Lipidic Cubic Phase-Induced Membrane Protein Crystallization

c Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research,. Parkville, VIC 3052, Australia d Department of Medical Biolog...
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Article Cite This: Cryst. Growth Des. 2017, 17, 5667-5674

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Lipidic Cubic Phase-Induced Membrane Protein Crystallization: Interplay Between Lipid Molecular Structure, Mesophase Structure and Properties, and Crystallogenesis Alexandru Zabara,†,#,▽ Thomas G. Meikle,‡,⊥,▽ Raphael Trenker,§,∥ Shenggen Yao,⊥ Janet Newman,# Thomas S. Peat,# Frances Separovic,‡,⊥ Charlotte E. Conn,† Melissa J. Call,§,∥ Matthew E. Call,§,∥ Ehud M. Landau,¶ and Calum J. Drummond*,† †

School of Science, College of Science, Engineering and Health, RMIT University, 124 La Trobe Street, Melbourne, VIC 3000, Australia ‡ School of Chemistry, ∥Department of Medical Biology, and ⊥Bio21 Institute, University of Melbourne, Melbourne, VIC 3010, Australia § Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia # Biomedical Manufacturing Program, Commonwealth Scientific and Industrial Research Organization (CSIRO), 343 Royal Parade, Parkville, VIC 3052, Australia ¶ Department of Chemistry, University of Zurich, 8057 Zurich, Switzerland S Supporting Information *

ABSTRACT: Obtaining well-diffracting crystals of membrane proteins, which is crucial to the molecular-level understanding of their intrinsic three-dimensional structure, dynamics, and function, represents a fundamental bottleneck in the field of structural biology. One of the major advances in the field of membrane protein structural determination was the realization that the nanostructured lipidic cubic phase (LCP) environment constitutes a membrane mimetic matrix that promotes solubilization, stabilization, and crystallization of specific membrane proteins. Despite two decades passing since the introduction of LCP-based membrane protein crystallization, the research community’s understanding of the processes that drive protein nucleation and macromolecular assembly in the LCP generally remains limited. In the current study, we present a detailed, systematic investigation into the relationship between the chemical structure of the lipid, the physical properties of the ensuing mesophase, the translational diffusion of the encapsulated membrane protein, and the resulting protein crystallization. Importantly, we show for the first time that cubic phase transport properties directly modulate the size and morphology of fully grown protein crystals without affecting their crystallographic space group. These findings provide a deeper understanding of the LCP-based crystal growth process, setting the stage for the engineering of membrane protein crystals at the molecular level via intentional design of the host cubic phase, and, ultimately, accelerating progress in the field of membrane protein structural biology.

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INTRODUCTION Research into the structure, function, and dynamics of membrane proteins and their complexes has skyrocketed in the past few decades due to their inherent biomedical significance, great diversity and ubiquitous occurrence, and importance as drug targets. They represent the target for more than 40% of all commercially available drugs.1 This class of biomolecules, comprising roughly 20−30% of the total protein population in most organisms,2 has proven significantly more difficult to address experimentally than the complementary class of soluble proteins, presenting various challenges and hurdles in the quest to determine their structure, dynamics, and intricate molecular mechanisms. To date, close to 650 unique © 2017 American Chemical Society

three-dimensional structures of these biomolecules have been deposited in the protein data bank3 as compared with approximately 100,000 structures of soluble proteins. The unique function of membrane proteins, as well as their interactions with various other cellular players in health and disease, are directly governed by, and can be elucidated from, their three-dimensional structures, which are adopted during biosynthesis and insertion into the cellular membrane. A detailed atomic understanding of a protein’s structure can, Received: April 12, 2017 Revised: June 30, 2017 Published: August 16, 2017 5667

