www.sciencemag.org/content/347/6226/1113/suppl/DC1

Supplementary Materials for

Structural basis for chemokine recognition and activation of a viral

G protein–coupled receptor

John S. Burg, Jessica R. Ingram, A. J. Venkatakrishnan, Kevin M. Jude, Abhiram

Dukkipati, Evan N. Feinberg, Alessandro Angelini, Deepa Waghray, Ron O. Dror, Hidde

L. Ploegh, K. Christopher Garcia*

*Corresponding author. E-mail: kcgarcia@stanford.edu

Published 6 March 2015, Science 347, 1113 (2015)

DOI: 10.1126/science.aaa5026

This PDF file includes:

Materials and Methods

Figs. S1 to S10

Tables S1 and S2

Full Reference List

!

Materials and methods:

Protein engineering

CX3CL1 (amino acid residues 1 – 77) was expressed with its natural signal peptide under

control of the CMV promoter in the vector pVLAD6 (31). The C-terminal mucin-like

stalk was replaced with a flexible linker (SGSGSAAA) followed by a 3C protease site

(LEVLFQGP) and human Fc. US28 was expressed from pVLAD6 as a wild-type protein

with no mutations or truncations, except addition of an N-terminal Flag tag to facilitate

purification (Fig. S5A). For co-crystallization with the nanobody Nb7, US28 was

truncated by 10 amino acids at the N-terminus and 44 amino acids at the C-terminus

(US28∆N∆C) (Fig. S5B); N-terminal truncation by 10 amino acids does not diminish

chemokine binding or signaling (32). For crystallization with US28∆N∆C and Nb7, the

CX3CL1 Asn-linked glycosylation site at position 9 was changed to alanine

(CX3CL1N9A). Nanobody 7 (Nb7) was expressed as a fusion protein with an amino-

terminal maltose-binding protein (MBP) and a carboxy-terminal hexahistidine tag in the

pMal vector (New England Biolabs). A 3C protease site was inserted between MBP and

Nb7.

Protein purification

Nb7 was expressed in the periplasm of E. coli. Transformed cells were grown in Terrific

Broth supplemented with 100 µg/ml carbenicillin and 0.1% (w/v) glucose to a density of

OD600 = 0.8. Expression was induced overnight at 25˚C by addition of 1 mM IPTG. Cells

were harvested by centrifugation then resuspended in a solution of 0.2 M Tris-Cl pH 8.0,

0.5 mM EDTA and 0.5 M sucrose. After stirring for 1 h the cell suspension was diluted

4-fold with water and stirred for an additional 2 h, followed by centrifugation at 7000 x g

for 1 h. The supernatant was adjusted to 20 mM imidazole and passed through a nickel-

NTA column. The nickel column was washed with a solution of 10 mM HEPES pH 7.4

and 150 mM NaCl (HBS) supplemented with 20 mM imidazole. Bound MBP-3C-Nb7-

6xHis was eluted with 200 mM imidazole in HBS. After incubating overnight with 3C

protease, the protein solution was precipitated with 33% (w/v) ammonium sulfate,

isolated by centrifugation and resuspended in HBS. The re-dissolved protein was passed

!

through a 0.2 µm filter, desalted on a sepharose G25 column and passed over amylose

resin (New England Biolabs) to remove residual MBP. Analytical gel filtration confirmed

size monodispersity.

CX3CL1-3C-Fc was expressed in HEK293s GnTI- cells with BacMam baculovirus

transduction (31). Baculovirus was added to cells at a density of 4 x 106 cells per ml and

culture bottles were shaken for 72 h at 37 ˚C with 5% CO2. Cells were removed by

centrifugation and the culture supernatant was filtered and then stirred at 4 ˚C overnight

with 10 ml protein A agarose (Sigma). The protein A agarose was then collected by

filtration and washed HBS.

US28 was expressed in HEK293s GnTI- cells using BacMam baculovirus transduction

(31). Baculovirus was added to cells at a density of 4 x 106 cells per ml and culture

bottles were shaken for 48 h at 37 ˚C with 5% CO2. After harvesting, cells were washed

with a solution of 10 mM HEPES pH 7.4, 150 mM NaCl and 5 mM EDTA and stored at -

20˚C. Cell pellets were thawed and lysed with a Dounce homogenizer in a solution

comprised of 20 mM Tris-Cl pH 8.0, 5 mM EDTA and 2 mg ml-1 iodoacetamide. The

lysate was centrifuged at 40,000 x g for 1 h, and the membrane pellet was resuspended

and stirred for 2 h at 4 ˚C in a solubilization buffer consisting of 1% (w/v)

dodecylmaltoside (DDM), 0.2% (w/v) cholesteryl hemisuccinate (CHS), 10 mM HEPES

pH 7.4, 150 mM NaCl, 20% (v/v) glycerol and 2 mg ml-1 iodoacetamide. After

centrifugation, protein A-immobilized CX3CL1-Fc was added to the solubilized lysate

and mixed by rotating overnight at 4 ˚C. The protein A resin was collected by filtration,

washed with HBS and incubated at room temperature with 3C protease for 1 h to release

the US28-CX3CL1 complex. We used anti-Flag M1 affinity resin to further purify the

complex and remove excess CX3CL1. The complex was eluted from the anti-Flag M1

affinity resin with 0.2 mg ml-1 Flag peptide and 5 mM EDTA. In the case of the wild-type

US28/CX3CL1 complex, size exclusion chromatography was conducted at this point

using a buffer containing HBS, 0.02% (w/v) DDM and 0.004% (w/v) CHS. The wild-

type US28/CX3CL1 complex was concentrated to 20 mg ml-1 for crystal trials.

!

To form the US28∆N∆C/CX3CL1N9A/Nb7 ternary complex, Flag-purified

US28∆N∆C/CX3CL1N9A was mixed with Nb7 at a molar ratio of 1:1.9. The protein

surface was then modified by reductive methylation with formaldehyde and

dimethylaminoborane (33). US28∆N∆C/CX3CL1N9A/Nb7 was isolated and desalted by

size exclusion chromatography, and then concentrated to 34 mg ml-1 for crystal trials. An

overview of the US28∆N∆C/CX3CL1N9A/Nb7 purification is shown in Figure S10A.

Alpaca immunization

A three-year-old female alpaca (Lama pacos) was maintained in pasture, and immunized

following a protocol authorized by the Camelid Immunogenics (Belchertown, MA) and

MIT IACUC committees. The alpaca was immunized by subcutaneous injection of a 1:1

mixture of Imject alum (Thermo Scientific) and 200 mg of recombinant US28/CX3CL1

reconstituted in phospholipid vesicles. After a total of five injections (200 mg each)

spaced two weeks apart, 50 mL of blood was harvested by venipuncture and collected

into heparinized tubes.

