Chemically Stable Lipids for Membrane Protein Crystallization

Article abstract of DOI:10.1021/acs.cgd.7b00458

The lipidic cubic phase (LCP) has been widely recognized as a promising membrane-mimicking matrix for biophysical studies of membrane proteins and their crystallization in a lipidic environment. Application of this material to a wide variety of membrane proteins, however, is hindered due to a limited number of available host lipids, mostly monoacylglycerols (MAGs). Here, we designed, synthesized, and characterized a series of chemically stable lipids resistant to hydrolysis, with properties complementary to the widely used MAGs. In order to assess their potential to serve as host lipids for crystallization, we characterized the phase properties and lattice parameters of mesophases made of two most promising lipids at a variety of different conditions by polarized light microscopy and small-angle X-ray scattering. Both lipids showed remarkable chemical stability and an extended LCP region in the phase diagram covering a wide range of temperatures down to 4 °C. One of these lipids has been used for crystallization and structure determination of a prototypical membrane protein bacteriorhodopsin at 4 and 20 °C.

Full text of DOI:10.1021/acs.cgd.7b00458

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Article
Chemically Stable Lipids for Membrane Protein Crystallization
Andrii Ishchenko, Lingling Peng, Egor Zinovev, Alexey Vlasov, Sung Chang Lee, Alexander
Kuklin, Alexey Mishin, Valentin Borshchevskiy, Qinghai Zhang, and Vadim Cherezov
Cryst. Growth Des., Just Accepted Manuscript • Publication Date (Web): 12 May 2017
Downloaded from http://pubs.acs.org on May 16, 2017
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Chemically Stable Lipids for Membrane Protein Crystallization.
Andrii Ishchenko1, Lingling Peng2, Egor Zinovev3, Alexey Vlasov3, Sung Chang Lee2, Alexander
Kuklin3,4, Alexey Mishin5, Valentin Borshchevskiy3,6, Qinghai Zhang2*, Vadim Cherezov1,5*
1 Department of Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA 90089, USA
2 Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
3 Laboratory for Advanced Studies of Membrane Proteins, Moscow Institute of Physics and Technology (MIPT), Dolgoprudny, Russia
4 Frank Laboratory of Neutron Physics, Joint Institute for Nuclear Research, Dubna, Russia
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5 Laboratory for Structural Biology of GPCRs, Moscow Institute of Physics and Technology, Dolgoprudny, Russia
6 Institute of Complex Systems (ICSꢀ6): Structural Biochemistry, Research Centre Jülich, Germany
* Authors for correspondence: Q.Z. (qinghai@scripps.edu) and V.C. (cherezov@usc.edu)
Lipidic cubic phase (LCP) has been widely successful as a matrix for membrane protein
crystallization in a lipidic environment. Application of this material to a wide variety of membrane
proteins is hindered due to a limited number of available host lipids, mostly monoacylglycerols
(MAGs). In this work, we designed, synthesized and characterized a series of chemically stable
lipids resistant to hydrolysis, with properties complementary to the widely used MAGs. In order to
assess their potential to serve as host lipids for crystallization, we characterized the phase properties
and lattice parameters of mesophases made of two most promising lipids at a variety of different
conditions by polarized light microscopy and smallꢀangle Xꢀray scattering. One of these lipids has
been used for crystallization and structure determination of a prototypical membrane protein
bacteriorhodopsin at 4 °C and 20 °C.
O
HO
HO
N
H
OH
Monoolein
(9.9 MAG)
N = T = 9
GlyNCOC15+4
OH
O
HO
O
N
O
OH Me
GlyNMeCOC15+4
Chemical structures of monoolein (9.9 MAG) and two new LCP lipids characterized in this study: GlyNCOC15+4 and
GlyNMeCOC15+4
.
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Chemically Stable Lipids for Membrane Protein Crystallization.
Andrii Ishchenko1, Lingling Peng2, Egor Zinovev3, Alexey Vlasov3, Sung Chang Lee2, Alexander
Kuklin3,4, Alexey Mishin5, Valentin Borshchevskiy3,6, Qinghai Zhang2*, Vadim Cherezov1,5*
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Department of Chemistry, Bridge Institute, University of Southern California, Los Angeles, CA
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90089, USA
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Department of Integrative Structural and Computational Biology, The Scripps Research Institute,
La Jolla, CA 92037, USA
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Laboratory for Advanced Studies of Membrane Proteins, Moscow Institute of Physics and
Technology, Dolgoprudny, Russia
4 Frank Laboratory of Neutron Physics, Joint Institute for Nuclear Research, Dubna, Russia
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Laboratory for Structural Biology of GPCRs, Moscow Institute of Physics and Technology,
Dolgoprudny, Russia
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Institute of Complex Systems (ICS), ICSꢀ6: Structural Biochemistry, Research Centre Jülich,
Germany
* Authors for correspondence: Q.Z. (qinghai@scripps.edu) and V.C. (cherezov@usc.edu)
ABSTRACT
Lipidic cubic phase (LCP) has been widely recognized as a promising membraneꢀmimicking matrix
for biophysical studies of membrane proteins and their crystallization in a lipidic environment.
Application of this material to a wide variety of membrane proteins, however, is hindered due to a
limited number of available host lipids, mostly monoacylglycerols (MAGs). Here, we designed,
synthesized and characterized a series of chemically stable lipids resistant to hydrolysis, with
properties complementary to the widely used MAGs. In order to assess their potential to serve as
host lipids for crystallization, we characterized the phase properties and lattice parameters of
mesophases made of two most promising lipids at a variety of different conditions by polarized light
microscopy and smallꢀangle Xꢀray scattering. Both lipids showed remarkable chemical stability and
an extended LCP region in the phase diagram covering a wide range of temperatures down to 4 °C.
One of these lipids has been used for crystallization and structure determination of a prototypical
membrane protein bacteriorhodopsin at 4 °C and 20 °C.