DOI: 10.1021/acs.cgd.7b00519 Cryst. Growth Des. 2017, 17, 5667−5674

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lipid nanostructure and phase in the crystallization of membrane proteins, using synchrotron SAXS to probe phase changes occurring over the first hours of crystallization in meso.38 Multiple studies now point to the mechanistic importance of a localized lamellar phase, which forms at the site of crystal nucleation,38,39 but the properties and conditions contributing to protein crystal quality, size, and speed of growth remain poorly defined. In this study we aim to elucidate the interplay between the lipid chemical and nanoscale structure, the ensuing cubic phase transport properties (both within the hydrophilic aqueous channels and the hydrophobic lipid bilayer) and the process of membrane protein crystal nucleation and growth, examining factors such as crystal size, speed of formation, and morphology. To achieve this, we have investigated a range of monoacylglycerols (MAGs) with various chain lengths and position of the cis double bond, as well as an isoprenoid-type branched chain lipid, phytantriol (PT), as building blocks of the host matrices for crystallization of membrane proteins. The membrane protein used in this study was the E. coli virulence factor intimin, selected for its ease of expression and purification, and previous successful crystallization from LCPs. Physicochemical characterization techniques, including pulsed-field gradient NMR, fluorescence recovery after photobleaching (FRAP), and small-angle X-ray scattering (SAXS) with microscopy-based visual inspections, were combined with single crystal synchrotron X-ray diffraction in order to achieve a comprehensive understanding of how the crystallization process is affected by the molecular and bulk properties of the host phase. The results provide a significant advancement in our fundamental understanding of LCP-based crystallization and, by extension, assist the ongoing quest to increase the number of high-resolution membrane protein structures.

therefore, offer the possibility of deliberately altering its functionality to either implicitly negate or promote its molecular activity. X-ray crystallography has been the method of choice in obtaining such high-resolution structures, but this method requires the availability of well-ordered three-dimensional crystals, which has been the major bottleneck slowing progress in the field. The initial approach toward overcoming this problem employed the use of detergents, aimed at shielding the hydrophobic trans-membrane domain of the protein molecules during vapor diffusion-based crystallization experiments, and has yielded the first crystals and high resolution structures of membrane proteins.4,5 Two decades ago the fields of lyotropic liquid crystal (LLC) mesophase chemistry and membrane protein structural biology converged, leading to the utilization of the structural and dynamic properties of lipidic cubic phases (LCPs) to provide a membrane-mimetic host matrix for the stabilization, solubilization, and crystallization of these inherently unstable biomacromolecules.6 This novel approach exploits the innate ability of the LLC bilayer to provide optimal shielding for the protein hydrophobic domain, as well as providing viscoelastic and structural properties similar to the cellular membrane,7,8 and hence a closer biomimetic environment than detergent micelles for membrane protein reconstitution. The LCP is a highly curved, three-dimensional periodic liquid crystalline structure that spontaneously self-assembles across a range of lipid hydrations, and has shown great potential for applications in a number of fields ranging from drug delivery and food science to structural biology.9−14 To this point, the relationship between the composition and structure of the cubic phase and the diffusion of soluble bioactives has been well established,15−17 while the cubic phase bilayer has been shown to assist retention of functionality for large integral membrane proteins.18,19 Moreover, as was demonstrated for hydrophilic proteins, by overcoming the intrinsically small aqueous channel size of the cubic phase and thereby altering the phase transport properties, one can potentially allow for a much wider array of proteins to be crystallized as well as potentially engineer crystal growth at the molecular level.20,21 Since its initial utilization, the field of LCP-based crystallization of membrane proteins has thrived,22−24 transforming the field and enabling the determination of ∼260 highresolution structures3 for a number of previously undetermined membrane proteins,25,26 including the pharmacologically relevant class of G-Protein Coupled Receptors.27−29 A related, alternative in meso crystallization approach employed the use of a disordered sponge phase for successful membrane protein crystal growth.30−32 The sponge phase retains the fundamental bilayer lipid membrane structure but with the loss of the periodic order in three dimensions associated with the LCP. Moreover, crystallization of small hydrophilic proteins from the aqueous channels of two distinct LLC symmetries, the double diamond cubic phase and the inverse hexagonal phase, revealed the possibility of employing the various existing symmetries in order to engineer crystal growth of soluble species at the molecular level.33−35 Despite significant progress in developing high-throughput methodologies for sample preparation, LLC structural analysis, and crystal growth,36,37 our fundamental understanding of the direct interplay between the structural and molecular properties of the lipid mesophase and the process of membrane protein crystal growth remains poorly developed and calls for further investigation. In previous work, we have examined the role of

