www.sciencemag.org/cgi/content/full/science.1241475/DC1

Supplementary Materials for

Structure of the CCR5 Chemokine Receptor–HIV Entry Inhibitor Maraviroc Complex

Qiuxiang Tan, Ya Zhu, Jian Li, Zhuxi Chen, Gye Won Han, Irina Kufareva, Tingting Li,

Limin Ma, Gustavo Fenalti, Jing Li, Wenru Zhang, Xin Xie, Huaiyu Yang, Hualiang Jiang, Vadim Cherezov, Hong Liu, Raymond C. Stevens, Qiang Zhao, Beili Wu*

*Corresponding author. E-mail: beiliwu@simm.ac.cn

Published 12 September 2013 on Science Express

DOI: 10.1126/science.1241475

This PDF file includes:

Materials and Methods Supplementary Text Figs. S1 to S8 Table S1 References (31–40)

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Materials and Methods:

Cloning and expression. The wild type human CCR5 cDNA was synthesized with sequence

optimization for insect cell expression by DNA2.0 and then cloned into a modified pFastBac1

vector (Invitrogen), which contained an HA signal sequence at the N-terminus prior to the

receptor sequence, and a PreScission protease site followed by a FLAG tag and a 10xHis tag at

the C-terminus. Residues Cys224-Asn226 in the ICL3 of CCR5 were deleted using standard

QuickChange PCR, and Met1-Glu54 of rubredoxin (31) were inserted between Arg223 and

Glu227 instead. Thirty-three amino acids were truncated from the C-terminus (residues Phe320-

Leu352) to improve protein thermo-stability. The CCR5 gene was further modified by

introducing four rationally designed mutations, Cys581.60

Tyr, Gly1634.60

Asn, Ala2336.33

Asp and

Lys303Glu, using standard QuickChange PCR. The mutation Ala2336.33

Asp was designed to

selectively stabilize the inactive state, while the other three mutations were rationally designed to

improve general stability and homogeneity of the detergent-solubilized CCR5 (figure S1 and

figure S2). High-titer recombinant baculovirus (>109 viral particles per ml) was generated using

the Bac-to-Bac Baculovirus Expression System (Invitrogen) and used to infect Sf9 insect cells at

a density of 2-3 × 106 cells per ml at MOI (multiplicity of infection) of 5. Culture flasks were

shaken at 27 °C for 48 h, then cells were harvested by centrifugation and stored at -80 °C until

use.

Purification of Sf9-expressed CCR5 construct for crystallization. Cells were lysed by

thawing frozen cell pellets in a hypotonic buffer containing 10 mM HEPES, pH 7.5, 10 mM

MgCl2, 20 mM KCl and EDTA-free complete protease inhibitor cocktail (Roche), and cell

membranes were disrupted by dounce homogenization. Extensive washing of the membranes

was performed by repeated dounce homogenization and centrifugation in the same hypotonic

buffer (one more time), followed by a high osmotic buffer supplemented with 1 M NaCl (three

times), and then the hypotonic buffer (one more time) to remove the high concentration of NaCl.

Purified membranes were resuspended in 10 mM HEPES, pH 7.5, 30% (v/v) glycerol, 10 mM

MgCl2, 20 mM KCl and EDTA-free complete protease inhibitor cocktail, flash-frozen with

liquid nitrogen, and stored at -80 °C until use.

Purified membranes were thawed on ice and resuspended into a buffer containing 200

μM Maraviroc [synthesized as previously described (32)], 2 mg/ml iodoacetamide and EDTA-

free complete protease inhibitor cocktail, then incubated at 4 °C for 1 h prior to solubilization.