INTRODUCTION
Membrane proteins reside in lipid bilayers of biological membranes, where they play critical roles in
mediating cellular processes. The knowledge of their threeꢀdimensional structures is essential for
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deciphering the underlying mechanisms of intercellular interactions, signal transduction, transport of
ions and molecules, and other functions. Xꢀray crystallography represents the most successful
technique for structure determination, however, it requires the availability of highly ordered protein
crystals, growing which in case of membrane proteins is challenging. Structural biology of
membrane proteins received a significant boost after the introduction of unique crystallization
methods using lipidic mesophases, such as a lipidic cubic phase (LCP)1. LCP is an artificial
membraneꢀmimicking gelꢀlike material that forms spontaneously upon mixing approximately equal
volumes of specific lipids and an aqueous buffer2. Solubilized membrane proteins can be
reconstituted into the lipid bilayer of LCP after purification relieving them from their detergent
micelle coating. Adding a precipitant to the lipid/protein mixture triggers crystal nucleation and
growth3. LCP crystallization offers several advantages over the conventional vaporꢀdiffusion
methods, such as an increased stability of proteins, a tighter crystal packing, and a better tolerance to
impurities4. As a result, LCP crystallization method often yields wellꢀordered crystals diffracting to
higher resolution compared to crystals grown in detergent micelle solutions5.
After the first demonstration of LCP crystallization with bacteriorhodopsin in 19961, the method has
undergone continuous development. Special tools, assays and instrumentation for handling of LCP
have been introduced2. Automation and miniaturization of the crystallization process enabled highꢀ
throughput screening of a vast range of precipitant conditions6. Advances in the LCP crystallization
method have contributed to the structure determinations of about a hundred different membrane
proteins from diverse families, including enzymes, transporters, ion channels and G proteinꢀcoupled
receptors7. It was shown that LCP can be used for direct crystallization of membrane proteins from
membranes8, amphipols9 and nanodiscs10,11. Future progress in the application of this method to
crystallization of a wider variety of membrane proteins, however, may be hindered due to a limited
number of available lipids that that support formation of a stable LCP at or below room temperature.
The identity of the host lipid affects such LCP parameters as the bilayer thickness, solvent channel
diameter, curvature and fluidity of the bilayer. Consequently, stability, diffusion, nucleation and
crystallization of the embedded membrane protein targets critically depend on the selection of the
LCP host lipid.
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Currently, monoacylglycerols (MAGs) represent the most widely used lipid class for crystallization
of membrane proteins by the LCP method12. MAGs are derivatives of the triꢀhydric alcohol
glycerol, in which the primary hydroxyl group is esterified with a longꢀchain fatty acid, typically
monounsaturated. Different monounsaturated MAGs are often described by an N.T MAG shorthand
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notation13, where N (Neck) is the number of carbon atoms between the lipid’s polar head and the
double bond, and T (Tail) is the number of carbon atoms from the double bond to the terminal
methyl. While serving successfully as LCP host lipids, MAGs are chemically unstable and
susceptible to hydrolysis of the ester linkage between the glycerol head group and the fatty acid
under alkaline and acidic conditions leading to production of fatty acid14,15. The same process occurs
slowly even at midꢀrange pH values, and can be accelerated by trace amounts of lipases, potentially
modifying the properties of LCP or even converting it into a nonꢀLCP phase16, thereby affecting
crystal nucleation and growth. Additionally, alkyl chain migration from the primary 1ꢀOH to the
secondary 2ꢀOH group also occurs in MAGs during incubation in aqueous solution, resulting in the
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accumulation of 2ꢀMAGs that modulate LCP properties17,18
.
The most commonly used MAG is monoolein (9.9 MAG) (Scheme 1), which supports
crystallization of many different membrane proteins19. Several other MAGs with varying N and T
numbers have been developed for low temperature crystallization and other specific crystallization
projects7,20,21. Recently, Salvati et al.22 substituted the double bond of monoolein with a cyclopropyl
group, which extended the cubic phase formation region to a lower temperature range and supported
the crystallization of bacteriorhodopsin (bR) at 4 °C.
In this study, we have designed, synthesized and tested a panel of 33 nonꢀhydrolysable amideꢀlinked
lipids with cisꢀolefinic or isoprenoid tails for their ability to form LCP and support crystallization.
Isoprenoid lipids have previously been successfully used in LCP crystallization studies23 and have
displayed distinct structural properties24,25 making them a useful compliment to the standard MAG
lipids set. Our goal was to develop chemically stable lipids that form a stable cubic phase under a
broad range of conditions including extreme pH and low temperature. We streamlined lipid
screening to quickly identify most promising LCP lipids. With two promising hits found
(GlyNCOC15+4 and GlyNMeCOC15+4, where Gly refers to the glycerol headgroup, NCO or NMeCO
– to the amideꢀ or Nꢀmethyl amideꢀ linkers and C15+4 – to the isoprenoid chain with 15 carbon
groups and 4 branched methyls), and one of them (GlyNCOC15+4) selected for further crystallization
studies, we crystallized a prototypical membrane protein bacteriorhodopsin26 at 20 °C and 4 °C and
solved the corresponding structures. Initial attempts to crystallize the A2A adenosine (A2AAR) G
proteinꢀcoupled receptor (GPCR) using the same host lipid produced small crystals. This work, thus,
contributes to the ongoing efforts of expanding the lipid toolbox for LCP crystallization of
membrane proteins.
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EXPERIMENTAL SECTION
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Synthesis of GlyNCOC15+4 and GlyNMeCOC15+4
Phytol (5.0 g, 16.9 mmol) and RaneyꢀNi (50% slurry in H2O, 1.0 g, 8.4 mmol) were mixed in 25 mL
ethanol (Scheme S1a). The reaction atmosphere was then exchanged to H2 and stirred for 3 days at
room temperature. The suspension was filtered through celite and washed with methanol. After the
removal of solvent, dihydroꢀphytol (3,7,11,15ꢀtetramethylhexadecanꢀ1ꢀol) was left as a colorless oil
(5.0 g, 99%). Dihydroꢀphytol (5.0 g, 16.8 mmol) was dissolved in 120 mL acetone and the solution
was cooled down to 0 oC. Then Jones reagent (2.7 M, 7.5 mL, 20.2 mmol) was added dropwise over
half an hour with stirring at the same temperature. The reaction was quenched in half an hour by
adding isopropanol. The mixture was then filtered through celite, washed with acetone, and
concentrated in vacuo. The residue was purified by silicaꢀgel column chromatography to give
phytanic acid (3,7,11,15ꢀtetramethylhexadecanoic acid) as a colorless oil (4.7 g, 91%).
