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In Meso Crystallization of the Integral Membrane Glycerol 3-phosphate Acyltransferase with Substrates Zhenjian Li, Yannan Tang, and Dianfan Li Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b01678 • Publication Date (Web): 12 Mar 2018 Downloaded from http://pubs.acs.org on March 12, 2018

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Crystal Growth & Design

Cover Page In Meso Crystallization of the Integral Membrane Glycerol 3-phosphate Acyltransferase with Substrates Zhenjian Li1, §, Yannan Tang1, 2, §, Dianfan Li1,* 1 National Center for Protein Science Shanghai, Shanghai Science Research Center, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 333 Haike Road, Shanghai 201210, China. 2University of Chinese Academy of Sciences, 333 Haike Road, Shanghai 201210, China. § These authors contributed equally to this work. Abstract: Here we retrospectively report the successful application of the “host lipid screening” strategy in the in meso crystallization of PlsY, a bacterial glycerol 3-phosphate (G3P) acyltransferase that catalyzes the committed step in phospholipid biosynthesis. Two monoacylglycerols (MAGs) with different chain length, namely the 9.9 MAG (monoolein, nine carbons on either side of the cis-double bond in the acyl chain) and the 7.8 MAG, were the two main host lipids used in this study. The native PlsY crystals growing in 9.9 MAG diffracted to 1.62 Å. However, the selenomethioninyl crystals growing in the same host lipid under similar precipitant conditions diffracted poorly (4.3 Å). Switching the host lipid to 7.8 MAG dramatically improved crystal quality in both size and diffraction, yielding a 2.0-Å dataset, with which the structure was solved. Along with low-temperature crystallization, 7.8 MAG was also critical for cocrystallization of PlsY with the unstable lipid substrate, acyl phosphate (acylP). Interestingly, however, 7.8 MAG was unsuitable for PlsY-G3P co-crystallization as multiple crystal forms obtained from an extensive screening did not reveal electron density for this water-soluble substrate. By contrast, 9.9 MAG supported the co-crystallization, yielding a 2.37 Å dataset for the PlsY-G3P complex. We hypothesize that, under crystallization conditions, G3P binding which occurs at the membrane boundary favors a more rigid mesophase lipid bilayer formed by the longer-chain 9.9 MAG.

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*

Corresponding Author Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 333 Haike Road, Shanghai 201210, China. Phone: +86 21 2077 8212 Email: [email protected]

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Crystal Growth & Design 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Title Page In Meso Crystallization of the Integral Membrane Glycerol 3-phosphate Acyltransferase with Substrates Zhenjian Li1, §, Yannan Tang1, 2, §, Dianfan Li1,* 1 National Center for Protein Science Shanghai, Shanghai Science Research Center, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 333 Haike Road, Shanghai 201210, China. 2 University of Chinese Academy of Sciences, 333 Haike Road, Shanghai 201210, China. § These authors contributed equally to this work. * Corresponding author. E-mail: [email protected] Abstract: Here we retrospectively report the successful application of the “host lipid screening” strategy in the in meso crystallization of PlsY, a bacterial glycerol 3-phosphate (G3P) acyltransferase that catalyzes the committed step in phospholipid biosynthesis. Two monoacylglycerols (MAGs) with different chain length, namely the 9.9 MAG (monoolein, nine carbons on either side of the cis-double bond in the acyl chain) and the 7.8 MAG, were the two main host lipids used in this study. The native PlsY crystals growing in 9.9 MAG diffracted to 1.62 Å. However, the selenomethioninyl crystals growing in the same host lipid under similar precipitant conditions diffracted poorly (4.3 Å). Switching the host lipid to 7.8 MAG dramatically improved crystal quality in both size and diffraction, yielding a 2.0-Å dataset, with which the structure was solved. Along with low-temperature crystallization, 7.8 MAG was also critical for cocrystallization of PlsY with the unstable lipid substrate, acyl phosphate (acylP). Interestingly, however, 7.8 MAG was unsuitable for PlsY-G3P co-crystallization as multiple crystal forms obtained from an extensive screening did not reveal electron density for this water-soluble substrate. By contrast, 9.9 MAG supported the co-crystallization, yielding a 2.37 Å dataset for the PlsY-G3P complex. We hypothesize that, under crystallization conditions, G3P binding which occurs at the membrane boundary favors a more rigid mesophase lipid bilayer formed by the longer-chain 9.9 MAG.