The membranes were solubilized in 50 mM HEPES, pH 7.5, 150 mM NaCl, 0.5% (w/v) n-

dodecyl-D-maltopyranoside (DDM, Anatrace), 0.1% (w/v) cholesteryl hemisuccinate (CHS,

Sigma), and 100 μM Maraviroc for 3 h at 4 °C. The supernatant was isolated by centrifugation at

160,000 × g for 35 min, supplemented with 5 mM buffered imidazole, pH 7.5, and incubated

with TALON Superflow Metal Affinity Resin (Clontech, 1 ml of resin per 500 ml of original

culture volume was used) overnight at 4 °C. After binding, the resin was poured into a column

and washed with ten column volumes of 25 mM HEPES, pH 7.5, 150 mM NaCl, 10% (v/v)

glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS, 30 mM imidazole and 100 μM Maraviroc,

3

followed by ten column volumes of 25 mM HEPES, pH 7.5, 150 mM NaCl, 10% (v/v) glycerol,

0.05% (w/v) DDM, 0.01% (w/v) CHS, 5 mM ATP (Sigma), 10 mM MgCl2 and 100 μM

Maraviroc, and five column volumes of 25 mM HEPES, pH 7.5, 150 mM NaCl, 10% (v/v)

glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS and 100 μM Maraviroc. The CCR5 protein was

then eluted by five column volumes of 25 mM HEPES, pH 7.5, 150 mM NaCl, 10% (v/v)

glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS, 300 mM imidazole and 300 μM Maraviroc, and

exchanged into 25 mM HEPES, pH 7.5, 150 mM NaCl, 10% (v/v) glycerol, 0.05% (w/v) DDM,

0.01% (w/v) CHS, 40 mM imidazole and 300 μM Maraviroc using a PD MiniTrap G-25 column

(GE Healthcare). The protein was then supplemented with Maraviroc to a final concentration of

1 mM, and treated with home-made His-tagged PreScission protease overnight to remove the C-

terminal FLAG and His tags. The protein was further purified by removing protease and cleaved

C-terminal fragment using Ni-NTA superflow resin (Qiagen) incubation at 4 °C for 1 h. The tag-

cleaved protein was collected as the Ni-NTA column flow-through, and concentrated to 40-50

mg/ml using a 100 kDa molecular weight cut-off Vivaspin concentrator (Sartorius Stedim

Biotech).

Lipidic cubic phase crystallization. Protein samples of CCR5 in complex with Maraviroc were

reconstituted into lipidic cubic phase (LCP) by mixing with molten lipid using a mechanical

syringe mixer (33). The protein-LCP mixture contained 40% (w/w) receptor solution, 54% (w/w)

monoolein, and 6% (w/w) cholesterol. Crystallization trials were performed in 96-well glass

sandwich plates (Shanghai FAstal BioTech, Inc.) using a Mosquito LCP robot (TTP Labtech),

dispensing 45 nL of protein-laden LCP and 800 nL of precipitant solution per well. Protein

reconstitution in LCP and crystallization trials were performed at room temperature (19-22 °C).

Plates were incubated and imaged at 20 °C using an automated incubator/imager (RockImager

1000, Formulatrix). Initial crystal hits were found from a precipitant condition containing 100

mM Tris, pH 8.5, 30% (v/v) PEG400, 100 mM NaCl. After extensive optimization, diffraction

quality crystals were obtained from 100 mM HEPES, pH 7.0, 32-38% (v/v) PEG400, 100-200

mM NaCl, 1 mM Maraviroc. Crystals usually grew to a maximum size of 100 μm × 20 μm × 15

μm in two weeks, and were harvested directly from the LCP matrix using MiTeGen

micromounts and flash frozen in liquid nitrogen.

Data collection, structure solution and refinement. Crystallographic data were collected on

the 23ID-D beamline (GM/CA CAT) of the Advanced Photon Source at the Argonne National

Laboratory using a 10 μm collimated minibeam. Among the several hundred crystal samples

screened, most crystals diffracted to 2.5-3.5 Å resolution when exposed to 1 s of unattenuated

beam using 1° oscillation. Data collection was limited to 6-13 frames per crystal, due to the fast

onset of radiation damage in the microcrystals. A 95.6% complete data set at 2.70 Å resolution

of CCR5/Maraviroc from 10 crystals were integrated, scaled and merged using HKL2000 (34).

