmor frequency of Binding affinity and kinetics of OXM1 were measured with the
600.1 MHz that was equipped with a 5 mm TCI helium cryoprobe using CCR6_Nα7.1 construct using a Surface Plasmon Resonance (SPR)-based
Topspin 3.2 at 300 K. The chemical shifts are referenced to residual binding assay on Biacore T200 instrument (Cytiva) equipped with a
solvent using two Bruker pulse sequences (selnogp & selrogp)29 in Biacore Series S NTA sensor chip (Cytiva cat no BR100532). The steps
CDCl3 or DMSO-d6. The absolute values for the ROE/NOE intensities of sensor preparation and protein capture were carried out at 25 °C

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and the binding interactions were measured at 10 °C. The CCR6 pro- total and non-specific binding conditions. After 3 h of incubation at
tein was initially captured via immobilized metal affinity chromato- room temperature, cells were washed 3 times with wash buffer
graphy (IMAC) followed by amine coupling onto an activated Ni-NTA (PBS + 1 mM CaCl2 + 0.1% BSA) before adding Microscint-20 (Perki-
sensor chip treated by a 1:1 mixture of 0.05 M n-hydroxysuccinimide nElmer) for tritium concentration measurement using a TriLux
(NHS) and 0.2 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide MicroBeta2 plate reader (PerkinElmer). Data was analysed with
(EDC). The experiments were carried out in 50 mM HEPES buffer (pH GraphPad Prism v9.5.1 software using a one-site saturation binding
7.5) containing 150 mM NaCl, 0.1% DDM, and 3% DMSO. OXM1 was equation.
injected in a concentration series consisting of 6-point, 3-fold dilutions
from 10 μM to 0.014 μM. The data were processed and analysed using CCR6 + T cell chemotaxis assay
Biacore Biaeval and Scrubber 2.0 software. The binding affinities and Human CD4+CCR6+CXCR3- T cells were isolated from human donor
on and off rates were calculated by globally fitting the sensorgrams to a leukopaks using EasySep™ Human Th17 Cell Enrichment Kit (Stem-
1:1 binding model. Cell Technologies, 18162). To obtain large quantities of cells,
CCR6 + T cells were activated with Dynabeads Human T-activator
Crosstalk study by SPR (Gibco, 11132D) at a density of 1 × 106 cell/ml in growth media
The crosstalk SPR experiments were performed using a Biacore T200 (RPMI1640 media with 10% serum, 4 ng/ml IL-2) with a 1:1.5 cell to
Instrument (Cytiva) equipped with a NiHC 200 M sensor chip (Xan- bead ratio. On day 4 post activation, Dynabeads were removed from
tec) at 25 °C. The run buffer for all experiments, unless otherwise the culture. The activated T cells were maintained at 1‒2 × 106 cells/ml
stated, was 50 mM HEPES pH 7.5, 500 mM NaCl, 1 mM MgCl2, 0.03% for 15 days by feeding fresh growth media when needed. The
DDM, 0.003% CHS, 5% DMSO. The CCR6_Nβ10.2 protein was CCR6 + T cell chemotaxis assay was carried out on day 12 to 15 post T
immobilized on the sensor chip by Ni-NTA pull down and amine cell activation using the 96 well ChemoTx® disposable chemotaxis
coupling. 0.5 M EDTA pH 8.0 was injected for 60 s at 5 µL/min fol- system (Neuroprobe 101-5) according to the manufacturer’s proto-
lowed by a 60 s injection of run buffer containing 0.5 mM NiCl2. The col. After one wash with assay buffer (1 × HBSS containing 20 mM
surface was activated with a 1:1 mixture of 0.1 M NHS and 0.5 M EDC HEPES and 0.25% BSA), cells were incubated with test compounds for
at a flow rate of 10 µL/min. The receptors were injected at a con- 30 min at room temperature prior to initiation of chemotaxis. CCL20
centration of 0.5 µM at 5 µL/min until a surface response of 2000 RU induced CCR6+ human T cell chemotaxis with an EC50 = 0.19 nM
was achieved. The surface was equilibrated for 10 h in run buffer (pEC50 = 9.7 ± 0.9, mean ± s.d., n = 5). CCL20 was used at 0.5 nM in all
before commencing experiments. OXM1 binding affinity was mea- measurements. For IC50 determination, the top and bottom of the
sured in a 5-point, 2-fold dilution series in single cycle kinetics format chemotaxis chamber contained the same concentration of com-
with a top concentration of 1 µM in run buffer with and without pound. DMSO was kept constant at 0.1% (v/v) in all wells. The final
100 nM SQA1. The flow rate of binding measurement was 50 µL/min. concentration of CCL20 (Peprotech, 300-29 A) in the bottom
The association phase was 120 s and dissociation phase was between chamber is 0.5 nM. The fully assembled chemotaxis plate was placed
1800 s. All experiments were run in triplicate. The data were pro- in a cell culture incubator at 37 °C, 5% CO2 for 1 h. After incubation,
cessed using Biacore T200 Evaluation Software (Version 2.0, Cytiva). the top filter was removed, followed by a quick freeze of the bottom
Responses were corrected for volume exclusion effects. Sensor- chamber at −80 °C for 1 h. The migrated cells in the bottom chamber
grams were reference and blank subtracted before fitting with 1:1 were stained with CyQUANT dye (Life Technologies, C7026) for cell
binding model which accounted for drift, bulk shift and mass number determination. Data was analysed with GraphPad Prism
transport. Sensorgram images were prepared using GraphPad Prism v9.5.1 Software using a non-linear regression analysis of dose
v9.5.1 software. response curves for IC50 determination.