Alpaca nanobody yeast display library construction

Peripheral blood lymphocytes (PBLs) were isolated from total blood by Ficoll-Paque

gradients (Ficoll-Paque Plus, GE Healthcare). Total RNA was isolated from ~107 fresh

PBLs using the RNeasy Plus Mini Kit (Qiagen), following the manufacturer’s guidelines.

First strand cDNA synthesis was performed using SuperScript III reverse transcriptase

(Life Technologies) and a combination of oligo dT, random hexamer or the

immunoglobulin-specific primers AlCH2 and AlCH2.2 (34). Nanobody sequences were

then amplified by PCR in two steps for the generation of a yeast display library, using the

following primer sets: For round 1: JRI_162(5’-

GTCGGCTAGCGCTCAGKTGCAGCTCGTGGAGWCNGG-3’ and JRI_163(5’-

TTTTGTTCGGATCCCCGGGAGGAGACGGTGACCTG-3’); for round 2: JRI_164

(5’-GGAGGCGGTAGCGGAGGCGGAGGGTCGGCTAGC-3’ and JRI_165 (5’-

GTCCTCTTCAGAAATAAGCTTTTGTTCGGAT-3’). PCR products were ethanol

precipitated, and 4 mg of resulting PCR product and 1 mg linearized pCTCON vector

(35) were co-transformed into electrocompetent EBY100 yeast (Invitrogen). Following

!

transformation, cells were recovered in 1 L synthetic defined medium (SDCAA) at 30 ˚C.

Ten-fold serial dilutions were plated onto SDCAA, grown for 3 days at 30 ˚C, and

colonies were counted to determine library size. The resulting library was determined to

contain 1 x 109 transformants, although the actual diversity was limited by the initial

number of lymphocytes to approximately 1 x 107.

Nanobody selection by yeast surface display

Yeast surface display nanobody selections were conducted as previously described (35-

37). For the first round of selection, 1 x 108 yeast displaying the nanobody library were

washed with PBE buffer (phosphate buffered saline with 0.5#mM EDTA and 0.5% BSA)

supplemented with 0.02% (w/v) dodecylmaltoside detergent (PBED buffer) and stained

with 1 µM purified Flag-US28/CX3CL1 for 1#h at 4#˚C. The yeast were then washed with

PBED buffer and stained with Alexa-647 conjugated anti-DYKDDDDK antibody (Cell

Signaling) for 15#min at 4 ˚C. Yeast were washed again with PBED buffer and

magnetically labeled with 250#µl anti-Alexa-647 microbeads (Miltenyi) in PBED buffer

for 15#min at 4 ˚C. Yeast were washed a final time and labeled yeast were isolated by

magnetic selection with an LS column (Miltenyi) pre-equilibrated with PBED buffer.

Magnetically sorted yeast were resuspended in SDCAA medium and cultured at 30 ˚C.

For the second round of selection, two-color FACS was performed. 1 x 107 induced yeast

were washed with PBED and stained with 200 nM Flag-US28/CX3CL1 for 1#h at 4#˚C.

The yeast were then washed with PBED buffer and stained with Alexa-647 conjugated

anti-DYKDDDDK and Alex-488 conjugated anti-Myc antibodies (Cell Signaling) for

15#min at 4#˚C. Yeast were washed again with PBED buffer. The Alexa-647 positive

yeast with the highest Alexa-647 / Alexa-488 ratios were purified using a FACS Jazz cell

sorter (BD Biosciences). Post-sorted yeast were resuspended in SDCAA medium and

cultured at 30 ˚C. Nanobody cDNA was prepared from the Round 2 library, transformed

into E. coli and sequenced.

!

Crystallization and data collection

Crystallization was performed by the in meso method (38). The wild-type US28/CX3CL1

complex was reconstituted in 10:1 monovaccenin:cholesterol (Nu-Chek Prep and Sigma,

respectively) in a ratio of 1:1 (w/w) protein solution:lipid. The ternary complex of

US28∆N∆C, CX3CL1N9A and Nb7 was reconstituted in 10:1 monoolein:cholesterol

(Sigma) in a ratio of 1:1.5 (w/w) protein solution:lipid. In both cases, it was necessary to

add 10 – 20% (v/v) 5 M NaCl to the protein/lipid mixture to achieve a clear cubic phase

(39). The lipidic cubic phase was dispensed onto glass sandwich plates in 30 nl drops,

overlaid with 700 nl precipitant solution using a Gryphon LCP robot (Art Robbins

Instruments) and incubated at 20 ˚C. Crystals of the wild-type US28/CX3CL1 complex

grew in a precipitant solution containing 35 – 45% (v/v) PEG 300, 100 mM HEPES pH

7.4, and 100 mM ammonium phosphate. Crystals of the US28∆N∆C/CX3CL1N9A/Nb7

complex grew in 32% (v/v) PEG 300, 100 mM sodium 2-(N-morpholino)ethanesulfonate

pH 6.3, 100 mM sodium succinate pH 7.0, and 1% (v/v) polypropylene glycol P 400.

Crystals were observed after 24 h and grew to full size in 3 – 5 d. Crystals were then

harvested with mesh grid loops (MiTeGen) and stored under liquid nitrogen. Typical

crystals are shown in Figure S10B.

Diffraction data were collected at the Advanced Photon Source GM/CA beamlines 23-

ID-B and 23-ID-D using a beam size of 10 µm. The wild-type US28/CX3CL1 crystals

were sensitive to radiation damage, causing decay of diffraction quality during exposure.

Wedges of 5 – 20 degrees were collected and merged from 26 crystals using XDS (40),

with the maximum resolution of each wedge ranging from 3.6 – 9 Å. Crystals of the

US28∆N∆C/CX3CL1N9A/Nb7 ternary complex diffracted to 2.9 Å, and it was possible

to collect complete datasets from single crystals. Data from two

US28∆N∆C/CX3CL1N9A/Nb7 crystals were processed and merged with HKL2000 (41).

The structure of the wild-type US28/CX3CL1 complex was solved by molecular

replacement using CCR5 (PDB ID: 4MBS) (2) and CX3CL1 (PDB IDs: 1F2L and

3ONA) (42, 43) as search models. We improved the model by manual building in Coot

(44) and reciprocal space refinement in Phenix (45), but it was not initially possible to

!

improve the model to an Rfree better than 37% due to the low resolution and the distantly

related molecular replacement model. We subsequently solved the 2.9 Å structure of

US28∆N∆C/CX3CL1N9A/Nb7 by molecular replacement using our partially refined 3.8

Å model and a nanobody (PDB ID: 4B41) as search models. Refinement was conducted

by manual building in Coot and reciprocal space refinement in Phenix and BUSTER (46)

with translation-liberation-screw motion (TLS) groups (47). Fourteen TLS groups were

chosen based on the inflection point of the plot of crystallographic residual versus

number of TLS groups as calculated using the TLSMD web server (48). The refined 2.9

Å structure of US28∆N∆C/CX3CL1N9A/Nb7 was used to re-solve the 3.8 Å wild-type

US28/CX3CL1 dataset. Simulated annealing composite omit maps were used to ensure

that the 3.8 Å structure was not unduly biased by the 2.9 Å molecular replacement model.