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MATERIALS AND METHODS

Materials. Monoolein (MO), monopalmitolein (MP), monovaccenin (MV), and monoeicosenoin (ME) were purchased from Nu-Chek Prep (Waterville, MN). 3,7,11,15-Tetramethyl-1,2,3-hexadecanetriol (phytantriol) (96%) was provided by DSM Nutritional Products, Germany. LCP glass plates with a 100 μm double-sided tape spacer and 200 μm plastic seals were purchased from Molecular Dimensions. Salts used for the preparation of crystallization buffers were purchased from Sigma-Aldrich (Sydney, Australia), unless otherwise stated. Expression and Purification of Intimin. Information detailing the construction of the plasmids containing the intimin E. coli O157:H7 gene has been provided previously.40 The construct was kindly provided by Dr Susan K. Buchanan from the National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, USA, and was used with no further modifications. An in depth description of the expression and purification of the membrane protein intimin is provided in the Supporting Information. Briefly, the vector containing the E. coli O157:H7 gene was transformed into BL21 (DE3) cells which were grown in Terrific Broth media (50 μg/mL kanamycin) and lysed using a probe sonicator (Misonix S4000), using 30 s bursts at 60% amplitude. Membranes containing the intimin protein were harvested using ultracentrifugation (160,000 g, 60 min, 4 °C) and resuspended in solubilization buffer (50 mM TRIS pH 8.0, 200 mM NaCl, 20 mM imidazole, 5% Elugent (Calbiochem)) by stirring overnight at 4 °C. Insoluble material was subsequently removed from the sample by ultracentrifugation (250,000 g, 120 min, 4 °C). Protein purification was conducted using a combination of affinity and ion exchange chromatography, with protein containing fractions concentrated using a YM30 Amicon Ultra concentrator (Millipore). 5668

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Table 1. Effect of Protein Reconstitution on the Symmetry and Structural Parameters of Various Host LCPsa Water

Protein (20 mg/mL)

Sample

Lattice Parameter (Å)

Water Channel Diameter (Å)

Bilayer Thickness (Å)

Lattice Parameter (Å)

Water Channel Diameter (Å)

Bilayer Thickness (Å)

ME 40% H2O MV 50% H2O MO 42% H2O MP 50% H2O PT 30% H2O

112.5 127.2 106.4 118.3 68.1

49.9 64.2 48.4 60.4 25.6

38.1 35.3 34.8 32.1 27.7

109.0 126.7 104.8 118.6 66.6

48.3 63.9 47.7 60.6 25.0

37.0 35.1 34.2 32.2 27.1

a All mesophase materials shown exhibited a QIID cubic phase (crystallographic space group Pn3m), and are listed in order of decreasing bilayer thickness.

Sample Preparation for Benchtop SAXS Experiments. SAXS samples were prepared by gently melting the lipids and mixing with a water/protein sample using a coupled syringe apparatus, resulting in formation of the mesophase. The water content used in SAXS samples was selected to approximate the excess water point for each lipid, ensuring formation of the QIID phase commonly used in crystallization experiments. Water contents were as follows: MO + 42 w/w% H2O, MP + 50 w/w% H2O, MV + 50 w/w% H2O, ME + 40 w/w% H2O, 7.7 MAG + 50 w/w% H2O, PT + 30 w/w% H2O. A needle was attached to the syringe and the mesophase injected into a 1.5 mm glass capillary, which was then sealed using wax. Samples were equilibrated overnight prior to performing measurements. SAXS diffraction patterns were recorded at 20 °C on a Bruker MicroCalix SAXS/ WAXS system and analyzed using the IDL based AXCESS software package, developed at Imperial College London.41 Pulsed-Field Gradient (PFG) NMR. Lipid mesophase samples used in PFG-NMR experiments were prepared in the same fashion as SAXS samples. The water content used in each NMR experiment was as follows: MO + 40 w/w% H2O, MP + 50 w/w% H2O, MV + 48 w/w % H2O, ME + 46 w/w% H2O, PT + 25 w/w% H2O. The mesophase was injected into a 2 mm OD glass capillary and allowed to equilibrate overnight before being placed into a 5 mm NMR tube filled with either D2O or CDCl3 and fixed in place using a Teflon adapter. The translational diffusion coefficients of lipids were measured on a Bruker Avance II 800 MHz spectrometer equipped with a TCI cryoprobe using a standard BPP-STE sequence without modification,42,43 as described previously.17 Samples were measured at 20 °C. The diffusion coefficient, D, was determined by fitting diffusion weighted intensities of well resolved peaks to the following equation using the T1/T2 relaxation module in TOPSPIN as well as OriginPro software