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Phytanic acid (160 mg, 0.51 mmol), 3ꢀaminopropaneꢀ1,2ꢀdiol (60 mg, 0.66 mmol), NꢀEthylꢀN′ꢀ(3ꢀ
dimethylaminopropyl)carbodiimide (EDC, 196 mg, 1.02 mmol) and 1ꢀhydroxybenzotriazole(HOBt,
156 mg, 1.02 mmol) were mixed in 3 mL DMF (Scheme S1b). N,Nꢀdiisopropylethyl amine (DIEA,
o
0.35 mL, 2.04 mmol) was then added at 0 C. The reaction mixture was stirred at room temperature
for 1 day. Then H2O and ethyl acetate were added. The reaction mixture was washed sequentially
with H2O and a saturated solution of sodium chloride. The organic layer was separated, dried over
Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silicaꢀgel column
chromatography to give GlyNCOC15+4 as a colorless oil (175 mg, 88%). GlyNMeCOC15+4 was
prepared similarly as GlyNCOC15+4 except for the using of 3ꢀ(methylamino)propaneꢀ1,2ꢀdiol as a
reactant (Scheme S1a,c).
13
The 1H and C NMR spectra for GlyNCOC15+4 and GlyNMeCOC15+4 are shown in Figure S1. Both
GlyNCOC15+4 and GlyNMeCOC15+4 could be efficiently scaled up for synthesis at multigrams scale.
GlyNCOC15+4: 1H NMR (600 MHz, CDCl3) δ 6.95 (s, br, 1H), 4.40 (s, br, 2H), 3.74ꢀ3.70 (m, 1H),
3.54ꢀ3.47 (m, 2H), 3.41ꢀ3.24 (m, 2H), 2.24ꢀ2.20 (m, 1H), 1.96ꢀ1.90 (m, 2H), 1.52ꢀ1.46 (m, 1H),
1.37ꢀ0.99 (m, 20H), 0.88 (d, J = 6.1 Hz, 3H), 0.84 (d, J = 6.6 Hz, 6H), 0.81 (d, J = 6.6 Hz, 6H). 13C
NMR (125 MHz, CDCl3) δ 175.1, 71.2, 63.9, 44.5, 44.4, 42.3, 39.6, 37.7, 37.6, 37.5, 33.0, 31.1,
28.2, 25.0, 24.7, 22.9, 22.8, 19.9, 19.8, 19.7, 19.6.
GlyNMeCOC15+4: 1H NMR (600 MHz, CDCl3) δ 3.96 (br, 2H), 3.81ꢀ3.77 (m, 1H), 3.52ꢀ3.41 (m,
4H), 3.06 (s, 2.7H), 2.94 (s, 0.3H), 2.33ꢀ2.27 (m, 1H), 2.12ꢀ2.08 (m, 1H), 1.96ꢀ1.92 (m, 1H), 1.51ꢀ
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1.44 (m, 1H), 1.34ꢀ0.98 (m, 20H), 0.90ꢀ0.88 (m, 3H), 0.82 (d, J = 6.7 Hz, 6H), 0.80 (d, J = 6.7 Hz,
6H). 13C NMR (125 MHz, CDCl3) δ 175.1, 70.5, 63.5, 51.0, 40.8, 40.7, 39.4, 37.8, 37.5, 37.4, 37.3,
32.8, 30.4, 28.0, 24.8, 24.5, 22.8, 22.7, 19.9, 19.9, 19.7, 19.6.
The amide bond in GlyNMeCOC15+4 exists in a mixture of cisꢀ and transꢀ configurations at an
approximate ratio of 1:9, based on the 1H NMR assignment.
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Small-angle X-ray scattering
Samples in capillaries
Initial SAXS samples were prepared by mixing corresponding lipids with an excess buffer (25 mM
HEPES pH 7.0; 2:3 v/v ratio, lipid:buffer) at room temperature using a syringe mixer27. The
resulting lipidic mesophases were then transferred from the Hamilton syringe into Xꢀrayꢀtransparent
capillaries (d = 1.5 mm) that were flameꢀsealed afterwards. The capillaries were placed in a
temperatureꢀcontrolled holder and SAXS data were collected at 4 °C, 10 °C, 20 °C, 30 °C and 40
°C. The samples were incubated at 4 °C for 1 h before collecting data at this temperature and for 30
min at subsequent temperatures in the heating direction. SAXS experiments were conducted on a
Rigaku SAXS instrument equipped with a pinhole camera attached to a rotating Cu anode Xꢀray
highꢀflux beam generator (MicroMax 007ꢀHF) which operated at 40 kV and 30 mA (1200 W). A
multiwire gasꢀfilled area detector Rigaku ASM DTR Triton 200 (diameter 200 mm, pixel size 200
ꢁm) was placed at a distance of 203 cm from the sample. The entire optical path of the Xꢀray beam
was kept in vacuum during data collection. The detector covered Qꢀrange was 0.006 – 0.2 Åꢀ1
(ꢀ = 4ꢁ ꢂꢃꢄꢅ/λ , where λ is the wavelength and 2θ is the scattering angle). The beam size was 400
ꢁm and the exposure time ranged from 1800 s to 2500 s.
Samples of GlyNCOC15+4 at different hydration levels were prepared the same way except for the
different lipid:water ratios. SAXS data for these samples were collected at room temperature.