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1. Introduction Membrane proteins carry out diverse and critical functions such as uptake of nutrients, extrusion of toxic compounds, conducting ions, transduction of extracellular signals, and generating lipids as biomembrane building blocks. They account for an approximate one-third of genomes and 60% of current drug targets.1 As such, understanding how membrane proteins work at the molecular level is very important for both fundamental biology and drug discovery. Being capable of providing atomic resolutions, macromolecule crystallography has been the main approach for such investigations.2 Essential to X-ray crystallography is crystallization, a challenging process for macromolecules, especially membrane proteins. The in meso method, which makes use of lipid cubic phase (LCP), has shown great promises for membrane protein crystallization.3-5 LCP is formed by mixing an aqueous solution with certain neutral lipids, such as monoacylglycerols (MAGs) and their derivatives. The transparent, viscous and sticky material composes a continuous lipid bilayer which separates two continuous water channels. Its bicontinuous structure facilitates diffusion of the reconstituted membrane proteins along the curved and open bilayer, as well as diffusion of hydrophilic molecules (such as precipitants) within the water channels, both of which are required for crystallization to occur. Proposedly, in a successful crystallization event, precipitants trigger the formation of local lamellar phases which contain stacked sheets of bilayers. Membrane protein packs orderly in the multi-layer phases to form nuclei, drawing more molecules from the bulk cubic phase to form crystals.6,7 The in meso method has been responsible for over 430 membrane protein structures since its first introduction8 two decades ago. Importantly, the in meso method was critical for the success of several projects where the traditional in surfo (in detergents)3 method produced either no crystals or crystals of poor diffraction quality. Such projects include the determination of the first highresolution structure of a G-protein coupled receptor (GPCR)9,10 in 2007 and most of GPCR structures ever since,5 the GPCR-Gs complex,11 the rhodopsin-arrestin complex,12,13 the microbial diacylglycerol kinase,14 and the caa3-type respiratory complex IV.15 The biomembrane alike structure probably provides a more stabilizing environment than monolayer micelles, and therefore in meso crystals generally diffract to higher resolutions than in surfo crystals. In addition, in meso crystals thus far all have type-I packing which generally features low solvent contents and high-resolution diffraction.5 The development of the in meso methodology have benefited from studies in the following aspects. The phase behavior of several MAGs (mostly 9.9 MAG) under different hydration levels and temperatures,16,17 and in the presence of a wide range of chemicals including native lipids,18 detergents,19,20 salts,21 and polymers21 has been characterized in detail, providing guidelines in choosing the right conditions for making LCP. The invention of mesophase-making devices,22 special glass plates,23 crystallization robots24,25 and imaging systems23,26-28 enabled easier handling, automation, miniaturization, and rapid screening. Special manipulation techniques and toolsets in crystal soaking,29 harvesting,29 centering30 and in situ synchrotron data collection31-33 have made the method more applicable. New lipids that were rationally designed for lowtemperature crystallization34,35 or higher stability34,36,37 have enriched the choices and opened new



dimensions for crystal optimization. The combination of the in meso method with serial femtosecond crystallography (SFX) has been very successful.12,38-44 High-resolution and radiation-damage-free diffraction data could be collected at room temperature from crystals that were of sub-micron sizes. Related techniques such as biochemical assay,45 refolding,46 and concentration of membrane proteins in meso46,47 offered new opportunities for preparation and characterization of challenging samples. In meso crystallization often generates microcrystals of only a few microns in size from initial wide-screening experiments.48-50 SFX is still far from routine and therefore optimization is often required to improve crystal sizes for data collection at synchrotron facilities. Apart from the common refinement protocols for protein/precipitant concentrations and pH, host lipids are important variables for optimization. The “host lipid screening” strategy was reported in 2011 with the carbohydrate transporter OprB from Pseudomonas aeruginosa.49 Thus, crystallization of the -barrel porin was carried out in a number of N.T MAGs45 (‘N’ represents the number of carbons in the ‘Neck’ region which is the glycerol end of the cis-double bond in the acyl chain, and ‘T’ represents the number of carbons in the opposite ‘Tail’ region), including 6.8 MAG, 6.9 MAG, 7.7 MAG, 7.8 MAG, 8.7 MAG and 8.8 MAG. 7.8 MAG performed the best as judged by both crystal size and diffraction quality. Later, the strategy was applied to the optimization of the microbial diacylglycerol kinase (DgkA), and 7.8 MAG again produced bigger crystals than other host lipids.50 In addition, the DgkA study suggested temperature and salts as two important variables for optimization. The combination of host-lipid/salt screening and low-temperature crystallization was later applied to the crystallization of the human prostaglandin E2 synthase 1 (mPGES1) whereby diffraction-quality crystals were obtained rapidly.48 These results encouraged the use of non-monoolein lipids in a number of projects with great success, including the caa3type oxidase,48 gramicidin,51 the alginate transporter AlgE,52 the peptide transporter PepT,53 the GPCR-Gs complex,11 and the rhodopsin-arrestin complex.12,13 Recently we solved the in meso structure of PlsY,54 an integral membrane enzyme that catalyzes the acylation of glycerol 3-phosphate (G3P) using acyl phosphate (acylP) as the donor.55,56 Here, we retrospectively report the design and rationale of the crystallization process. After failing in improving the crystal quality of PlsY from Escherichia coli, we switched to an ultra-stable ortholog from Aquifex aeolicus and obtained high-resolution diffraction data (1.62 Å) with crystals growing in 9.9 MAG. Subsequent optimization with the selenomethionine (SeMet)labeled protein identified 7.8 MAG and 7.9 MAG as key host lipids for generating high-quality anomalous signal for experimental phasing. In addition, we found that 7.8 MAG and lowtemperature crystallization were important for co-crystallization of PlsY with its unstable lipid substrate acylP, whereas 9.9 MAG was required for the co-crystallization of PlsY-G3P. Our experience once again demonstrates the importance of protein stability, temperature, and host lipids for in meso crystallization.