⎧ ⎛ δ τ⎞ ⎫ I = I0 exp⎨− γ 2s 2g 2δ 2⎜Δ − − ⎟D⎬ ⎝ ⎩ 3 2⎠ ⎭

experiments were performed on a Zeiss LSM 780 confocal microscope at 514 nm excitation bleaching a small region of interest (ROI) with a radius of 27 μm. Five prebleach images were recorded, the ROI was bleached, and recovery was recorded for a total of 6−7 min in 0.78 s intervals. The fractional fluorescence recovery was obtained by normalizing the mean fluorescence signal in the ROI to prebleach intensities and potential bleaching effects during the measurement were compensated by subtracting background fluorescence changes in nonbleached areas as previously described.46−48 High-Throughput Crystallization of Intimin. High-throughput LCP preparation and crystallization was carried out according to the literature.14 The protein solution was mixed with molten lipid to achieve a water content of 50% (v/v) for MP, 42% (v/v) for MO, 50% (v/v) for MV, 40% (v/v) for ME, 70% (v/v) for 7.7 MAG, and 30% (v/v) for PT. The LCP was dispensed onto the surface of a LCP glass plate with double-sided tape spacer (Laminex 100 μm spacer, Molecular Dimensions) in 200 nL aliquots, 1 μL of screen was dispensed on top, and a 100 μm plastic seal (Molecular Dimensions) was applied to seal the experiment. The screen was designed around three previously known conditions for intimin crystallization (Table S1), randomized to ensure reproducibility, and with the most successful conditions selected for presentation. Both the LCP and screen were dispensed using a Mosquito LCP machine equipped with a humidity chamber (TTPLabtech, UK). Sealed plates were incubated at 20 °C and imaged in a Minstrel HT/UV imaging system (Rigaku). The resulting crystals were harvested into Mylar loops and cryocooled using liquid nitrogen. Data were collected at the MX2 microfocus beamline at the Australian Synchrotron using one degree oscillations for a total of 360° of data. The data were indexed with XDS49 and scaled using Aimless.50 For the one data set with reflections beyond 3 Å (MP lipid), molecular replacement was performed with Phaser,51 using the PDB entry 4e1s as the search model. Manual building was done using Coot52 and refinement was performed using both Refmac53 and Phenix Refine54 with the dictionary cif file for MP generated by eLBOW. The PDB deposition code for this structure is 5g26.

(1)