Samples in 96ꢀwell sandwich plates
Samples were prepared in special Xꢀray transparent 96ꢀwell sandwich plates made of two 140 ꢁmꢀ
thick COC films (TOPAS 8007X4) with a 130 ꢁmꢀthick perforated doubleꢀsticky spacer 468MP
(3M) between them and mounted on a rigid PMMA frame (Figure S2). Lipids were first mixed with
water at a ratio of 3:2 v/v using a lipid syringe mixer to form LCP. Then LCP was distributed by 50
nL per well in 96ꢀwell sandwich plates and covered with 800 nL of precipitant solutions using an
NT8ꢀLCP crystallization robot (Formulatrix). Analysis was made with a homeꢀmade “FRAP pH 7”
screen28, and commercial “MemStart & MemSys”, “MemGold” (Molecular Dimensions) and
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“Wizard Cubic LCP” (Rigaku) screens. For each combination of lipid and screen two crystallization
plates were prepared that differed in the direction of sample dispensing (from A1 to H12 and from
H12 to A1) to reduce potential effects of sample dehydration29. Coordinates of LCP drops in each
plate were manually identified after taking their images with an automatic FRAP imager
(Formulatrix) and then translated to the SAXS holder coordinates. SAXS data for each well were
collected by the Rigaku instrument. 2D scattering profiles were collected using automatic sample
holder. The beam size of 400 ꢁm and 100 s exposure time per well were used for this data
collection. All experiments were performed at 20 °C. The scattering data were analyzed with the
SAXSGUI package (Rigaku) to convert 2D scattering images into 1D radiallyꢀaveraged scattered
curves. Phase identification and lattice parameters determination were performed as previously
described29.
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Polarized Light Microscopy (PLM)
Lipids were first mixed with water at a ratio of 1:2 (v/v) using a lipid syringe mixer to form a
homogenous phase. The resultant material was deposited on a microscope glass slide and covered
with a cover slip to form a thin layer. Samples were tested for birefringence using Nikon SMZ1500
microscope equipped with two rotating linear polarizing filters placed before and after the sample.
The samples that didn’t change light polarization were likely isotropic and formed either LCP or
sponge phase.
Expression, purification and crystallization of bR and A2AAR
bR
Wild type bR was expressed in Halobacterium salinarum, isolated and purified using the standard
protocol30. In brief, bR was solubilized from purple membranes of Halobacterium salinarum with
50 mM sodium phosphate buffer (pH 6.9) containing 1.2% (w/v) βꢀoctylglucoside (OG) and purified
by SEC using a Superdex 200 16/60 column (GE Healthcare). The protein was concentrated using a
50 kDa MWCO Amicon Centrifugal Device (Millipore). bR concentration was determined by
absorbance at 550 nm on a NanoDrop spectrophotometer using the extinction coefficient ε550 = 5.8 ×
104 M−1cm−1. For crystallization, 30 mg/ml of bR in OG was mixed with GlyNCOC15+4 in 2:3 ratio
(v/v) using coupled syringes device27 to create LCP. LCP drops of 200 nl volume were dispensed in
each well of 96ꢀwell glass sandwich plates (Marienfeld) using a manual LCP dispenser (Hamilton)
and covered with 0.8 ꢁl of precipitant. The precipitant screen was based on literature conditions1
using a concentration gradient around the condition: 1.9ꢀ3.0 M sodium/potassium phosphate buffer
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(pH5.6), 3.5% v/v methylpentanediol and 0.5% w/v OG. No further screening and optimization of
crystallization conditions were performed.
A2AAR
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A2AAR was expressed and purified according to previously published protocol31. In short, the third
intracellular loop of A2AAR between residues L209 and G218 was replaced with a thermostabilized
apocytochrome b562RIL (BRIL) and the Cꢀterminal residues 317ꢀ412 were truncated. HA and Flag
tags were added to the Nꢀterminus of the protein and 10xHis tag was placed on the Cꢀterminus. The
protein was expressed in insect sf9 cells using BacꢀtoꢀBac Baculovirus Expression System
(Invitrogen). Insect cell membranes were disrupted by thawing frozen cell pellets in a hypotonic
buffer containing an EDTAꢀfree complete protease inhibitor cocktail (Roche). Extensive washing of
the isolated raw membranes was performed by repeated dounce homogenization and centrifugation
in the same hypotonic buffer (~2ꢀ3 times), and then in a high osmotic buffer (~3ꢀ4 times) to remove
soluble and membrane associated proteins. Prior to solubilization, purified membranes were thawed
on ice in the presence of 4 mM theophylline (Sigma), 2.0 mg/ml iodoacetamide (Sigma), and an
EDTAꢀfree complete protease inhibitor cocktail (Roche). After incubation for 30 min at 4 °C,
membranes were solubilized by incubation in the presence of 0.5% (w/v) nꢀdodecylꢀβꢀDꢀ
maltopyranoside (DDM) (Anatrace) and 0.1% (w/v) cholesteryl hemisuccinate (CHS) (Sigma) for
2.5 h at 4 °C. The unsolubilized material was removed by centrifugation at 150,000 ×g for 45 min.
The supernatant was incubated with TALON IMAC resin (Clontech) in the buffer containing 50
mM HEPES (pH 7.5), 800 mM NaCl, 0.5% (w/v) DDM, 0.1% (w/v) CHS, and 20 mM imidazole.
The protein was purified on TALON resin using 10 column volumes (CV) of wash buffer 1 (50 mM
HEPES (pH 7.5), 800 mM NaCl, 10% (v/v) glycerol, 25 mM imidazole, 0.1% (w/v) DDM, 0.02%
(w/v) CHS, 10 mM MgCl2, 8 mM ATP (Sigma) and 25 µM ZM241385) followed by 5 CV of wash
buffer 2 (50 mM HEPES (pH 7.5), 800 mM NaCl, 10% (v/v) glycerol, 50 mM imidazole, 0.05%
(w/v) DDM, 0.01% (w/v) CHS and 25 µM ZM241385). The receptor was eluted with 25 mM
HEPES (pH 7.5), 800 mM NaCl, 10% (v/v) glycerol, 220 mM imidazole, 0.025% (w/v) DDM,
0.005% (w/v) CHS and 25 µM ZM241385.