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2. Materials and Methods 2.1 Materials Monoolein (9.9 MAG, Cat. M239) was purchased from Nu-Chek Prep (Elysian, MN). Nonmonoolein lipids 7.7 MAG (Cat. 850530O), 7.8 MAG (Cat. 850531O), 7.9 MAG (Cat. 850534O), and 8.8 MAG (Cat. 850536O) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). ndodecyl--D-maltoside (DDM, Cat. C24H46011) for solubilization was purchased from Exceed Bio Inc (Shanghai, China). DDM (Cat. D310S) for size exclusion chromatography, n-decyl--Dmaltoside (DM, Cat. D322S) and n-octyl--D-glucopyranoside (OG, Cat. O311) were from Anatrace (Maumee, OH, USA). Polyethylene glycol (PEG) 400 (Cat. HR2-603) was sourced from Hampton Research (Aliso Viejo, CA, USA). Triethylene glycol (TEG, Cat. 95126) was purchased from Sigma (St. Louis, MO, USA). Crystallization kits were obtained as follows: JBScreen Membrane HTS (Cat. CS-310), Jena Biosciences (Jena, Germany); MbClass Suite (Cat. 130711), Qiagen (Hilden, Germany); MemStart & MemSys HT-96 (Cat. MD1-33), MemGold (Cat. MD139), and MemGold2 (MD1-86), Molecular Dimensions (Newmarket, Suffolk, UK); MemFac HT (Cat. HR2-137) and StockOptions Salt (Cat. HR2-245), Hampton Research. SeMet (Cat. 114587) was purchased from J&K Scientific (Beijing, China). Palmitoyl phosphate was synthesized as described.54,55 Salts and buffer were from Sigma and Ameresco (Solon, OH, USA). 2.2 Methods 2.2.1 PlsY purification The cloning, expression, and purification of PlsY from E. coli (ecPlsY) and A. aeolicus (aaPlsY) have been reported.54 Briefly, plsY from E. coli was amplified using genomic DNA as template and cloned into a modified pET vector containing the encoding sequence of, from 5’-3’, the 3C protease cleavage site, the green fluorescence protein (GFP) and the 8 × His tag. plsY from A. aeolicus was codon-optimized and synthesized by overlapping polymerase chain reaction (PCR) using primers of 59-nt or shorter. Both constructs were verified by sequencing. E. coli BL21 (DE3) cells carrying the plasmids above were grown to OD600 of 0.6-0.8 at 20 °C in M9 minimum medium before induced with 0.05 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 20-22 h. For SeMet labeling, methionine biosynthesis was suppressed prior to induction by the addition of 100 mg L-1 of Lys, Phe, and Thr, and 50 mg L-1 of Ile, Leu, and Val.57 L-SeMet was added at the time of IPTG induction. PlsY purification was performed at 4 °C unless specified otherwise. Cells were lysed by passing through a cell disruptor at 25 kpsi for three times. Cellular debris and remaining intact cells were removed by centrifugation at 20,000 g for 30 min. The supernatant was centrifuged further at 150,000 g for 1.5 h. Membrane pellets from the ultra-centrifugation were solubilized with 1 %(w/v) of DDM for 1.5 h. Insoluble materials were removed by centrifugation at 48,000 g for 1 h. The supernatant containing DDM-solubilized PlsY was heated at 65 °C for 10 min (for aaPlsY only), and the precipitates were removed by centrifugation at 20,000 g for 30 min. The PlsY-GFP fusion protein was purified using immobilized metal affinity chromatography (IMAC) as follows. The supernatant was mixed with Ni-NTA resin for 2 h for batch-binding. The resin was packed to a gravity column and washed successively with 10 mM and 45 mM imidazole. The fusion protein was then eluted with 0.25 M imidazole and digested with 3C protease to generate tag-free PlsY which was separated from His-tagged GFP and the protease by a second IMAC. PlsY was