Initial phase information was obtained by molecular replacement (MR) with the program Phaser

(35) using a polyalanine model of the 7 TM α-helices of the CXCR4/IT1t structure (PDB ID:

3ODU) and rubredoxin structure (PDB ID: 1IRO) as search models. The correct MR solution

(TFZ=6.9) contained two CCR5-rubredoxin molecules in the asymmetric unit, related by a

4

pseudo-translational symmetry. Refinement was performed with REFMAC5 (36) and

autoBUSTER (37) followed by manual examination and rebuilding of the refined coordinates in

the program COOT (38) using both |2Fo| - |Fc| and |Fo| - |Fc| maps, as well as omit maps. The

final model includes 295 residues (19 to 313) of the 352 residues of CCR5 and residues 1 to 54

of rubredoxin. The remaining N- and C-terminal residues are disordered and were not refined.

The two CCR5-rubredoxin protomers are very similar (Cα RMSD = 0.46 Å for all residues).

Strong electron density was observed for one metal ion bound to the four-Cysteine motif in each

rubredoxin fusion protein, which was determined to be Zinc by X-ray fluorescence scans (figure

S4).

Modeling of CCR5/R5-V3 and CXCR4/X4-V3 complexes. Full atom docking of the isolated

peptide of 20 amino acids from the V3 loop of a T-tropic isolate (HXBc2 strain, residues 303-

322, figure S6B) was performed in the Internal Coordinate Mechanics (ICM) molecular

modeling package (39). The receptor pocket from the CXCR4/CVX15 crystal structure (PDB ID:

3OE0) was converted into a set of soft potential grid maps representing van der Waals,

electrostatic, hydrophobic, and hydrogen bonding potentials. Prior to map construction, flexible

side-chains of residues Lys25, Glu26, Phe29, Glu179-Arg183, Ile185, Asp187, Phe189, and

Asp193 were assigned artificial occupancy of 0 to eliminate their contribution to the nature of

the maps. The peptide was built as a polypeptide chain with ideal covalent geometry and then

assigned multiple starting conformations observed in NMR and X-ray structures of V3 loops in

context of the full gp120 protein or as isolated peptides. Chemical field maps were built from the

CVX15 peptide in its crystallographic position. The initial conformational stack for the peptides

was generated by placing the multiple peptide conformations tip-down in the CXCR4 binding

pocket and restraining peptide termini to each other in four possible ways, so that the peptide

maintains an approximate β-hairpin fold. Biased probability Monte Carlo sampling of the peptide

was performed with the multiple starting conformations simultaneously optimizing the internal

energy of the peptide, its grid map energy, chemical field fit and the terminal restraints. The best

scoring conformation was selected and optimized in the presence of the full-atom model of the

receptor including the side-chains omitted at the sampling stage. The model of R5-V3 loop (YU2

strain, residues 303-322, figure S6B) was constructed by homology with the CXCR4-peptide

complex. The side-chains and the backbone of the peptide, as well as the side-chains in the

CCR5 pocket, were optimized in the full-atom model with soft restraints imposed on the peptide

backbone to keep it in place during the optimization.