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To make 9:1 (w/w) GlyNCOC15+4/cholesterol mixture, the lipids were added together and dissolved
in a minimal volume of chloroform. The excess of chloroform was removed by evaporation under a
stream of nitrogen gas, and the lipid mixture was fully dried under vacuum (<0.1 Torr) for over 12
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Protein samples of A2AAR in complex with ZM241385 were reconstituted into lipidic cubic phase
(LCP) by mixing it with molten GlyNCOC15+4/cholesterol at a 2:3 (v/v, protein/lipid) ratio using a
syringe mixer. Due to a relatively high viscosity of the lipid mixture, it was transferred into the
syringe mixer using a spatula. Crystallization trials were performed in 96ꢀwell glass sandwich plates
(Marienfeld) by an NT8ꢀLCP crystallization robot (Formulatrix) using 50 nl proteinꢀladen LCP
overlaid with 0.8 µl precipitant solution in each well. Crystals were obtained overnight in 25ꢀ28%
(v/v) PEG 400, 0.04 to 0.06 M sodium thiocyanate, 2% (v/v) 2,5ꢀhexanediol, 100 mM sodium
citrate pH 5.0.
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Crystallographic data collection and structure determination.
Synchrotron diffraction data was collected on the 23 IDꢀD beamline at the Advanced Photon Source
equipped with a Pilatus3 6M detector. Datasets were collected using 20×20 ꢁm2 beam, 0.2 sec
exposure time and oscillation angle 0.2° without attenuation of the beam intensity. bR_20C dataset
was obtained by merging the data collected on 4 crystals and bR_4C dataset was obtained by
merging the data from 5 crystals. The data was indexed, merged and integrated with XDS32,
molecular replacement solutions were found with Phaser33 using PDB ID 1XJI as a model. The
models were refined using multiple cycles of refinement with phenix.refine34 using NCS restraints
followed by manual rebuilding in Coot35.
Initially, bR crystals grown at 4 °C were indexed in C2221 space group, however the initial Rfree
after molecular replacement was above 40% and didn’t improve after rebuilding and refinement.
Subsequently, the data was reꢀindexed in P21, where the Lꢀtest has shown a perfect pseudoꢀ
merohedral twinning with a twin law h,ꢀk,ꢀhꢀl. After reꢀindexing the correct MR solution could be
found and the structure was refined to Rfree = 26.5% using phenix.refine with the twin law h,ꢀk,ꢀhꢀl.
The data obtained from the crystals grown at 20 °C was indexed in R3 space group with no twinning
detected by the L and NZ tests performed in phenix.Xtriage. Data processing and refinement
statistics is shown in Table 1.
RESULTS AND DISCUSSION
Design and synthesis of novel lipids
Currently, only several lipids have been extensively characterized for their ability to form LCP
under conditions amenable to membrane protein crystallization14,20,21,24,25,36,37. The complexity of
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lipidꢀsolvent interactions and a poor understanding of the relationship between the lipid structure
and its phase behavior makes it challenging to rationally design a lipid with specific physicalꢀ
chemical and phase properties suitable for LCP crystallization. Given the labile nature of the ester
bond in MAGs, we set our goals to discover more chemically stable LCP lipids as part of our efforts
to advance the utility of the LCP technology. Based on our prior experience, simple substitution of
the ester group of monoolein with a nonꢀhydrolysable amide or ether group results in formation of
nonꢀLCP liquid crystalline phases at room temperature and excess hydration conditions. We
speculated that the change of the ester linkage to the amide or ether group may lead to altered lipid
hydration and hydrogenꢀbond patterns. A subsequent search for new lipids was therefore mainly
focused on the alteration of the polar groups while using traditional cisꢀolefinic and isoprenoid
chains. Following this approach, we have synthetized and evaluated 33 new amideꢀlinked lipids
(Table S1) with two of them found to form stable LCP suitable for membrane protein crystallization
studies.
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Polarized Light Microscopy and SAXS analysis of the designed lipids
To quickly identify lipids suitable for membrane protein crystallization, all synthesized lipids were
first preꢀscreened for their ability to form LCP at excess hydration and 20 °C by mixing them with
water (lipid:water = 1:2 v/v) using a syringe mixer27 and analyzing the samples under crossꢀ
polarized light (Table S1). Two lipids containing a branched isoprenoid chain, GlyNCOC15+4 (lipid
16) and GlyNMeCOC15+4 (lipid 18) (Scheme 1), formed a gelꢀlike phase and showed no
birefringence under crossꢀpolarized light microscopy, indicative of a cubic phase formation. Both of
these lipids contain glycerol as their polar head group, similar to monoolein, connected to an
isoprenoid acyl chain through an amide bond. To determine the phase state, these two lipids were
further subjected to SAXS analysis at five different temperatures between 4 °С and 40 °С (Figure
S3). SAXS measurements also allow to obtain lattice parameters for each identified phase. We
observed that samples made of GlyNCOC15+4 formed cubicꢀPn3m phase in the entire temperature
range from 4 to 40 °С, while samples made of GlyNMeCOC15+4 formed mixtures of lamellar and
cubic phases at low temperatures, which turned into a single cubicꢀPn3m phase at 30 °С and 40 °С.
The full hydration boundary for the cubicꢀPn3m phase made of GlyNCOC15+4 at 20 °С was found to
be 44±2% (v/v water) and the lattice parameter of the fully hydrated cubicꢀPn3m phase 106.2±1.0 Å
(Table S2).
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Both lipids that formed LCP in excess hydration were further subjected to an extensive screening for
their compatibility with typical conditions encountered during crystallization trials using a highꢀ
throughput LCPꢀSAXS method29. For these tests we used three commercial screens:
“MemStart&MemSys” and “MemGold” screens from Molecular Dimensions and “Emerald Cubic
LCP” screen from Rigaku, as well as an inꢀhouse made “FRAP pH 7” screen (see Methods section).