The overall quality of the models was assessed with MolProbity (49). Contacts were

analyzed using CONTACT (50) and PISA (51) with manual inspection. Figures were

prepared in PyMOL (52). Software used in this project was installed and configured by

SBGrid (53). Representative electron density maps are shown in Figure S7. A list of

contacts between CX3CL1 and US28 in the 2.9 Å structure is presented in Table S1. The

statistics for data collection and refinement are presented in Table S2.

System setup for molecular dynamics simulations

Simulations of the human cytomegalovirus-encoded chemokine receptor US28 (US28

receptor) were based on the 2.9 Å crystal structure described here. Coordinates were

prepared by first removing the nanobody, as well as non-protein molecules, from the

structure. Prime (Schrödinger) was used within Maestro (Schrödinger) to model atoms

missing from the crystal structure, including portions of certain side chains (specifically

Arg62ICL1, Arg63ICL1, Gln65ICL1, Gln912.62, Arg137ICL2, and Lys297Helix8) and of the

extracellular loop between TM2 and TM3. Histidine residues were simulated as the

neutral Nε tautomer. Other titratable residues were simulated in their dominant

protonation state at pH 7, except for Asp792.50 and Asp1283.49, which were neutral in

some simulations and charged in others, as described below.

!

The resulting prepared US28 receptor structure was then aligned to the Orientations of

Proteins in Membranes (OPM) (54) entry for the CXCR4 chemokine receptor using

PyMOL (52). The US28 receptor was modified with disulfide bridges and inserted into a

hydrated, equilibrated palmitoyloleoylphosphatidylcholine bilayer using the CHARMM-

GUI interface (55-58). Sodium and chloride ions were added to neutralize the system,

and extra NaCl was added to reach a final concentration of approximately 150 mM. In

each simulation without CX3CL1, the simulation box initially measured approximately

82#×#82#×#99#Å3, and contained one US28 receptor, approximately 160

palmitoyloleoylphosphatidylcholine molecules, approximately 34 sodium ions,

approximately 38 chloride ions, and approximately 11,500 water molecules, for a total of

approximately 61,000 atoms. In each simulation with CX3CL1, the simulation box

measured approximately 82#×#82#×#106#Å3, and contained one US28 receptor,

approximately 160 palmitoyloleoylphosphatidylcholine molecules, approximately 36

sodium ions, approximately 45 chloride ions, and approximately 13,000 water molecules,

for a total of approximately 66,000 atoms.

Molecular dynamics simulation protocol

Each simulation was performed on two GPUs using the CUDA version of PMEMD

(Particle Mesh Ewald Molecular Dynamics) Amber14 (59-61) with 2.5 fs time steps.

Simulations were heated from 0 K to 100 K in the NVT ensemble and then from 100 K to

310 K in the NPT ensemble, both with 10.0 kcal#mol−1#Å−2 harmonic restraints applied to

the protein and to the lipids. Initial velocities were assigned randomly at the first heating

step with Langevin dynamics. Subsequently, simulations were equilibrated in the NPT

ensemble at 310 K (controlled with a Langevin thermostat) with pressure of 1 bar

(controlled with anisotropic Berendsen weak-coupling barostat), with harmonic restraints

on all protein atoms tapered off 1.0 kcal#mol−1#Å−2 in a stepwise fashion every 2.0 ns

starting at 5.0 kcal#mol−1#Å−2 to 0.0 kcal#mol−1#Å−2, for a total of 12.0 ns of equilibration.

Bond lengths to hydrogen atoms were constrained using SHAKE. Production simulations

used a Langevin thermostat for temperature coupling and a Monte Carlo barostat for

pressure coupling, and were initiated from the final snapshot of the corresponding

equilibration simulation. Nonbonded interactions were cutoff at 9.0 Å, and long-range

!

electrostatic interactions were computed using the particle mesh Ewald (PME) method,

with an Ewald coefficient � of approximately 0.31 Å−1 and B-spline interpolation of

order 4. The FFT grid size was 84#×#84#×#100 for simulations without CX3CL1 and 84 ×

84 × 108 for simulations with CX3CL1. We performed a total of eight simulations. In

two of these, of lengths 0.78! μs! and! 0.92! μs, CX3CL1 was present and bound to the

receptor in its crystallographic pose. In these two simulations, Asp792.50! was! neutral!and!Asp1283.49!was!charged.!In!the!other!six!simulations,!CX3CL1 was removed. Two

of these simulations, of lengths 2.01!μs!and!1.68!μs, had both Asp792.50!and!Asp1283.49!charged.! Two! others,! of! lengths! 1.68! μs! and! 1.78! μs,! had! both Asp792.50! and!Asp1283.49! neutral.! The! final! two,! of! lengths! 1.93! μs! and! 1.63! μs,! had! Asp792.50!neutral!and!Asp1283.49!charged.!

Force field parameters

We used the CHARMM36 parameter set for protein molecules, lipid molecules, and salt

ions, and the CHARMM TIP3P model for water; protein parameters incorporated CMAP

terms (62-64). These parameters were assigned using the ParmEd implementation of

Chamber, provided with AmberTools14.

Parameters for the pyroglutamate (PCA) residue at the N-terminus of CX3CL1

were based on results from the ParamChem web server, version 0.9.7.1 (65). A

PCA–His dipeptide was submitted to ParamChem, and the results were used to

determine parameters for PCA and to modify charges near the PCA–His peptide

bond.

Sequence analysis

Analysis of amino acid conservation was performed using GPCR Motif Searcher

(GMoS). GMoS searches for conserved sequence motifs in a sequence alignment of Class

A GPCRs obtained from GPCRDB (66).

!

Table S1. US28-CX3CL1 contacts. Interface contacts are listed by residue number, atom name and interaction type (VDW: van der Waals, H-bond: hydrogen bond). Interactions are further specified by indicating whether they are between main chain (mc) or side chain (sc) atoms by listing the US28 residue contact followed by the CX3CL1 residue.