where γ is the gyromagnetic ratio of protons and s, g, δ, and Δ are the shape factor, amplitude, duration, and separation, respectively, of the single pair of gradient pulses, while τ is the time interval within the bipolar pulse pair. Sinusoidal shaped gradient pulses, with δ of 2.5 ms (except for the phytantriol sample where δ = 4 ms), Δ of 50 ms and the amplitude of the field Gz ranging from 2.8 to 53.1 (G cm−1) were used. To calibrate the strength of pulsed-field gradient, Gz, the selfdiffusion coefficient of residual H2O in a 100% 2H2O sample was measured at 298.13 K.44,45 Fluorescence Recovery after Photobleaching (FRAP). Purified intimin was labeled with Sulfo-Cyanine3 NHS ester (lumiprobe) in 0.1 M sodium phosphate pH 8.4 according to the manufacturer’s protocol with a protein concentration of 2 mg/mL. Free dye was removed using a Micro Biospin 6 column (Bio-Rad) following the buffer exchange protocol (50 mM Tris pH 8, 0.1% n-dodecyl-β-D-maltopyranoside). The cubic phases were prepared as described above using a final labeled protein concentration of 1 mg/mL. The samples were prepared on microscopy slides with 140-μm-thick double-sided adhesive spacers, commonly used as LCP crystallization sandwich plates, and sealed with glass coverslips. The buffer mentioned above was used as precipitant solution for each measurement. FRAP

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RESULTS Small Angle X-ray Scattering Investigation of the Cubic Phase Nanostructure under Crystallization Conditions. Mismatch between the length of the lipid bilayer and the membrane protein hydrophobic domain can potentially induce irreversible structural changes within the mesophase symmetry. Therefore, in order to correlate the specific diffusive properties of the various phases with protein crystal nucleation and growth, retention of the double diamond LCP structural symmetry subsequent to protein reconstitution needs to be initially confirmed for each lipidic system. To this end, we chose specific formulations for each system whereby the respective binary lipid:water phase diagrams (i.e., devoid of added membrane protein) would yield a LCP with QIID architecture. 5669

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SAXS data for pure lipid QIID excess water mesophase systems. Values of MO and PT lipid diffusion closely match those observed for similar LCP samples in a previous study.17 The diffusion of PT was found to be significantly slower than that observed for MAG lipids, while diffusion coefficients for the MAGs decreased in the order MP > MO ≈ MV > ME. Among MAG lipids, we note an inverse correlation between the bilayer thickness of the lipid mesophase and the translational diffusion coefficient of the lipid; with the shortest chain lipids diffusing fastest. To the best of our knowledge, this is the first time that this effect has been reported for these lipid systems. Monitoring Diffusion of Membrane Protein Molecules in the LCP via FRAP. Cy3-labeled intimin was reconstituted into the LCPs of five different host lipids under otherwise identical conditions, and the diffusion characteristics of the protein in the various LCP media compared by FRAP. All recovery curves (Figure S7) could be described using a onecomponent diffusion model with mean diffusible fractions between 72% and 88%. The fastest recovery rate was observed in MP with a mean diffusion coefficient of 7.5 × 10−12 m2/s, which is higher than the diffusion coefficients of 7 × 10−12 and 6.9 × 10−12 m2/s obtained in MO and MV, respectively (Figure 2). Protein diffusion in ME and PT were significantly slower with mean values for the diffusion coefficient of 5.2 × 10−12 and 2 × 10−12 m2/s, respectively.

The structure of the ensuing mesophase, with and without reconstituted intimin, was investigated by SAXS. In all cases investigated, peaks were observed in the specific spacing ratio √2, √3, √4, √6, √8, √9, representative of the QIID diamond cubic phase (crystallographic space group Pn3m see SI Figures S1−S5). Structural parameters, including the bilayer thickness and water channel diameter, determined from SAXS (see SI for method) for the various LCPs with and without addition of intimin protein in solution at a concentration of 20 mg/mL are shown in Table 1. Regardless of the choice of lipid, addition of 20 mg/mL intimin solution did not have a significant effect on the lattice parameter, water channel diameter, and bilayer length of the host cubic phase, despite the bilayer thickness varying between 27 Å (phytantriol) to 37 Å (ME). The retention of the cubic phase nanostructure following protein reconstitution, coupled with the lack of variability of the structural parameters, suggests that hydrophobic mismatch between the protein hydrophobic domain and the lipid bilayer thickness in these systems has less of an effect than observed previously for other peptides and proteins.18,55 Thus, the reconstitution of intimin did not destabilize the structure and symmetry of the host mesophase, despite the different degrees of hydrophobic mismatch between the bilayer thickness and the protein hydrophobic core. Cubic symmetry, with or without addition of protein, was not observed in samples consisting of intimin reconstituted within the bilayer of 7.7 MAG, the MAG lipid with the shortest aliphatic tail and bilayer thickness. The system exhibited a lamellar or disordered sponge phase (depending on temperature and hydration) which cannot be directly compared with the QIID phase adopted by the other lipid systems (Figure S6). Exploring Lipid Self-Diffusion via Pulsed-Field Gradient NMR. Pulsed-field gradient NMR was used to determine the lipid translational diffusion coefficients of MP, MO, MV, ME, and PT when each was reconstituted with pure water to form the QIID cubic phase. Sample inhomogeneity precluded PFG-NMR measurements in the case of 7.7 MAG. The apparent diffusion coefficients range from 2.0 × 10−12 m2 s−1 (PT) to 1.8 × 10−11 m2 s−1 (MP), and are presented in Figure 1 as a function of bilayer thickness, which was calculated from

