Host Lipid and Temperature as Important Screening Variables for Crystallizing Integral Membrane Proteins in Lipidic Mesophases. Trials with Diacylglycerol Kinase

Abstract

A systematic study of the crystallization of an α-helical, integral membrane enzyme, diacylglycerol kinase, DgkA, using the lipidic cubic mesophase or in meso method is described. These trials have resulted in the production of blocky, rhombohedron-shaped crystals of diffraction quality currently in use for structure determination. Dramatic improvements in crystal quality were obtained when the identity of the lipid used to form the mesophase bilayer into which the protein was reconstituted as a prelude to crystallogenesis was varied. These monoacylglycerol lipids incorporated fatty acyl chains ranging from 14 to 18 carbon atoms long with cis olefinic bonds located toward the middle of the chain. Best crystals were obtained with a lipid that had an acyl chain 15 carbon atoms long with the double bond between carbons 7 and 8. It is speculated that the effectiveness of this lipid derives from hydrophobic mismatch between the target integral membrane protein and the bilayer of the host mesophase. Low temperature (4 °C) worked in concert with the short chain lipid to provide high quality crystals. Recommended screening strategies for crystallizing membrane proteins that include host lipid type and low temperature are made on the basis of this and related in meso crystallization trials.

Keywords: DAGK; DgkA; X-ray crystallography; integral membrane enzyme; lipid cubic phase; mesophase; screen; sponge phase; α-helical membrane protein structure.

Fig. 1

Cartoon representation of the events proposed to take place during the crystallization of an integral membrane protein from the lipidic cubic mesophase. The process begins with the protein reconstituted into the curved bilayer of the ‘bicontinuous’ cubic phase (tan). Added ‘precipitants’ shift the equilibrium away from stability in the cubic membrane. This leads to phase separation wherein protein molecules diffuse from the bicontinuous bilayered reservoir of the cubic phase into a sheet-like or lamellar domain (A) and locally concentrate therein in a process that progresses to nucleation and crystal growth (B). Cocrystallization of the protein with native lipid (cholesterol) is shown in this illustration. As much as possible, the dimensions of the lipid (tan oval with tail), detergent (pink oval with tail), cholesterol (purple), protein (blue and green; β₂-adrenergic receptor-T4 lysozyme fusion; PDB code 2RH1), bilayer and aqueous channels (dark blue) have been drawn to scale. The lipid bilayer is ~40 Å thick. From reference with permission. An expanded view of the various components in the system is shown in (C).

Fig. 2

Purification of WT and Δ4 DgkA. Size exclusion chromatographic analysis of WT DgkA (A) and Δ4 DgkA (B). V_o and V_t mark the void and total column volumes, respectively. The elution volume (V_e) for both WT and Δ4 DgkA is 76.8 mL. The near Gaussian shaped elution profiles are consistent with a monodisperse protein preparation suitable for crystallization trials. Protein purity of the WT DgkA (C) and Δ4 DgkA (D) post-size exclusion chromatography was estimated at >95 % on the basis of a loading series analysis by SDS-PAGE and Coomassie Blue staining. The high molecular weight bands observed at the 50 μg loading in (C) are likely oligomers of DgkA. In (C), WT DgkA migrates as a monomer at the expected molecular weight of ~14 kDa at low loading. At higher loading, it migrates progressively slower. This may relate to the fact that the lower loading samples were prepared by diluting concentrated samples with SDS loading buffer. Thus, the SDS/DM ratio in the latter samples and thus bound to the protein is less. This reduces the associated negative charge on the protein accounting for its slower migration on the gel. Interestingly, Δ4 DgkA was prepared in the same way as WT DgkA but did not display the same behaviour. By contrast, it migrates as a trimer with anomalously lower apparent molecular weight, as reported previously.

Figure 3

In surfo-grown crystal of WT DgkA. Crystals were grown for 10 days at 20 °C by the hanging drop vapour diffusion method using 100 nL each of reservoir and protein solutions. The reservoir solution consisted of 30 %(v/v) PEG 400, 0.1 M HEPES/Na pH 7.5. The protein solution included 16 mg DgkA/mL, 4.6 %(w/v) DM, 1 mM TCEP, 150 mM NaCl, 10 mM Tris HCl pH 7.4. Images were recorded with normal light (A) and between crossed-polarizers (B).