"SITE 1" (US28 + CX3CL1 residues 8-62)

US28 CX3CL1 distance (Å) type interaction ASP15 CG SER17 CB 3.55 VDW sc-sc ASP15 OD1 SER17 CB 3.68 VDW sc-sc ASP15 OD2 LYS18 N 3.10 H-bond sc-mc ASP15 OD1 LYS18 N 3.11 H-bond sc-mc ASP15 OD1 LYS18 CA 3.40 VDW sc-mc ASP15 CG LYS18 N 3.48 VDW sc-mc ASP15 OD2 LYS18 CB 3.52 VDW sc-sc ASP15 OD2 LYS18 CG 3.55 VDW sc-sc ASP15 CG LYS18 CB 3.70 VDW sc-sc GLU18 OE2 MET15 O 2.98 H-bond sc-mc GLU18 O PHE49 CE1 3.76 VDW mc-sc ALA20 CB LYS14 O 3.52 VDW sc-mc ALA20 CB LYS14 C 3.67 VDW sc-mc ALA20 O PHE49 CD1 3.74 VDW mc-sc ALA20 O PHE49 CA 3.79 VDW mc-mc ALA20 O CYS50 N 3.21 H-bond mc-mc THR21 CG2 LEU48 O 3.71 VDW sc-mc PRO22 CB ILE10 CG2 3.72 VDW sc-sc PRO22 CA THR11 O 3.69 VDW mc-mc PRO22 CD LEU48 O 3.40 VDW sc-mc PRO22 CD LEU48 C 3.72 VDW sc-mc PRO22 CG LEU48 CB 3.72 VDW sc-sc CYS23 O ALA9 O 3.59 VDW mc-mc CYS23 N THR11 O 3.15 H-bond mc-mc CYS23 O THR11 N 3.37 H-bond mc-mc CYS23 O THR11 OG1 3.38 H-bond mc-sc CYS23 CB THR11 OG1 3.67 VDW sc-sc VAL24 C ALA9 O 3.42 VDW mc-mc VAL24 CG1 ILE10 CD1 3.68 VDW sc-sc PHE25 N ALA9 O 2.72 H-bond mc-mc PHE25 CD2 ALA9 O 3.52 VDW sc-mc PHE25 CA ALA9 O 3.71 VDW mc-mc PHE25 N ALA9 C 3.74 VDW mc-mc PHE25 CB ALA9 O 3.79 VDW sc-mc

!

PHE25 CE2 THR11 CG2 3.6 VDW sc-sc THR26 N ALA9 CB 3.76 VDW mc-sc LEU29 CD2 CYS8 O 3.71 VDW sc-mc LYS169 O ALA32 CB 3.45 VDW mc-sc GLN172 NE2 GLN31 OE1 3.47 H-bond sc-sc GLN172 OE1 ALA32 N 3.37 H-bond sc-mc GLN172 OE1 ALA32 CB 3.46 VDW sc-sc GLN172 OE1 ALA32 CA 3.73 VDW sc-mc GLN172 OE1 SER33 N 3.14 H-bond sc-mc GLN172 OE1 SER33 CB 3.78 VDW sc-sc MET174 CG ALA32 O 3.65 VDW sc-mc MET174 CB SER33 CA 3.80 VDW sc-mc MET174 CE GLY35 CA 3.71 VDW sc-mc TYR177 CD2 CYS34 O 3.69 VDW sc-mc TYR177 N GLY35 O 3.18 H-bond mc-mc ASP178 OD2 GLY35 O 3.74 VDW sc-mc ASP178 N GLY35 O 3.76 VDW mc-mc ASP178 OD2 LYS36 CA 3.41 VDW sc-mc ASP178 OD2 LYS36 C 3.58 VDW sc-mc ASP178 OD2 ARG37 N 2.81 H-bond sc-mc ASP178 OD2 ARG37 CB 3.66 VDW sc-sc GLU266 OE1 THR11 OG1 3.72 VDW sc-sc LYS270 NZ CYS8 O 3.90 VDW sc-mc

"SITE 2" (US28 + CX3CL1 residues 1-7)

US28 CX3CL1 distance (Å) type interaction TYR40 OH HIS2 NE2 2.89 H-bond sc-sc TRP89 NE1 HIS2 CE1 3.61 VDW sc-sc TRP89 CE2 HIS2 CE1 3.64 VDW sc-sc TRP89 CE3 VAL5 CG1 3.53 VDW sc-sc TRP89 CE3 VAL5 CG2 3.68 VDW sc-sc TRP89 CZ3 VAL5 CG2 3.77 VDW sc-sc TYR92 O LYS7 NZ 3.43 H-bond mc-sc TYR92 O LYS7 CE 3.74 VDW mc-sc LEU93 CD2 VAL5 CG1 3.68 VDW sc-sc LEU93 CG VAL5 CG1 3.72 VDW sc-sc ASP95 C LYS7 NZ 3.72 VDW mc-sc PHE111 CE2 HIS2 ND1 3.44 VDW sc-sc PHE111 CE1 HIS2 ND1 3.52 VDW sc-sc PHE111 CE1 HIS2 CE1 3.56 VDW sc-sc PHE111 CZ HIS2 CG 3.57 VDW sc-sc

!

PHE111 CZ HIS2 CE1 3.77 VDW sc-sc TYR112 OH PCA1 O 3.13 H-bond sc-mc TYR112 OH PCA1 C 3.57 VDW sc-mc VAL166 CG2 PCA1 N 3.44 VDW sc-mc VAL166 CG2 PCA1 OE 3.76 VDW sc-sc GLN172 NE2 LYS7 CD 3.67 VDW sc-sc CYS173 O PCA1 OE 3.69 VDW mc-sc THR175 OG1 PCA1 OE 3.45 H-bond sc-sc LEU273 CD1 HIS3 CD2 3.50 VDW sc-sc LEU273 CD1 HIS3 NE2 3.62 VDW sc-sc ILE274 CG1 HIS3 O 3.43 VDW sc-mc ILE274 CD1 HIS3 O 3.78 VDW sc-mc GLU277 OE1 HIS2 CB 3.43 VDW sc-sc GLU277 OE1 HIS2 CG 3.56 VDW sc-sc GLU277 CD HIS2 CB 3.59 VDW sc-sc GLU277 OE2 HIS3 N 3.06 H-bond sc-mc GLU277 OE2 HIS3 ND1 3.17 H-bond sc-sc GLU277 OE1 HIS3 N 3.35 H-bond sc-mc GLU277 CD HIS3 N 3.59 VDW sc-mc GLU277 OE2 HIS3 CB 3.69 VDW sc-sc GLU277 OE1 HIS3 C 3.73 VDW sc-mc GLU277 OE1 GLY4 N 2.84 H-bond sc-mc GLU277 OE1 GLY4 CA 3.54 VDW sc-mc

!

Table S2. Data collection and refinement statistics.

Statistics for the highest-resolution shell are shown in parentheses.