Protein surface expression measurement GPCR β-arrestin assay panel

HEK293 cells (Charles River, CTN6199) transiently expressing CCR6 OXM1 was tested at 1 μM for agonistic and antagonistic activity on a
constructs or mock transfected control after 72 h post transfection panel of GPCRs in gpcrMAX℠ GPCR assay panel using PathHunter®
were detached using an enzyme-free dissociation buffer. Cells were β-arrestin enzyme complementation technology as described in
blocked with 0.1 mg/ml (1:40 dilution) mouse IgG (Sigma, Cat. No. I- Eurofins Discovery website (gpcrMAX- (discoverx.com)).
5381) prior to staining with 1.25μg/ml BV-421 labelled anti-human CCR6 Preparation of [3H]-SQA1 and synthesis of different OXM ana-
antibody (BD Biosciences, Cat. No. 562515, 1:30 dilution) or 1.25 μg/ml logues, OXM1, OXM2, OXM3, and OXM4 are provided as Supple-
BV-421 isotype (control antibody, BD Biosciences, Cat. No. 562438, mentary Methods in the Supplementary Information file.
1:312 dilution) for 1 h at 4 °C. After washing, cells were analysed on a BD
LSRFortessa cytometer (BD Biosciences) for cell surface expression of Reporting summary
CCR6 by determining the mean fluorescence intensity (MFI) of BV-421 Further information on research design is available in the Nature
using FlowJo™ Software (Becton, Dickinson and Company). Portfolio Reporting Summary linked to this article.

Radioligand saturation binding in HEK293 or CHO-K1 cells Data availability

Saturation binding experiments were performed on HEK293 cells The cryo-EM density maps for the complexes have been deposited into
(Charles River, CTN6199) transiently expressing human CCR6 48 h the Electron Microscopy Data Bank (EMDB) under accession codes
post transfection, or CHO-K1 cells (Eurofins, CYL3038) stably expres- EMD-46534 for CCR6/SQA1/OXM1 and EMD-46533 for CCR6/SQA1/
sing human CCR6. HEK293 cells were plated at 80,000/well in growth OXM2. The coordinates for the models have been deposited into the
media (DMEM + 10% Serum + 20 mM HEPES) in white/clear bottom Worldwide Protein Data Bank (wwPDB) under accession codes 9D3G
PDL coated 96 well plates (Corning); CHO-K1 cells were plated at for CCR6/SQA1/OXM1 and 9D3E for CCR6/SQA1/OXM2. Source data
50,000/well in white/clear bottom 96 well Isoplates (PerkinElmer) in are provided with this paper. Solution conformer library files of OXM1-
growth media a day before the experiments. On the day of assay, cells 4 in.sdf format and the corresponding input file to allow RDC data fit
in the assay well were treated with either 0.3–0.4% DMSO (for total using Mestre MSpin/Stereofitter software are provided as Source Data
binding) or 90–120 μM of unlabelled SQA1 (for non-specific binding), files with this paper. All the other data supporting the findings of this
followed by addition of a serial dilution of [3H]-SQA1 in growth media. study are provided with the article and the Supplementary Information
For each dose of [3H]-SQA1, triplicate reactions were prepared for both file. Source data are provided with this paper.