Phase identities and unit cell parameters were determined from SAXS patterns for each screened
condition. Lipids GlyNCOC15+4 and GlyNMeCOC15+4 formed LCP in a wide range of precipitant
conditions with predominantly Pn3m and Ia3d symmetries and cell parameters ranging from 68 Å to
128 Å for cubicꢀPn3m and from 106 Å to 156 Å for cubicꢀIa3d phases (Figure S4). The LCP
composed of GlyNCOC15+4 was found to be compatible with most conditions from the commercial
screens, which were not designed specifically for LCP crystallization (MemStart&MemSys and
MemGold), and thus contain many conditions not compatible with a monooleinꢀbased LCP29,38. The
LCP composed of GlyNMeCOC15+4 was generally much less stable, being compatible with around
50% conditions of the MemStart&MemSys and 25% conditions of the MemGold screens (solutions
containing PEG with molecular weight 4,000 Da and higher turned the phase into lamellar). While
both monoolein and GlyNCOC15+4 are fully compatible with the Wizard Cubic LCP screen,
GlyNMeCOC15+4 displayed low compatibility mostly due to the presence of high molecular weight
PEGs in this screen. Overall, GlyNCOC15+4 exhibited superior LCP compatibility with a broader
variety of crystallization screening conditions compared to both GlyNMeCOC15+4 and monoolein38.
In general, we observed an increased stability of GlyNCOC15+4 and GlyNMeCOC15+4 towards
highly alkaline and acidic buffers. Monoolein, the most widely used lipid for LCP crystallization,
can be typically used only in the pH range between 2ꢀ3 and 8ꢀ9 (depending on the nature of the
precipitant salt used), limiting its application for the proteins that favor crystallization conditions
outside of this range. We have tested GlyNCOC15+4 with different buffers in the range from pH=2.2
to pH=11, where it demonstrated a remarkable stability and LCP remained intact for at least four
weeks (Figure 1). Therefore, substitution of the ester linkage with an amide greatly improved the
stability of lipids both at harsh chemical conditions and during long incubations at normal
conditions.
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LCP lattice parameters of GlyNCOC15+4
We used the SAXS data for GlyNCOC15+4 to estimate the lipid membrane thickness and the
diameter of the water channels for the cubicꢀPn3m phase at 20 °C and maximum hydration level.
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The lipid length, l, is related to the LCP lattice parameter, a, and the volume fraction of lipids in
LCP, Φl, through the following equation39:
Φꢆ = 2Aꢇ ꢈꢉꢆꢊ + ꢋπꢌ (ꢉꢆ)ꢍ,
ꢍ
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where A0 is the area of the membrane surface in the unitary unit cell, and χ is the EulerꢀPoincare
parameter. In case of the cubicꢀPn3m phase A0=1.919, χ=ꢀ2. Using the estimated Φl of 0.56±0.01 at
the full hydration boundary and the measured lattice parameter of 106.2±1.0 Å, we derived the lipid
length l=16.3±0.3 Å and the bilayer thickness d=2·l=32.6±0.6 Å. The radius of the aqueous channel
in LCP can then be further estimated from these parameters using the following formula36:
rw=0.391aꢀl,
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resulting in rw = 25.2±0.5 Å.
Comparing these numbers with the bilayer thickness, d=34.6 Å, and the radius of the aqueous
channel, rw=25.3 Å, reported previously for the monooleinꢀbased cubic Pn3m phase36,40 we conclude
that the LCP formed by GlyNCOC15+4 has a slightly smaller bilayer thickness and a similar water
channel diameter.
Crystallization of membrane proteins using designed lipid
The choice of a host lipid is undoubtedly an important parameter in LCP crystallization trials41. We
tested the newly synthesized lipids for their ability to support crystallization of membrane proteins.
GlyNCOC15+4 was chosen for the crystallization trials, as it formed stable LCP in the widest
temperature and precipitant composition space. bR was used for initial crystallization tests, as it is a
wellꢀcharacterized membrane protein, for which highꢀresolution structures are available in the
Protein Data Bank (PDB). Crystals of bR were obtained after incubating LCP crystallization plates
at two different temperatures: 4 °C and at 20 °C (Figure 2), simply by precipitant screening around
conditions that are known to support bR crystallization in monoolein1 (see Methods). With this
observation, we did not attempt to further extend and optimize the screens. Crystals appeared in 3ꢀ5
days and continued to grow for several weeks at both crystallization temperatures. At 20 °C crystals
grew to a maximal size of 40 µm and had a hexagonal shape, similar to bR crystals grown in
monoolein42,43. The crystals grown at 4 °C were rhombic and, generally, smaller in size (~20ꢀ30
µm).
Xꢀray crystallographic data for both types of crystals were collected at the GM/CA facility at the
Advanced Photon Source, Argonne, IL (Table 1). Data obtained from four hexagonal crystals grown
at 20 °C were indexed in the R3 space group and merged together at the resolution of 2.7 Å. A PDB
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search indicated that bR has never been previously crystallized in such a space group. Typically,
hexagonal crystals of bR belong to the space group P63, where bR molecules are packed as trimers
in the plane of the lipidic membrane, with one molecule per asymmetric unit (ASU) (Figure 3a, d).
In our case, however, there are two bR molecules per ASU, one of which forming a trimer together
with its symmetry mates around the threeꢀfold symmetry axis similar to the trimers in the P63 space
group bR structures. Meanwhile, the second bR molecule is attached peripherally to the trimer at all
three sides (Figure 3b, e), thus forming a hexamer of bR molecules. There are no crystal contacts
between hexamers in the membrane plane resulting in a loose crystal packing with the Matthews
coefficient Vm of 3.08 Å3/Da and an estimated solvent content of 60% compared to 2.21 Å3/Da and
45% correspondingly for bR crystallized in the P63 space group (PDB ID 1MD2)43. Consistent with
the absence of crystal contacts in the membrane plane, the outer parts of the hexamerꢀforming
proteins have increased Bꢀfactors compared to the hexamer core (Figure 3h). In the stacking
direction, on the contrary, the crystal packing is very tight with a minimal spacing between protein
layers.