US28∆N∆C-CX3CL1N9A-Nb7 US28-CX3CL1 Data Collection Number of crystals 2 26 Wavelength (Å) 1.033 0.9795 Resolution range 35.8 - 2.886 (2.989 - 2.886) 47.2 - 3.801 (3.937 - 3.801) Space group I 4 C 2 2 21 Unit cell (Å) a = b = 81.024, c = 231.303 a = 59.9, b = 192.7, c = 94.4 Total reflections 141,947 26,869 Unique reflections 16,646 (1421) 4834 (577) Multiplicity 8.5 (7.5) 5.6 (2.9) Completeness (%) 98.5 (85.0) 85.0 (55.5) Mean I/sigma(I) 10.7 (1.6) 4.1 (1.6) Resolution at I/sigma(I)=2.0 (Å) 3.0 4.1 Wilson B-factor 58.12 94.10 R-merge 0.189 (1.206) 0.275 (0.555) R-pim 0.085 (0.478) 0.096 (0.330) CC1/2 0.99 (0.43) 0.97 (0.77) Refinement Reflections used in refinement 16,646 (1596) 4343 (1079) Reflections used for R-free 1659 (154) 484 (120) R-work 0.1991 (0.2706) 0.2814 (0.2989) R-free 0.2492 (0.3315) 0.3223 (0.3665) Number of non-hydrogen atoms 3978 2774 macromolecules 3700 2718 Protein residues 475 357 RMS(bonds) 0.002 0.003 RMS(angles) 0.60 0.88 Ramachandran favored (%) 96 95 Ramachandran allowed (%) 3.4 4.0 Ramachandran outliers (%) 0.21 0.57 Rotamer outliers (%) 0.77 0.36 Clashscore 4.01 6.25 Average B-factor 67.01 93.81 macromolecules 66.35 93.10 solvent 40.45 n/a Number of TLS groups 14 n/a

!

Supplementary Figure Legends

Figure S1. Production of alpaca nanobodies specific for the US28/CX3CL1 complex.

A) A nanobody yeast display library was constructed from immunized alpaca peripheral

blood lymphocyte mRNA, and the US28/CX3CL1 complex was used to select the

highest affinity binders. B) Enrichment of the nanobody library after two rounds of

selection with US28/CX3CL1 was measured by flow cytometry. C) Competitive binding

titrations of two nanobodies. Yeast displaying Nb7 were stained with Flag-

US28/CX3CL1 and a fluorescent anti-Flag antibody. Soluble Nb7 or Nb11 were added as

competitors at varying concentrations. D) Size exclusion chromatography purification of

Nb7 bound to the US28/CX3CL1 complex.

Figure S2. Binding interface between Nb7 and US28. Nb7 (green) occupies the

intracellular cavity of US28 (orange), forming contacts with all three intracellular loops

and Helix 8.

Figure S3. Crystal packing constraints on the position of US28 Helix 8 in the C2221

lattice. Helix 8 is prevented from assuming the outward-facing conformation observed in

the I4 crystal form (drawn in orange) by a steric clash with a symmetry-related US28

molecule.

Figure S4. Comparison of crystal packing between I4 and C2221 crystals. A)

Flexibility in the position of the CX3CL1 body in the two different crystal forms. The

position of the chemokine domain of CX3CL1 differs by approximately 2.5Å between

the two different crystal forms. B) Crystal packing of the nanobody-bound I4 crystal form

(left) and nanobody-free C2221 crystal form (right).

Figure S5. Snake diagrams of US28. A) Amino acid snake representation of the

crystallization construct for wild-type US28. CX3CL1-interacting residues are indicated

in blue. B) Amino acid snake representation of the US28∆N∆C crystallization construct.

!

Figure S6. Electrostatic surfaces of chemokines. CX3CL1, CCL5 (RANTES),

CXCL12 (SDF1) and CCL2 (MCP1) are shown with electrostatic surfaces colored as

blue (basic), red (acidic) and white (neutral). The N-terminus of US28 is shown as an

orange tube with CX3CL1. PDB IDs: 3ONA, 1EQT, 2J7Z, 1DOL. Electrostatic surfaces

were calculated using PyMOL (Schrödinger, LLC).

Figure S7. Representative electron density maps. A) Cα trace of TM helices in 2.9 Å

structure. 2mFobs-DFcalc map contoured at 2.0 σ. B) Interaction between N-loop of

CX3CL1 (blue) and us28 site 1 (orange) at 2.9 Å resolution. 2mFobs-DFcalc map

contoured at 1.0 σ. C) Extracellular view of the CX3CL1N-terminal “hook” viewed

within the ligand binding pocket of US28 at 2.9 Å resolution. Simulated annealing omit

mFobs – DFcalc electron density map (green) is contoured at 3σ. 2mFobs – DFcalc electron

density map (gray) is contoured at 1.8 σ. D) Detail of monoolein lipids, cholesterol

(CLR), and succinate (SIN) interacting with us28 (orange) bound to CX3CL1 (blue) at

2.9 Å resolution. mFobs-DFcalc composite omit map contoured at 1.0 σ around ligands. E)

Detail of (NAG)3 glycosylation of CX3CL1 Asn9 at 3.8 Å resolution. mFobs-DFcalc

composite omit map contoured at 2.0 σ around glycan. CX3CL1 is colored light green;

wild-type US28 is colored magenta. F) Carbon-α trace of TM helices of US28 in the 3.8

Å structure. 2mFobs-DFcalc map contoured at 2.0 σ.

Figure S8. Mass spectrometry analysis of CX3CL1. A) Chemical diagram of CX3CL1

residues 1-4 depicting a 17 dalton mass decrease upon pyroglutamate formation from the

N-terminal glutamine. B) MALDI-TOF analysis of CX3CL1 N9A indicates a molecular

weight of 9954.5 daltons. The predicted molecular weight is 9971.5, giving a -17 dalton

difference consistent with pyroglutamate formation. Mass spectrometry was performed

by the Protein and Nucleic Acid Facility at the Stanford University School of Medicine.

Figure S9. Crystal lattice contacts involving US28 Arg139ICL2. A) Overview of crystal

packing in the US28∆N∆C/CX3CL1N9A/Nb7 complex. CX3CL1 is shown in blue,

US28 in orange and Nb7 in green. Symmetry-related molecules are shown in gray.

Packing between ICL2 and Helix8 of an adjacent symmetry mates is marked with a black

!

box. B) Detail of boxed area in (A). Arg139ICL2 makes multiple polar contacts with Helix

8 of an adjacent symmetry mate involving the carboxylic acid of Arg310 and the

carbonyl of Glu308. In MD simulations, Arg139ICL2 reorients and assumes its most

favored rotamer, forming polar interactions with Glu1243.45.

Figure S10. US28/CX3CL1 purification and crystallization. A) Diagram of the

purification scheme for US28, CX3CL1 and Nb7. B) Representative crystal images of

US28∆N∆C/CX3CL1N9A/Nb7 (left) and wild-type US28/CX3CL1 (right). Scale bar

indicates 50 µm.