Ligand binding assay and functional assay. Chemokine binding assay: Full-length cDNAs

encoding human CCR5 and CCR5 mutants were cloned into the pSNAP vector (Cisbio

Bioassays) in-frame with SNAP-tag attached at the N terminus. HEK293 cells were transfected

with plasmids encoding CCR5 or CCR5 mutants by electroporation and seeded onto 35 mm

dishes at a density of 1 × 105 cells/dish and cultured overnight. After removal of the cell culture

medium, 1 ml of Tag-lite labeling medium (Cisbio Bioassays) containing 100 nM SNAP-Lumi4-

Tb was added to the dish and incubated for 1 h at 37 °C under 5% CO2. After removal of SNAP-

Lumi4-Tb, cells were washed three times with Tag-lite labeling medium. Cells were then

5

detached and resuspended in Tag-lite labeling medium. A density of 4000 cells per well was

used to carry out binding assays in suspension in 384-well plates. Then cells were incubated with

various concentrations of d2 fluorescent probe-labeled MIP-1α (CCL3, Cisbio Bioassays) for 20

h at room temperature before signal detection. To detect non-specific binding signals, 100 µM of

Maraviroc was added to the assay. HTRF signal was detected using the Envision (PerkinElmer)

multi-plate reader according to the manufacturer’s instructions.

Calcium Flux assay: HEK293 cells were co-transfected with plasmids encoding

CCR5/CCR5 mutants and G 16 by electroporation. After transfection, cells were seeded onto a

96-well plate at a density of 3 × 104 cells per well and cultured overnight. Cells were then

incubated with 2 μM Fluo-4 AM in HBSS (5.4 mM KCl, 300 μM Na2HPO4, 400 μM KH2PO4,

4.2 mM NaHCO3, 1.3 mM CaCl2, 500 μM MgCl2, 600 μM MgSO4, 137 mM NaCl, 5.6 mM D-

glucose and 250 μM sulfinpyrazone, pH 7.4) at 37 °C for 45 min. After a thorough wash, 50 μL

of HBSS was added. After incubation at room temperature for 10 min, 25 μL of RANTES at

various concentrations were dispensed into the well using a FlexStation III microplate reader

(Molecular Devices), and intracellular calcium change was recorded at an excitation wavelength

of 485 nm and an emission wavelength of 525 nm.

Protein stability assays. Protein thermostability was tested by a microscale fluorescent thermal

stability assay using the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-

coumarinyl)phenyl]maleimide (CPM), which reacts with the native cysteines embedded in the

protein interior as a sensor for the overall integrity of the folded state. The CPM dye (Invitrogen)

was dissolved in DMSO at 4 mg/ml and stored at -80 °C. Prior to use the dye stock was diluted

1:20 in buffer 20 mM HEPES, pH 7.5, 150 mM NaCl, 10% Glycerol, 0.05% (w/v) DDM and

0.01% (w/v) CHS. The tested protein (~ 5 μg) was diluted in the same buffer to a final volume of

120 μl. 1 μl of the diluted dye was added and thoroughly mixed with the protein. The reaction

mixture was incubated at room temperature for 15 min, and subsequently transferred to a sub-

micro quartz fluorometer cuvette (Starna Cells, Inc.) and heated in a controlled way with a ramp

rate of 1 °C/min over a temperature range from 10-90 °C in a Cary Eclipse Fluorescence

Spectrophotometer (Agilent Technologies). The excitation wavelength was set at 387 nm, while

the emission wavelength was 463 nm. Protein homogeneity was also tested by analytical size-

exclusion chromatography (aSEC) using a 1260 Infinity HPLC system (Agilent Technologies).

Supplementary Text:

Models of CCR5/R5-V3 and CXCR4/X4-V3 complexes. Binding of co-receptor to HIV-1

gp120 is mediated by the V3 loop and a co-receptor binding site in and around the bridging sheet

of gp120. Simply switching the V3 loop between an R5 and an X4 virus could alter co-receptor

choice, which indicates that the receptor selectivity is determined by the V3 loop (25). Within

the V3 loop, mutagenesis studies have demonstrated that some basic residues in both R5 and X4

viruses play important roles on co-receptor binding, including Arg298, Arg308, Arg315 and

Arg327. Substitutions of two conserved hydrophobic residues, Phe317 and Ile323, also

dramatically reduced co-receptor binding. Additionally, residues Asn302, Asp325 and Ile326 are