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The overall bR structure within the trimer core of the hexamer is very similar to the previous bR
structures (Cα atoms RMSD = 0.40 Å with PDB ID 1M0L). However, the tight packing between
crystal layers has a big impact on the structure of the extracellular loop between helices B and C
(BC loop) of the distal protomers in the hexamer. This BC loop interacts with the same protomers of
the neighboring layer in a crystal and adopts a distinct conformation compared to the core protomers
and all the previously published structures (Figure 4). Phe71 plays a crucial role in this interaction
protruding out into the adjacent membrane layer and anchoring the loop to the cytoplasmic side of
the neighboring protein.
Data collected from bR crystals grown at 4 °C were indexed in P21 space group. A full Xꢀray
diffraction dataset in this case was obtained by combining data from five crystals grown in similar
conditions at 2.6 Å resolution. Interestingly, the crystal packing is very similar to that observed for
bR crystallized from bicelles at 37 °C44. The ASU contains two molecules of bR assembled in an
antiparallel manner. The overall structure of both molecules is similar to that of published before
(Cα atoms RMSD = 0.49 Å with PDB ID 1KME).
After successful bR crystallization, we tested GlyNCOC15+4 for its ability to support crystallization
of a GPCR: A2A adenosine receptor (A2AAR). Several structures of this receptor containing a fusion
protein, either a cysteineꢀless lysozyme from T4 phage (T4L) or a thermostabilized apoꢀcytochrome
b562 (BRIL), have been previously obtained using LCP crystallization method with monoolein (9.9
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MAG) doped with cholesterol as the host lipid45–47. Since GPCRs typically require cholesterol
binding for their stability, we mixed GlyNCOC15+4 with 10% cholesterol by weight to ensure typical
crystallization conditions for this family of receptors. We used the same engineered A2AARꢀBRIL
construct as in our previous work31 and obtained hits (Figure 5) with very small (~1 ꢁm) crystals
forming with a high nucleation rate and in similar precipitant conditions as previously published31.
Despite the efforts to optimize crystallization conditions by varying PEG400 and salt concentrations,
the crystal size could not be further improved. Although such crystals are too small for data
collection at a synchrotron source, they could potentially be suitable for serial femtosecond
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crystallography (SFX) with an Xꢀray free electron laser (XFEL)48,49
.
In this work, we synthesized dozens of nonꢀhydrolysable lipids and characterized them for their
potential to be used as host lipids for LCP crystallization of membrane proteins. By examining
subgroups of structures containing an identical aliphatic chain, we showed that minor structural
variation in the polar region have a dramatic impact on the lipid hydration and corresponding phase
properties. Two of these lipids, GlyNCOC15+4 and GlyNMeCOC15+4, formed stable LCP at room
temperature and excess hydration. Substitution of the ester bond between the lipid head and the tail
with an amide bond as expected greatly improved the overall resistance towards hydrolysis during
prolonged incubation. While different by only a single methyl, GlyNCOC15+4 demonstrated the
ability to sustain LCP in a wider range of conditions, and was further used as a host lipid for
crystallization of two membrane proteins.
Analysis of the phase behavior with various screens typically employed for membrane protein
crystallization confirmed that both GlyNCOC15+4 and GlyNMeCOC15+4 are suitable to be used as
host lipids for LCP crystallization, although the latter lipid forms LCP under substantially fewer
conditions than the former one. In contrast to monooleinꢀbased LCP, most conditions induced
shrinkage of the cubicꢀPn3m phase for both tested lipids. For example, in case of the FRAP pH 7
screen the lattice parameter of the cubicꢀPn3m phase was on averages smaller by 15 Å for
GlyNCOC15+4 and by 20 Å for GlyNMeCOC15+4 (Figure S4). Few conditions, especially those with
high concentrations of alcohols swelled the cubicꢀPn3m phase and occasionally transformed it into a
sponge phase. We expect that new and a broader range of screens could be developed for further
evaluation of these lipids, especially for GlyNCOC15+4
.
Since LCP made of GlyNCOC15+4 was found to be compatible with a wider array of conditions
compared to GlyNMeCOC15+4, we tested whether this lipid can support crystallization of a
prototypical membrane protein bR and A2AAR. The crystals of bR were grown both at 20 °C and 4
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°C and the respective structures were solved at 2.7 Å and 2.6 Å resolution. The structure of bR at 20
°C revealed novel crystal packing in the R3 space group, while at 4 °C crystal packing was very
similar to that previously obtained for bR crystallized in bicelles at 37 °C. These observations
emphasized the importance of the host lipid structure and properties on the mechanism and outcome
of membrane protein crystallization in LCP.
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Therefore, based on the SAXS results and crystallization experiments we conclude that the new
lipids can be a useful alternative to MAGs as the host lipids for LCP crystallization, in particularly,
at harsh conditions and at low temperatures.
ASSOCIATED CONTENT
Supporting information
The Supporting information is available free of charge on the ACS Publications website at DOI:
Chemical structures of synthesized lipids, physical properties of lipidic mesophases, synthesis
schemes for GlyNMeCOC15+4 and GlyNCOC15+4, NMR spectra, assembly of SAXS sandwich
plates, SAXS patterns, phase identities and lattice parameters of lipidic mesophases.
Accession codes
The coordinates and structure factors were deposited in the Protein Data Bank with the accession
codes 5VN7 (bR at 20 °C) and 5VN9 (bR at 4 °C).
ACKNOWLEDGMENTS
This work was supported by the Ministry of Education and Science of the Russian Federation
(RFMEFI58716X0026) and the National Institutes of Health (P50 GM073197 and R01 GM098538).
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tailored bilayers. Structure 2004, 12, 2113–2124.
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Siegel, J. S.; Mezzenga, R.; Landau, E. M. Phase behavior of a designed yclopropyl analogue
of monoolein: implications for lowꢀtemperature membrane protein crystallization. Angew.
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of lipid matrices for membrane protein crystallization by highꢀthroughput small angle Xꢀray
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allosteric regulation of GPCRs by sodium ions. Science 2012, 337, 232–236.