References and Notes

1. V. Katritch, V. Cherezov, R. C. Stevens, Structure-function of the G protein-coupled receptor

superfamily. Annu. Rev. Pharmacol. Toxicol. 53, 531–556 (2013). Medline

doi:10.1146/annurev-pharmtox-032112-135923

2. Q. Tan, Y. Zhu, J. Li, Z. Chen, G. W. Han, I. Kufareva, T. Li, L. Ma, G. Fenalti, J. Li, W.

Zhang, X. Xie, H. Yang, H. Jiang, V. Cherezov, H. Liu, R. C. Stevens, Q. Zhao, B. Wu,

Structure of the CCR5 chemokine receptor-HIV entry inhibitor maraviroc complex.

Science 341, 1387–1390 (2013). Medline doi:10.1126/science.1241475

3. B. Wu, E. Y. Chien, C. D. Mol, G. Fenalti, W. Liu, V. Katritch, R. Abagyan, A. Brooun, P.

Wells, F. C. Bi, D. J. Hamel, P. Kuhn, T. M. Handel, V. Cherezov, R. C. Stevens,

Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide

antagonists. Science 330, 1066–1071 (2010). Medline doi:10.1126/science.1194396

4. D. M. Rosenbaum, S. G. Rasmussen, B. K. Kobilka, The structure and function of G-protein-

coupled receptors. Nature 459, 356–363 (2009). Medline doi:10.1038/nature08144

5. I. F. Charo, R. M. Ransohoff, The many roles of chemokines and chemokine receptors in

inflammation. N. Engl. J. Med. 354, 610–621 (2006). Medline

doi:10.1056/NEJMra052723

6. A. E. Proudfoot, Chemokine receptors: Multifaceted therapeutic targets. Nat. Rev. Immunol. 2,

106–115 (2002). Medline doi:10.1038/nri722

7. A. Sodhi, S. Montaner, J. S. Gutkind, Viral hijacking of G-protein-coupled-receptor signalling

networks. Nat. Rev. Mol. Cell Biol. 5, 998–1012 (2004). Medline doi:10.1038/nrm1529

8. M. S. Chee, S. C. Satchwell, E. Preddie, K. M. Weston, B. G. Barrell, Human

cytomegalovirus encodes three G protein-coupled receptor homologues. Nature 344,

774–777 (1990). Medline doi:10.1038/344774a0

9. J. F. Bazan, K. B. Bacon, G. Hardiman, W. Wang, K. Soo, D. Rossi, D. R. Greaves, A.

Zlotnik, T. J. Schall, A new class of membrane-bound chemokine with a CX3C motif.

Nature 385, 640–644 (1997). Medline doi:10.1038/385640a0

10. S. Thiele, M. M. Rosenkilde, Interaction of chemokines with their receptors—from initial

chemokine binding to receptor activating steps. Curr. Med. Chem. 21, 3594–3614 (2014).

Medline doi:10.2174/0929867321666140716093155

11. M. Szpakowska, V. Fievez, K. Arumugan, N. van Nuland, J. C. Schmit, A. Chevigné,

Function, diversity and therapeutic potential of the N-terminal domain of human

chemokine receptors. Biochem. Pharmacol. 84, 1366–1380 (2012). Medline

doi:10.1016/j.bcp.2012.08.008

12. C. J. Millard, J. P. Ludeman, M. Canals, J. L. Bridgford, M. G. Hinds, D. J. Clayton, A.

Christopoulos, R. J. Payne, M. J. Stone, Structural basis of receptor sulfotyrosine

recognition by a CC chemokine: The N-terminal region of CCR3 bound to

CCL11/eotaxin-1. Structure 22, 1571–1581 (2014). Medline

doi:10.1016/j.str.2014.08.023

13. C. T. Veldkamp, C. Seibert, F. C. Peterson, N. B. De la Cruz, J. C. Haugner III, H. Basnet, T.

P. Sakmar, B. F. Volkman, Structural basis of CXCR4 sulfotyrosine recognition by the

chemokine SDF-1/CXCL12. Sci. Signal. 1, ra4 (2008). Medline

doi:10.1126/scisignal.1160755

14. L. S. Mizoue, J. F. Bazan, E. C. Johnson, T. M. Handel, Solution structure and dynamics of

the CX3C chemokine domain of fractalkine and its interaction with an N-terminal

fragment of CX3CR1. Biochemistry 38, 1402–1414 (1999). Medline

doi:10.1021/bi9820614

15. J. A. Ballesteros, H. Weinstein, Integrated methods for the construction of three-dimensional

models and computational probing of structure-function relations in G protein-coupled

receptors. Methods Neurosci. 25, 366–428 (1995). doi:10.1016/S1043-9471(05)80049-7

16. A. Brelot, N. Heveker, M. Montes, M. Alizon, Identification of residues of CXCR4 critical

for human immunodeficiency virus coreceptor and chemokine receptor activities. J. Biol.

Chem. 275, 23736–23744 (2000). Medline doi:10.1074/jbc.M000776200

17. P. Casarosa, W. M. Menge, R. Minisini, C. Otto, J. van Heteren, A. Jongejan, H.

Timmerman, B. Moepps, F. Kirchhoff, T. Mertens, M. J. Smit, R. Leurs, Identification of

the first nonpeptidergic inverse agonist for a constitutively active viral-encoded G

protein-coupled receptor. J. Biol. Chem. 278, 5172–5178 (2003). Medline

doi:10.1074/jbc.M210033200

18. H. F. Vischer, M. Siderius, R. Leurs, M. J. Smit, Herpesvirus-encoded GPCRs: Neglected

players in inflammatory and proliferative diseases? Nat. Rev. Drug Discov. 13, 123–139

(2014). Medline doi:10.1038/nrd4189

19. H. F. Vischer, R. Leurs, M. J. Smit, HCMV-encoded G-protein-coupled receptors as

constitutively active modulators of cellular signaling networks. Trends Pharmacol. Sci.

27, 56–63 (2006). Medline doi:10.1016/j.tips.2005.11.006

20. A. J. Venkatakrishnan, X. Deupi, G. Lebon, C. G. Tate, G. F. Schertler, M. M. Babu,

Molecular signatures of G-protein-coupled receptors. Nature 494, 185–194 (2013).

Medline doi:10.1038/nature11896

21. S. G. Rasmussen, B. T. DeVree, Y. Zou, A. C. Kruse, K. Y. Chung, T. S. Kobilka, F. S.

Thian, P. S. Chae, E. Pardon, D. Calinski, J. M. Mathiesen, S. T. Shah, J. A. Lyons, M.

Caffrey, S. H. Gellman, J. Steyaert, G. Skiniotis, W. I. Weis, R. K. Sunahara, B. K.

Kobilka, Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477,

549–555 (2011). Medline doi:10.1038/nature10361

22. K. Palczewski, T. Kumasaka, T. Hori, C. A. Behnke, H. Motoshima, B. A. Fox, I. Le Trong,

D. C. Teller, T. Okada, R. E. Stenkamp, M. Yamamoto, M. Miyano, Crystal structure of

rhodopsin: A G protein–coupled receptor. Science 289, 739–745 (2000). Medline

doi:10.1126/science.289.5480.739

23. M. Waldhoer, P. Casarosa, M. M. Rosenkilde, M. J. Smit, R. Leurs, J. L. Whistler, T. W.

Schwartz, The carboxyl terminus of human cytomegalovirus-encoded 7 transmembrane

receptor US28 camouflages agonism by mediating constitutive endocytosis. J. Biol.