6

important to R5 tropism, while two basic residues Arg306 and Lys322 are critical for X4 tropism

(29, 30, 40). Given similarities between the gp120 V3 loop and the CVX15 peptide ligand in the

CXCR4 structure, we proposed that the V3 loop could also penetrate the ligand binding pocket

of its co-receptor (5). The ability of a panel of CXCR4 mutants to mediate gp120 binding was

tested, and the importance of residues in CXCR4 N-terminus (Tyr7, Tyr10) and ECLs (Asp187,

Asp193 and Glu268) for the HIV-1 co-receptor activity was confirmed, as well as the essential

role of some negatively charged residues in the 7TM helical bundle (Asp972.63

, Asp1714.60

,

Asp2626.58

and Glu2887.39

) in gp120 binding (28). In CCR5, the N-terminal sulfated tyrosine

residues, Tyr10 and Tyr14, have been shown to be crucial for gp120 binding by structural and

mutagenesis studies (24, 25). It was also reported that the binding to CCR5 of gp120 was

sensitive to mutations of some uncharged residues of CCR5, such as Trp862.60

, Trp94, Tyr1083.32

,

Thr177, Trp2486.48

and Tyr2516.51

etc. (11) (figure S7). These previous data provide clues for

modeling the CCR5/R5-V3 and CXCR4/X4-V3 complexes.

The putatively orthosteric CXCR4 inhibitor, CVX15, is similar to the V3 loop on

sequence and structural conformation, which makes the CXCR4/CVX15 crystal structure a good

candidate for docking X4-V3 peptide. In contrast, Maraviroc in the CCR5 structure is an

allosteric inhibitor, which may induce conformation change that impedes gp120 binding. To

avoid the bias caused by the different nature of the ligands, the CCR5/Maraviroc structure is

compared with the CXCR4/CVX15 structure and a CCR5 model based on the CXCR4/CVX15

structure reported by J. Garcia-Perez et al. (11). The CCR5 structure has a more open ligand

binding pocket, which is closer to the one in the CXCR4/CVX15 structure compared to the

CXCR4/IT1t structure (Cα RMSD within the 7TM bundle, N-terminus and ECL2 between

CCR5/Maraviroc and CXCR4/CVX15 is 1.64 Å). The Garcia-Perez’s CCR5 model is also very

similar to the CCR5 crystal structure (Cα RMSD within the 7TM bundle, N-terminus and ECL2

is 1.39 Å). These comparisons indicate that the conformational bias induced by different ligand

is minimal and the CCR5 crystal structure is appropriate for R5-V3 peptide docking.

In the models of the CCR5/R5-V3 and CXCR4/X4-V3 complexes, the V3 peptides form

similar β-hairpin structures, interacting with the co-receptor’s ECL2 and transmembrane helices

(figure S8A). The V3 peptide backbone of residues 318-320 engages in hydrogen bond

interactions with the backbone of ECL2, and Pro313 in the GPGR motif at the V3 tip makes

hydrophobic contacts with helices I and II of the co-receptor. Since the N-terminal fragments of

CCR5 and CXCR4 are absent in the crystal structures, interactions involving co-receptor N-

termini are not discussed here. The most striking differences in the binding modes between these

two models are the following: i) in the CXCR4/X4-V3 model, Asp1935.32

forms salt-bridges with

V3 Arg306 and Lys322. By contrast, substitutions to Gln1885.32

in CCR5, and Ser306, Glu322 in

R5-V3, respectively, weaken the interactions of CCR5/X4-V3 and CXCR4/R5-V3. In the

CCR5/R5-V3 model, V3 Ser306 hydrogen bonds with Tyr1875.31

in CCR5 (Asn1925.31

in

CXCR4), which may clash into the side chain of Arg306 in X4-V3, decreasing the ability of