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cubic phase for in meso crystallization of membrane proteins. Biophys. J. 2001, 81, 225–242.
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variables for crystallizing integral membrane proteins in lipidic mesophases. Trials with
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proteins yields a monomeric bacteriorhodopsin structure. J. Mol. Biol. 2002, 316, 1–6.
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Ijzerman, A. P.; Stevens, R. C. The 2.6 Angstrom Crystal Structure of a Human A2A
Adenosine Receptor Bound to an Antagonist. Science 2008, 322, 1211–1217.
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(47) Wang, C.; Jiang, Y.; Ma, J.; Wu, H.; Wacker, D.; Katritch, V.; Han, G. W.; Liu, W.; Huang,
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Rendek, K. N.; Grotjohann, I.; Fromme, P.; Kirian, R. A.; Beyerlein, K. R.; White, T. A.;
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serial femtosecond crystallography. Nat. Protoc. 2014, 9, 2123–2134.
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Scheme 1
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Chemical structures of monoolein (9.9 MAG) and two new lipids (GlyNCOC15+4 and
GlyNMeCOC15+4) characterized in this study.
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Figure 1
Stability of LCP made of GlyNCOC15+4 and monoolein under a range of pH values (2.2ꢀ11) over
time. Both lipids were mixed with water to produce LCP, and the LCP boluses were covered with
solutions containing 150 mM NaCl, 30% PEG400 and 100 mM of the respective buffer. The buffer
solutions were taken from the StockOptions pH screen (Hampton).
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Figure 2
Crystals of bacteriorhodopsin obtained by LCP crystallization using GlyNCOC15+4 as the host lipid.
(a) Crystals grown at 20 °C. (b) Crystals grown at 4 °C.
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Figure 3
Crystal packing of bR crystallized using monoolein, PDB ID 1M0L (a, d, g) and GlyNCOC15+4 as a
host lipid for LCP at 4 °C (b, e, h) and 20 °C (c, f, i) as viewed
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(a, b, c) in the membrane plane,
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(d, e, f) along the membrane,
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(g, h, i) in the membrane plane with B factors mapped as both thickness of the lines and as a
gradient coloring with the blue color corresponding to the lowest values and the red color
corresponding to the highest values.
The core bR trimer for the 20 °C structure is shown in green and the peripheral protomers are shown
in cyan (panels c and f). Unit cells are outlined with solid black lines.
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Figure 4
Crystal contacts between bR molecules crystallized at 20 °C. A bR protomer from the trimeric core
(shown in green) was superimposed with a peripheral protomer (shown in dark blue). The structure
of the BC loop is clearly different in the respective protomers. Molecules of the core bR trimer show
a typical configuration of the BC loop containing a βꢀhairpin motif. On the other hand, the
peripheral bR protomers exhibit an extended loop conformation without the βꢀhairpin, which would
clash with its crystal neighbor, and instead with F71 positioned to potentially stabilize the packing
by a hydrophobic interaction with the next membrane layer. The symmetry mate molecules are
shown in light grey and the membrane boundary is shown as a dashed line. The membrane boundary
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was
calculated
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the
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of
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in
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Server
(http://opm.phar.umich.edu/).
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Figure 5
Crystal hits obtained from crystallization of A2AAR using GlyNCOC15+4 as the LCP host lipid.
a. Brightfield mode
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b. Crossꢀpolarized light
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c. Twoꢀphoton UV fluorescence mode
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d. SHG (second harmonic generation) mode
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Table 1
Crystallographic data collection and structure refinement statistics. Values in parentheses
correspond to the highest resolution shell.
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BR_20C (5VN7)
BR_4C (5VN9)
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Data collection
Wavelength (Å)
1.033
1.033
Number of crystals
Resolution range (Å)
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28.52 - 2.70 (2.80 - 2.70)
28.34 - 2.59 (2.69 - 2.59)
Space group
Unit cell, a b c (Å)
α β γ (°)
R 3
P 21
107.05 107.05 149.86
90 90 120
50.07 109.63 56.56
90 116.3 90
Total reflections
Unique reflections
Multiplicity
51,454 (4,126)
17,181 (1,716)
3.0 (2.4)
40,533 (1,396)
15,034 (833)
2.7 (1.7)
Completeness (%)
Mean I/sigma(I)
Wilson B-factor (Å2)
R-merge
97 (96)
90 (49)
7.4 (1.8)
5.3 (1.7)
52.8
46.5
0.151 (0.645)
0.988 (0.476)
0.143 (0.317)
0.987 (0.76)
CC1/2
Refinement
R-work/ R-free
Clashscore
0.196 / 0.225
4.4
0.223 / 0.265
5.5
A
1,748
1,728
20
B
1,740
1,720
20
A
B
Number of non-hydrogen atoms
1,766
1,735
20
1,746
1,720
20
protein
retinal
water
0
0
11
6
Average B-factor
protein
51.8
51.9
42.5
-
67.5
67.5
49.5
-
39.3
39.4
35.4
38.1
36.4
36.5
33.2
33.7
retinal
water
RMS bonds (Å)
RMS angles (°)
0.004
0.64
0.003
0.58
A
99.1
0.9
0
B
97.3
2.7
0
A
98.6
1.4
0
B
98.6
1.4
0
Ramachandran favored (%)
Ramachandran allowed (%)
Ramachandran outliers (%)
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For Table of Contents Use Only
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Chemically Stable Lipids for Membrane Protein Crystallization.
Andrii Ishchenko1, Lingling Peng2, Egor Zinovev3, Alexey Vlasov3, Sung Chang Lee2, Alexander
Kuklin3,4, Alexey Mishin5, Valentin Borshchevskiy3,6, Qinghai Zhang2*, Vadim Cherezov1,5*
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We have designed, synthesized and characterized a series of chemically stable lipids resistant to
hydrolysis capable of forming a lipidic cubic phase. The phase properties and lattice parameters of
mesophases made of two most promising lipids were characterized. One of these lipids has been
used for crystallization and structure determination of a prototypical membrane protein
bacteriorhodopsin at 4 °C and 20 °C.
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