Chem. 278, 19473–19482 (2003). Medline doi:10.1074/jbc.M213179200

24. C. T. Gilliland, C. L. Salanga, T. Kawamura, J. Trejo, T. M. Handel, The chemokine receptor

CCR1 is constitutively active, which leads to G protein-independent, β-arrestin-mediated

internalization. J. Biol. Chem. 288, 32194–32210 (2013). Medline

doi:10.1074/jbc.M113.503797

25. X. Han, Constitutively active chemokine CXC receptors. Adv. Pharmacol. 70, 265–301

(2014). Medline doi:10.1016/B978-0-12-417197-8.00009-2

26. X. Deupi, P. Edwards, A. Singhal, B. Nickle, D. Oprian, G. Schertler, J. Standfuss, Stabilized

G protein binding site in the structure of constitutively active metarhodopsin-II. Proc.

Natl. Acad. Sci. U.S.A. 109, 119–124 (2012). Medline doi:10.1073/pnas.1114089108

27. J. Standfuss, P. C. Edwards, A. D’Antona, M. Fransen, G. Xie, D. D. Oprian, G. F. Schertler,

The structural basis of agonist-induced activation in constitutively active rhodopsin.

Nature 471, 656–660 (2011). Medline doi:10.1038/nature09795

28. R. O. Dror, D. H. Arlow, P. Maragakis, T. J. Mildorf, A. C. Pan, H. Xu, D. W. Borhani, D. E.

Shaw, Activation mechanism of the β2-adrenergic receptor. Proc. Natl. Acad. Sci. U.S.A.

108, 18684–18689 (2011). Medline doi:10.1073/pnas.1110499108

29. R. O. Dror, H. F. Green, C. Valant, D. W. Borhani, J. R. Valcourt, A. C. Pan, D. H. Arlow,

M. Canals, J. R. Lane, R. Rahmani, J. B. Baell, P. M. Sexton, A. Christopoulos, D. E.

Shaw, Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs.

Nature 503, 295–299 (2013). Medline

30. E. S. Burstein, T. A. Spalding, M. R. Brann, The second intracellular loop of the m5

muscarinic receptor is the switch which enables G-protein coupling. J. Biol. Chem. 273,

24322–24327 (1998). Medline doi:10.1074/jbc.273.38.24322

31. A. Dukkipati, H. H. Park, D. Waghray, S. Fischer, K. C. Garcia, BacMam system for high-

level expression of recombinant soluble and membrane glycoproteins for structural

studies. Protein Expr. Purif. 62, 160–170 (2008). Medline doi:10.1016/j.pep.2008.08.004

32. P. Casarosa, M. Waldhoer, P. J. LiWang, H. F. Vischer, T. Kledal, H. Timmerman, T. W.

Schwartz, M. J. Smit, R. Leurs, CC and CX3C chemokines differentially interact with the

N terminus of the human cytomegalovirus-encoded US28 receptor. J. Biol. Chem. 280,

3275–3285 (2005). Medline doi:10.1074/jbc.M407536200

33. Y. Kim, P. Quartey, H. Li, L. Volkart, C. Hatzos, C. Chang, B. Nocek, M. Cuff, J. Osipiuk,

K. Tan, Y. Fan, L. Bigelow, N. Maltseva, R. Wu, M. Borovilos, E. Duggan, M. Zhou, T.

A. Binkowski, R. G. Zhang, A. Joachimiak, Large-scale evaluation of protein reductive

methylation for improving protein crystallization. Nat. Methods 5, 853–854 (2008).

Medline doi:10.1038/nmeth1008-853

34. D. R. Maass, J. Sepulveda, A. Pernthaner, C. B. Shoemaker, Alpaca (Lama pacos) as a

convenient source of recombinant camelid heavy chain antibodies (VHHs). J. Immunol.

Methods 324, 13–25 (2007). Medline doi:10.1016/j.jim.2007.04.008

35. D. W. Colby, B. A. Kellogg, C. P. Graff, Y. A. Yeung, J. S. Swers, K. D. Wittrup,

Engineering antibody affinity by yeast surface display. Methods Enzymol. 388, 348–358

(2004). Medline doi:10.1016/S0076-6879(04)88027-3

36. G. Chao, W. L. Lau, B. J. Hackel, S. L. Sazinsky, S. M. Lippow, K. D. Wittrup, Isolating and

engineering human antibodies using yeast surface display. Nat. Protoc. 1, 755–768

(2006). Medline doi:10.1038/nprot.2006.94

37. A. M. Ring, A. Manglik, A. C. Kruse, M. D. Enos, W. I. Weis, K. C. Garcia, B. K. Kobilka,

Adrenaline-activated structure of β2-adrenoceptor stabilized by an engineered nanobody.

Nature 502, 575–579 (2013). Medline doi:10.1038/nature12572

38. M. Caffrey, V. Cherezov, Crystallizing membrane proteins using lipidic mesophases. Nat.

Protoc. 4, 706–731 (2009). Medline doi:10.1038/nprot.2009.31

39. Y. Misquitta, M. Caffrey, Detergents destabilize the cubic phase of monoolein: Implications

for membrane protein crystallization. Biophys. J. 85, 3084–3096 (2003). Medline

doi:10.1016/S0006-3495(03)74727-4

40. W. Kabsch, XDS. Acta Crystallogr. D 66, 125–132 (2010). Medline

doi:10.1107/S0907444909047337

41. Z. Otwinowski, W. Minor, Processing of x-ray diffraction data collected in oscillation mode.

Methods Enzymol. 276, 307–326 (1997). doi:10.1016/S0076-6879(97)76066-X

42. X. Xue, Q. Lu, H. Wei, D. Wang, D. Chen, G. He, L. Huang, H. Wang, X. Wang, Structural

basis of chemokine sequestration by CrmD, a poxvirus-encoded tumor necrosis factor

receptor. PLOS Pathog. 7, e1002162 (2011). Medline doi:10.1371/journal.ppat.1002162

43. D. M. Hoover, L. S. Mizoue, T. M. Handel, J. Lubkowski, The crystal structure of the

chemokine domain of fractalkine shows a novel quaternary arrangement. J. Biol. Chem.