CCR5 binding to the X4-V3 (figure S8, B, E, H and K). This difference is consistent with

previous studies that have shown that residues 306 and 322 are important for determining co-

7

receptor preference (29) (figure S6B). ii) In the CXCR4/X4-V3 model, Arg308 engages in

interactions with Asp2626.58

and Glu2777.28

of CXCR4, which are substituted by Asn258

6.58 and

Ser2727.28

in CCR5, leading to a weaker binding between CCR5 and the X4-V3. Additionally,

Arg308 of the X4-V3 may clash with the side chain of Lys1915.35

in CCR5 (Val1965.35

in

CXCR4), which thereby weakens the binding between the X4-V3 and CCR5 (figure S8, C, F, I

and L). iii) Gln310-Arg311 at the V3 tip are commonly absent in most HIV-1 isolates, except

some X4-tropic viruses (figure S6B). Removal of these two residues generates a dual-tropic

HIV-1 strain derived from the prototypical X4 isolate HXBc2, which indicates that the deletion

of residues 310-311 are necessary for efficient CCR5 binding and utilization (30). In the

CXCR4/X4-V3 model, Arg311 makes a salt bridge with Asp972.63

in CXCR4, while this residue

is substituted by bulkier Tyr892.63

in CCR5, which may sterically clash with the Arg311 in X4-

V3, but accommodates well Ile309 in R5-V3. In addition, Arg311 forms another salt bridge with

Asp187 from CXCR4’s ECL2, and substitution to Ser179 in CCR5 weakens the CCR5/X4-V3

interaction (figure S8, D, G, J and M). Taken together, the models of CCR5/R5-V3 and

CXCR4/X4-V3 complexes suggest that the different charge distributions in the co-receptor

ligand binding pockets and steric hindrances caused by residue substitutions in their side chains

may be major determinants of HIV-1 co-receptor selectivity. However, given the consideration

that the ligands in the two co-receptor crystal structures used in modeling, Maraviroc in CCR5

and CVX15 in CXCR4, are different in size and inhibitory mechanism, we cannot rule out that

the conformational differences of the co-receptors between the two complexes have arisen from

the nature of the different ligands. Additional structural information, such as complexes of both

CCR5 and CXCR4 with the same or similar allosteric or orthosteric ligands, especially peptides,

are needed to further improve the accuracy of the models.

8

Supplementary Figures:

9

Supplementary Figure S1. Stability assays of CCR5 mutants. (A) aSEC of CCR5-fusion

proteins showing the CCR5-rubredoxin fusion protein (red trace) has higher

monomer:aggregation ratio, which indicates better protein homogeneity, compared to wild-type

CCR5 protein (CCR5-WT) and two other fusion proteins, CCR5-T4 lysozyme (CCR5-T4L) and

CCR5-apocytochrome b562RIL (CCR5-Bril). (B) CPM ramping assay of CCR5-fusion proteins

showing the CCR5-rubredoxin fusion protein (red trace) has higher melting temperature (Tm),

which indicates better protein thermostability, compared to CCR5-WT, CCR5-T4L and CCR5-

Bril. (C, D) aSEC of CCR5 mutants testing the effects of mutations and mutation combinations

on protein homogeneity. C, the raw data show that the CCR5 mutant containing all the four

mutations (red trace) has the highest protein yield. D, the normalized data show that the mutation

combination (red trace) improves protein homogeneity. All of these CCR5 mutants contain

rubredoxin fusion. (E, F) aSEC of CCR5 mutants testing the effects of C-terminal truncations on

protein homogeneity. E, the raw data show that the CCR5 protein with the truncation of 33 C-

terminal residues (blue trace) has the highest protein yield. F, the normalized data show the

truncation of 33 C-terminal residues (blue trace) improves protein homogeneity. All of these C-

terminus truncated mutants contain rubredoxin fusion and all the four mutations. (G) CPM

ramping assay of CCR5 mutants testing the effects of mutations and mutation combinations on

protein thermostability. The Tm of the CCR5 mutant containing all the four mutations (red trace)

is higher than the other mutants, indicating that this mutation combination improves protein

thermostability. (H) CPM ramping assay of the crystallized CCR5 variant (CCR5v) in an apo

state (black) or complemented with different ligands, Maraviroc (red), Ancriviroc (green) and