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References of structure-function relations in G protein-coupled receptors.

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Article https://doi.org/10.1038/s41467-024-52045-7

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tion. J. Chem. Inf. Model 46, 665–676 (2006). vised all structural and biophysical studies, determined the structures,
47. Groom, C. R., Bruno, I. J., Lightfoot, M. P. & Ward, S. C. The Cam- analysed the results and wrote the manuscript.
bridge structural database. Acta Crystallogr B Struct. Sci. Cryst. Eng.
Mater. 72, 171–179 (2016). Competing interests
48. Gil-Silva, L. F., Santamaria-Fernandez, R., Navarro-Vazquez, A. & Gil, All authors were either employees of Pfizer Inc. or were employees of
R. R. Collection of NMR scalar and residual dipolar couplings using Sosei Heptares at the time the work was performed.
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schrodinger.com> (2011).
52. Molecular Operating Environment (MOE), 2022.02 Chemical Peer review information Nature Communications thanks Elias Lolis, who
Computing Group ULC, 910-1010 Sherbrooke St. W., Montreal, QC co-reviewed with Manjula Ramu and the other, anonymous, reviewer(s)
H3A 2R7, Canada. (2023). for their contribution to the peer review of this work. A peer review file is
available.
Acknowledgements
Cryo-EM data for the CCR6/SQA1/OXM2 complex was collected at the Reprints and permissions information is available at
Cryo-EM Facility at MIT.nano. We thank K. Fennell and H. Zhao for http://www.nature.com/reprints
recombinant protein expression support; K. Schildknegt, T. Kenakin, and
E.J. Corey for consultancy support for the CCR6 antagonist project; C. Publisher’s note Springer Nature remains neutral with regard to
Tang, Q. Jin, and N. Fadeyi for synthesis support; X. Qiu and D. Hepworth, jurisdictional claims in published maps and institutional affiliations.
for project management support. We thank all members of Pfizer CCR6
research project team for suggestions and comments. Open Access This article is licensed under a Creative Commons
Attribution-NonCommercial-NoDerivatives 4.0 International License,
Author contributions which permits any non-commercial use, sharing, distribution and
D.J.W. performed protein purification, thermal shift experiments, pre- reproduction in any medium or format, as long as you give appropriate
pared cryo-EM grids, collected and processed cryo-EM data, deter- credit to the original author(s) and the source, provide a link to the
mined the structures, and assisted with preparing the manuscript; B.S.G. Creative Commons licence, and indicate if you modified the licensed
supervised discovery and design of the OXM analogues; K.A.F. per- material. You do not have permission under this licence to share adapted
formed NMR studies; W.L. designed and supervised all pharmacology material derived from this article or parts of it. The images or other third
experiments; J.A. performed radioligand binding and CCR6 mutagen- party material in this article are included in the article’s Creative
esis studies; M.E.S. supervised discovery of the SQA analogue; R.J.U. Commons licence, unless indicated otherwise in a credit line to the
performed ligand-based virtual screening and generated conformer material. If material is not included in the article’s Creative Commons
libraries for the OXM analogues; J.V. performed protein purification; licence and your intended use is not permitted by statutory regulation or
K.K.C. performed T cell chemotaxis experiments; R.D. performed protein exceeds the permitted use, you will need to obtain permission directly
surface expression measurements; P.V.S performed SPR experiments; from the copyright holder. To view a copy of this licence, visit http://
F.V. and R.K.F. performed T cell chemotaxis experiments; A.D., A.F., C.C., creativecommons.org/licenses/by-nc-nd/4.0/.
G.C., J.J.M., J.I.T., P.N., P.M., and V.L. performed synthesis; D.L., E.S., and
C.O. performed StaR engineering, characterization, and saturation © The Author(s) 2024
bindings; B.J.H. performed crosstalk SPR studies; G.S.M. purified CCR6
protein for crosstalk SPR studies. I.W.W. collected cryo-EM data of the

Nature Communications | (2024)15:7574 13