275, 23187–23193 (2000). Medline doi:10.1074/jbc.M002584200

44. P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Features and development of Coot. Acta

Crystallogr. D 66, 486–501 (2010). Medline

45. P. V. Afonine, R. W. Grosse-Kunstleve, N. Echols, J. J. Headd, N. W. Moriarty, M.

Mustyakimov, T. C. Terwilliger, A. Urzhumtsev, P. H. Zwart, P. D. Adams, Towards

automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D

68, 352–367 (2012). Medline doi:10.1107/S0907444912001308

46. G. Bricogne, E. Blanc, M. Brandl, C. Flensburg, P. Keller, W. Paciorek, P. Roversi, A.

Sharff, O. Smart, C. Vonrhein, T. Womack, BUSTER, version 2.10.2, Global Phasing

Limited, Cambridge, UK.

47. J. Painter, E. A. Merritt, Optimal description of a protein structure in terms of multiple

groups undergoing TLS motion. Acta Crystallogr. D 62, 439–450 (2006). Medline

doi:10.1107/S0907444906005270

48. J. Painter, E. A. Merritt, TLSMD web server for the generation of multi-group TLS models.

J. Appl. Cryst. 39, 109–111 (2006). doi:10.1107/S0021889805038987

49. V. B. Chen, W. B. Arendall III, J. J. Headd, D. A. Keedy, R. M. Immormino, G. J. Kapral, L.

W. Murray, J. S. Richardson, D. C. Richardson, MolProbity: All-atom structure

validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).

Medline doi:10.1107/S0907444909042073

50. Collaborative Computational Project, Number 4, The CCP4 suite: Programs for protein

crystallography. Acta Crystallogr. D 50, 760–763 (1994). Medline

doi:10.1107/S0907444994003112

51. E. Krissinel, K. Henrick, Inference of macromolecular assemblies from crystalline state. J.

Mol. Biol. 372, 774–797 (2007). Medline doi:10.1016/j.jmb.2007.05.022

52. The PyMOL Molecular Graphics System, version 1.7.4.0, Schrödinger, LLC.

53. A. Morin, B. Eisenbraun, J. Key, P. C. Sanschagrin, M. A. Timony, M. Ottaviano, P. Sliz,

Collaboration gets the most out of software. eLife 2, e01456 (2013). Medline

doi:10.7554/eLife.01456

54. M. A. Lomize, A. L. Lomize, I. D. Pogozheva, H. I. Mosberg, OPM: Orientations of proteins

in membranes database. Bioinformatics 22, 623–625 (2006). Medline

doi:10.1093/bioinformatics/btk023

55. S. Jo, T. Kim, V. G. Iyer, W. Im, CHARMM-GUI: A web-based graphical user interface for

CHARMM. J. Comput. Chem. 29, 1859–1865 (2008). Medline doi:10.1002/jcc.20945

56. B. R. Brooks, C. L. Brooks III, A. D. Mackerell Jr., L. Nilsson, R. J. Petrella, B. Roux, Y.

Won, G. Archontis, C. Bartels, S. Boresch, A. Caflisch, L. Caves, Q. Cui, A. R. Dinner,

M. Feig, S. Fischer, J. Gao, M. Hodoscek, W. Im, K. Kuczera, T. Lazaridis, J. Ma, V.

Ovchinnikov, E. Paci, R. W. Pastor, C. B. Post, J. Z. Pu, M. Schaefer, B. Tidor, R. M.

Venable, H. L. Woodcock, X. Wu, W. Yang, D. M. York, M. Karplus, CHARMM: The

biomolecular simulation program. J. Comput. Chem. 30, 1545–1614 (2009). Medline

doi:10.1002/jcc.21287

57. E. L. Wu, X. Cheng, S. Jo, H. Rui, K. C. Song, E. M. Dávila-Contreras, Y. Qi, J. Lee, V.

Monje-Galvan, R. M. Venable, J. B. Klauda, W. Im, CHARMM-GUI Membrane Builder

toward realistic biological membrane simulations. J. Comput. Chem. 35, 1997–2004

(2014). Medline doi:10.1002/jcc.23702

58. S. Jo, T. Kim, W. Im, Automated builder and database of protein/membrane complexes for

molecular dynamics simulations. PLOS ONE 2, e880 (2007). Medline

doi:10.1371/journal.pone.0000880

59. D. A. Case et al., Amber 14, University of California, San Francisco, California.

60. R. Salomon-Ferrer, A. W. Götz, D. Poole, S. Le Grand, R. C. Walker, Routine microsecond

molecular dynamics simulations with AMBER on GPUs. 2. Explicit solvent particle

mesh Ewald. J. Chem. Theory Comput. 9, 3878–3888 (2013). doi:10.1021/ct400314y

61. S. Le Grand, A. W. Götz, R. C. Walker, SPFP: Speed without compromise—A mixed

precision model for GPU accelerated molecular dynamics simulations. Comput. Phys.

Commun. 184, 374–380 (2013). doi:10.1016/j.cpc.2012.09.022

62. A. D. MacKerell, D. Bashford, M. Bellott, R. L. Dunbrack, J. D. Evanseck, M. J. Field, S.

Fischer, J. Gao, H. Guo, S. Ha, D. Joseph-McCarthy, L. Kuchnir, K. Kuczera, F. T. Lau,

C. Mattos, S. Michnick, T. Ngo, D. T. Nguyen, B. Prodhom, W. E. Reiher, B. Roux, M.

Schlenkrich, J. C. Smith, R. Stote, J. Straub, M. Watanabe, J. Wiórkiewicz-Kuczera, D.

Yin, M. Karplus, All-atom empirical potential for molecular modeling and dynamics

studies of proteins. J. Phys. Chem. B 102, 3586–3616 (1998). Medline

doi:10.1021/jp973084f

63. A. D. Mackerell Jr., M. Feig, C. L. Brooks III, Extending the treatment of backbone

energetics in protein force fields: Limitations of gas-phase quantum mechanics in

reproducing protein conformational distributions in molecular dynamics simulations. J.

Comput. Chem. 25, 1400–1415 (2004). Medline doi:10.1002/jcc.20065

64. R. B. Best, X. Zhu, J. Shim, P. E. Lopes, J. Mittal, M. Feig, A. D. Mackerell Jr.,

Optimization of the additive CHARMM all-atom protein force field targeting improved

sampling of the backbone ϕ, ψ and side-chain χ1 and χ2 dihedral angles. J. Chem. Theory

Comput. 8, 3257–3273 (2012). Medline doi:10.1021/ct300400x

65. K. Vanommeslaeghe, E. Hatcher, C. Acharya, S. Kundu, S. Zhong, J. Shim, E. Darian, O.

Guvench, P. Lopes, I. Vorobyov, A. D. Mackerell Jr., CHARMM general force field: A

force field for drug-like molecules compatible with the CHARMM all-atom additive

biological force fields. J. Comput. Chem. 31, 671–690 (2010). Medline

66. V. Isberg, B. Vroling, R. van der Kant, K. Li, G. Vriend, D. Gloriam, GPCRDB: An

information system for G protein-coupled receptors. Nucleic Acids Res. 42, D422–D425

(2014). Medline doi:10.1093/nar/gkt1255