TAK-220 (blue). The Tm of the CCR5 variant complemented with either ligand is higher than

the apo protein, which demonstrates that these ligands bind to CCR5 and directly influence the

behavior of the variant receptor and improve the thermostability.

10

Supplementary Figure S2. Calcium flux assays and chemokine binding assays. (A-F) Calcium

flux assays in HEK293 cells transfected with different CCR5 mutants, showing the effects of the

single mutations, Cys581.60

Tyr (B), Gly1634.60

Asn (C), Ala2336.33

Asp (D) and Lys303Glu (E),

on signaling, compared to the wild type CCR5 (A) and the empty vector pcDNA3.0 (F). Only the

mutation Ala2336.33

Asp, which stabilizes the inactive conformation of the receptor, abolishes the

signaling. (G-I) MIP-1α (CCL3) binding assays of the wild type CCR5 (G), the crystallized

CCR5 variant (H, four mutations, rubredoxin fusion and C-terminal truncation), and another

engineered CCR5 protein without the Ala2336.33

Asp mutation (I, three mutations, rubredoxin

fusion and C-terminal truncation). The Ala2336.33

Asp mutation affects CCR5’s binding affinity

to CCL3, but the other three mutations, rubredoxin fusion and truncating 33 C-terminal residues

have little effect.

11

Supplementary Figure S3. CCR5-rubredoxin crystals and crystal packing. (A-C) Crystal

packing of CCR5-rubredoxin crystals. CCR5 is colored blue, and rubredoxin is shown in grey.

As in all crystals obtained by crystallization in LCP, CCR5-rubredoxin molecules are arranged in

a type I packing with layers of two dimensional crystals stacked through hydrophilic interactions

mediated by rubredoxins. The unit cell is shown as a red box. A, crystal packing in ac plane.

CCR5 molecules make abundant hydrophobic contacts with their two neighbors related by a

pseudo-translational symmetry forming arrays of receptors arranged within each layer in

alternating orientations and interacting with each other through rubredoxins. B, crystal packing

in bc plane. Contacts between receptors and rubredoxins involve ECL2 and the intracellular

surface of CCR5. C, crystal packing in ab plane. There are no direct interactions between

rubredoxins. (D) Crystals of CCR5-rubredoxin. Average crystals grew to 100 μm × 20 μm × 15

μm before harvesting.

12

13

Supplementary Figure S4. X-ray fluorescence scan of CCR5-rubredoxin/Maraviroc crystal.

Excitation X-ray energy is 12 keV. (A) Background signal by scanning an empty MiTeGen

micromount. The highlighted peak at 6.4 keV, which is from Fe, comes as a scattering signal

from surrounding metal objects. (B) Signal in a wide fluorescence range by scanning the CCR5-

rubredoxin/Maraviroc crystal. The arrow points to peaks at 8.6 keV and 9.6 keV from Zn bound

to rubredoxin. (C) Zoomed in view of signal from B in the range 7.0-10.0 keV.

14

Supplementary Figure S5. Electron density of Maraviroc. Maraviroc is shown in stick

representation with orange carbons. Electron density is contoured at 1.0σ from an |2Fo| - |Fc|

map.