Fig. 4

Initial crystallization hits observed with WT and Δ4 DgkA in 9.9 MAG at 20 °C using commercial screens. A: Day 0, WT DgkA. B: Day 1, WT DgkA. C: Expanded view of B. D: Day 0, Δ4 DgkA. E: Day 1, Δ4 DgkA. F: Expanded view of E. Brownish colored ‘aggregated’ protein formed almost immediately after the trials were set up. Micro-crystals appeared within about 16 h. The composition of the diluted precipitant solution is 7.8 %(v/v) MPD, 0.07 M sodium chloride, 0.07 M sodium citrate/HCl pH 5.6. Images were recorded using the RI-1500 imager (Formulatrix).

Fig. 5

Effect of temperature on the crystallization of WT DgkA in monoolein. A: 20 °C, B: 10 °C. C: 4 °C. The corresponding crystal sizes are A: 5 μm. B: 15 μm. C: 70 μm. Images were recorded 2 months post-setup. The composition of the precipitant solution is 7.8 %(v/v) MPD, 0.1 M sodium chloride, 0.1 M sodium citrate pH 5.6. Images in A and B were recorded with the RI-1500 imager at 20 °C; the image in C was recorded with the RI-54 imager at 4–6 °C (Formulatrix).

Fig. 6

Effect of nitrate salts on the in meso crystallization of WT and Δ4 DgkA at 4 °C in 9.9 MAG. Crystals of WT DgkA (A–D) and Δ4 DgkA (E–H) were grown in a precipitant consisting of 7.8 % MPD, 0.1 M sodium chloride, 0.1 M sodium citrate pH 5.6 supplemented with 0.1 M nitrate salts of potassium (A, E), lithium (B, F), sodium and ammonium (D, H). A small crystal in (A) resides at the tip of the solid arrow. The dotted arrow in (A) points to one of several droplets in the mesophase that should not be mistaken for crystals. Images were recorded using the RI-54 imager at 4–6 °C.

Fig. 7

In meso grown crystals of WT DgkA (A) and Δ4 DgkA (B) obtained at 4 °C in monoolein after extensive optimization. The precipitant in (A) contains 7.9 %(v/v) MPD, 0.08 M sodium chloride, 0.12 M potassium nitrate, 0.1 M sodium citrate pH 5.6. The condition in (B) contains 8.0 %(v/v) MPD, 0.11 M sodium chloride, 0.1 M lithium nitrate, 0.1 M sodium citrate pH 5.6. Images were recorded 60 days post-setup. Images were recorded using the RI-54 imager at 4–6 °C.

Fig. 8

Effect of hosting lipid and temperature on the in meso crystallization of Δ4 DgkA. Small crystals (arrows) were grown in 7.8 MAG (A), 8.9 MAG (B) and 9.7 MAG (C) at 20 °C. Relatively large, blocky crystals grew in 8.7 MAG (D), 8.9 MAG (E), 9.7 MAG (F) and 7.8 MAG (G) at 4 °C. Images were recorded in normal light (A–F) and between crossed polarizers (G). The following precipitant conditions apply for 8.9 MAG and 9.7 MAG: 6.5–7.5 %(v/v) MPD, 0.1 M sodium chloride, 0.1 M potassium nitrate, 0.1 M sodium citrate pH 5.6; for 7.8 MAG and 8.7 MAG: 4–6 %(v/v) MPD, 60 mM magnesium acetate, 0.05–0. 15 M sodium chloride, 0.1 M potassium nitrate, 0.05 M sodium citrate pH 5.6. Images in (A–C) were recorded using the RI-1500 imager at 20 °C while images in (D–G) were taken using a Nikon Coolpix 4500 camera attached to an Eclipse E 400 Pol microscope at 4–6 °C.

Fig. 9

X-ray diffraction from a Δ4 DgkA crystal grown in meso in 7.8 MAG at 4 °C. Data were recorded on a Pilatus 6M detector with 0.978 Å wavelength X-rays, a 0.2 ° oscillation and a 0.2 s exposure per image, a micro-focus beam size of 10 μm and a sample-to-detector distance of 645.6 mm.

Fig. 10

Steps taken and the order in which they were done to effect the production of diffraction quality crystals of DgkA by the in meso method. Wild-type and thermo-stabilized mutant variants of the kinase were used in the study. Square brackets refer to concentration.