Figure 2. Plot of experimentally determined diffusion coefficients of intimin reconstituted in a series of MAG lipids and phytantriol versus mesophase bilayer thickness, calculated from SAXS measurements of pure lipid excess water mesophase systems. Error bars indicate ±1 SD.

We note a strong correlation between the diffusion coefficient of lipid molecules and the protein diffusion coefficient, which decreases in the order MP > MO ≈ MV > ME > PT. This is not unexpected as intimin, a hydrophobic protein, should reside mainly within the bilayer. As with lipid molecule diffusion, the diffusion of intimin reconstituted in the various MAG bilayers could be directly linked with mesophase bilayer thicknesses; membrane protein molecules are found to diffuse faster within the shorter chained lipids such as MP, compared to lipids with longer aliphatic tails such as MV or ME. Diffusion within the bilayer composed of PT, which features a branched saturated hydrocarbon tail, was significantly slower than in MAG bilayers. This may result from increased

Figure 1. Plot of lipid diffusion coefficients determined experimentally using PFG-NMR versus bilayer thickness, calculated from SAXS measurements of pure lipid excess water mesophase systems. 5670

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crystallization; 142 mM MgCl2, 34.7% v/v PEG 400, 92 mM NaCl, 100 mM trisodium citrate-citric acid, pH 5.6 for MV crystallization; and 84 mM MgCl2, 26.2% v/v PEG 400, 99 mM NaCl, 100 mM trisodium citrate-citric acid, pH 4.92 for 7.7 MAG crystallization. In each lipid mesophase the screen conditions leading to optimal crystallization contained identical compounds present in similar concentrations. We assume, based upon previous studies,56,57 and the large difference in crystal morphology between lipids, that small variations in screen component concentrations exert only minor influence on the overall thermodynamics and kinetics of intimin protein crystallization, and thus allow for adequate comparison between different lipids. Specifically, thin, needle-shaped crystal clusters were consistently observed in both MP- and MO-based crystallization trials. The crystals growing from within the MP cubic phase were much longer and the time needed for nucleation and maximum crystal growth was considerably shorter (Table 2). These were first observed after less than 24 h and reached

steric interactions and lateral pressure within the bilayer, indicating the inefficacy of PT as a membrane protein hosting matrix throughout the process of LCP-based crystallization. Monitoring the Effects of Host LCP Properties on Intimin Crystallization. Crystallization trials were designed to assess the effect of lipid and protein diffusion on protein crystal growth and morphology. Crystal growth was also observed from the 7.7 MAG matrix, although we note that the results cannot be directly compared with the growth from the other lipid matrices due to the induced transition from a cubic to a disordered sponge phase. Representative images of intimin crystals, grown from the lipidic cubic mesophase systems, are presented in Figure 3.

Table 2. Overview of the Time Required by the Intimin Molecules within Each of the Six Tested Lipidic Phases from Protein Reconstitution to Microcrystal Formation and, Respectively, to Maximum Crystal Growtha

a

lipid

time for microcrystal formation

max. crystal size reached

7:7 MP MO MV ME PT