15

16

Supplementary Figure S6. Sequence alignments of HIV co-receptors and gp120 V3 loops. (A)

Sequence alignment between CCR5 and CXCR4. The α-helices are labeled as red and blue

cartoons. The most conserved residues in the 7TM helices of class A GPCRs, Asn1.50

, Asp2.50

,

Arg3.50

, Trp4.50

, Pro5.50

, Pro6.50

and Pro7.50

, are highlighted by black boxes. Colors represent

properties of the residues: blue, negatively charged; magenta, positively charged; red,

hydrophobic; green, polar. (B) Sequence alignment between gp120 V3 loops of X4-, R5X4- and

R5-tropic HIV-1 viruses. Residues 306, Gln310-Arg311, GPGR motif and residue 322 are

highlighted by black boxes. Colors represent similarities of residues in different V3 sequences:

red, identity; green, strongly similar; blue, weakly similar; black, different.

17

Supplementary figure S7. Top view of CCR5 ligand binding pocket, showing the key residues

of CCR5 involved in gp120 binding. CCR5 is shown in grey molecular surface representation.

The key residues, Tyr371.39

, Trp862.60

, Trp94, Leu1043.28

, Tyr1083.32

, Phe1093.33

, Phe1123.36

,

Thr177, Ile1985.42

, Trp2486.48

, Tyr2516.51

, Leu2556.55

and Glu2837.39

, are colored in blue.

18

Supplementary Figure S8. Models of CCR5/R5-V3 and CXCR4/X4-V3 complexes. (A)

Overall view of CCR5/R5-V3 and CXCR4/X4-V3 complexes. CCR5 is shown in blue, and

CXCR4 is in green. R5-V3 is colored in magenta, and X4-V3 is yellow. Pro313 in the GPGR

motif at the V3 tip is shown in stick presentation. (B-D) Proposed interactions between CXCR4

and X4-V3. The CXCR4/X4-V3 model was built based on the CXCR4/CVX15 crystal structure

(PDB ID: 3OE0). CXCR4 is colored in white, and X4-V3 is in yellow. Residues of CXCR4 are

shown in stick presentation with green carbons. Residues of X4-V3 are sticks with yellow

carbons. Salt bridges are represented as red dashed lines. (E-G) Proposed interactions between

CCR5 and R5-V3. CCR5 is colored in white, and R5-V3 is in magenta. Residues of CCR5 are

shown in stick presentation with blue carbons. Residues of R5-V3 are sticks with magenta

carbons. A hydrogen bond is represented as a blue dashed line. (H-J) Hypothesized interactions

between CCR5 and X4-V3, showing weaker interactions compared to the CXCR4/X4-V3

complex and steric hindrances caused by residue substitutions. (K-M) Hypothesized interactions

between CXCR4 and R5-V3, showing weaker interactions compared to the CXCR4/X4-V3

complex.

19

Supplementary Table:

Supplementary Table S1. Data collection and refinement statistics. Highest resolution shell is

shown in parentheses.

Data collection

Number of crystals used for data processing 10

Space group P212121

Cell dimensions a, b, c (Å) 72.92, 103.52, 137.47

Number of reflections processed 165,261

Number of unique reflections 27,674

Resolution (Å) 50.0-2.7 (2.8-2.7)

Rsym 14.4 (85.3)

Mean I/σ(I) 14.5 (2.0)

Completeness (%) 95.6 (92.3)

Redundancy 6.0 (6.2)

Refinement

Resolution (Å) 50.0-2.7

Number of reflections (test set) 27,630 (1,416)

Rwork / Rfree 0.217 / 0.263

Number of atoms

Protein

Ligand

Zn2+

A

2,742

37

1

B

2,744

37

1

Lipids and waters 91 111

Overall B values (Å2)

CCR5

Rubredoxin

Ligand

Zn2+

Lipids and waters

A

73.9

66.8

53.4

61.1

92.0

B

72.9

68.7

54.7

72.9

90.4

RMSD

Bond lengths (Å)

Bond angles (°)

0.010

0.99

Ramachandran plot statistics (%)*

Favored regions

Allowed regions

Disallowed regions

97.0

3.0

0.0

*As defined in MolProbity.

20

References and Notes

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21

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