concentrated and applied onto a Superdex 200 10/300 GL column for size exclusion chromatography (SEC). Pooled fractions were concentrated to 20 mg mL-1 for crystallization. For in surfo crystallization, DDM was exchanged to DM or OG as follows. After the washing step for the first IMAC, the column was washed further with either 1.2 %(w/v) OG or 0.25 %(w/v) DM in a Tris buffer containing 0.15 M NaCl and 10 mM Tris-HCl pH 8.0. DM or OG was included in place of DDM in the downstream elution and gel filtration steps. 2.2.2 Purification of 3C protease The detailed protocol for the purification of 3C protease was also published previously.54 Briefly, the protease was expressed in E. coli BL21 (DE3) cells as a fusion protein, from N- to C-terminus, as glutathione S-transferase (GST), 3C protease site (LEVLFQ^GP), 8×His tag and the protease, using a pET-backbone plasmid. The inclusion of GST improved the expression yield of the protease, and the GST tag was self-cleaved in vivo simultaneously. To purify the His-tagged protease, cell lysate was centrifuged to remove cell debris. The supernatant was applied to a NiNTA column. After two successive washing steps with 0 and 50 mM imidazole in a buffer containing 0.2 mM TCEP, 0.4 M of NaCl, and 20 mM Tris-HCl pH 8.0, the protease was eluted using 0.25 M imidazole in the buffer above, concentrated to 4 mg mL-1, flash frozen in liquid nitrogen and stored at -80 °C. 2.2.3 Detergent concentration measurement DDM concentrations were measured using the phenol-sulfuric acid method, as reported.54,58 For standard curve, 6 L of DDM solution ranging from 0.02 to 0.1 %(w/v) was mixed with 90 L of concentrated H2SO4. The mixture was added with 18 L of 5 %(w/v) phenol, heated at 90 °C for 5 min for colour development, and cooled down to room temperature (RT, 22-25°C). A490 of the standards was measured using a plate reader with 90 L of solution in a bottom-clear 384-well plate. The protein samples were diluted appropriately before the assay to ensure the A490 values were within those of the standards (typically 0.2-1.0). Measurements were performed in triplicates. 2.2.4 Detergent removal during concentration It has been reported that -cyclodextrin (-CD) bound DDM at an equal molar ratio and the complex could penetrate the concentrator membranes in spin-columns,59 reducing detergent concentration in the retained membrane protein fraction. This procedure was used in our recent paper.54 Briefly, A total volume of 4.2 mL of a pooled fraction of aaPlsY from SEC, which contained 0.77 mg mL-1 of protein and 0.5 %(w/v) of DDM, was mixed with 2.4 mL of 10 mM -CD. The protein was concentrated with a 50-kDa cut-off membrane to 0.9 mL before added with an additional 0.9 mL of 10 mM -CD. The diluted sample was re-concentrated to 0.38 mL, added with 0.43 mL of -CD and concentrated again to 22 mg mL-1. To wash out residual -CD which would otherwise interfere with the DDM concentration measurement, the sample was diluted 10 times and re-concentrated to 20 mg mL-1. The dilution and concentration process was repeated once. Using this method, the DDM concentration in a 20 mg mL-1 protein sample was reduced from 4.5-5.0 %(w/v) to 1.6-1.8 %(w/v). 2.2.5 Hydrolase activity assay of aaPlsY in LCP The hydrolase activity of aaPlsY against acylP was carried out as described.54 Briefly, aaPlsY (10


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mg-1 mL-1) was mixed with 9.9 MAG doped with 0.67 mol% acylP to form LCP. The LCP was transferred into a 50-L syringe attached to a 50-step dispenser (Cat. PB600-1, Hamilton). A 2cm needle (22 gauge, Cat. 7770-02, Hamilton) was fitted to the syringe. One microliter of LCP was dispensed onto the wall of a black, bottom clear half-area plate (Cat. CLS3994, Sigma) and soaked three times with 0.16 mL phosphate-free buffer for 5 min. Subsequently, the well was added with 50 L of pre-warmed (30 °C) assay buffer containing 8 M phosphate-biosensor54 to monitor the phosphate-releasing hydrolase activity by recording fluorescence (excitation wavelength 445 nm, emission wavelength 500 nm) for 1 h at 30 sec intervals in a SpectraMax M5 (Molecular Devices) plate reader at 30 °C. Control experiments were carried out exactly in the same manner but omitting aaPlsY. Three independent experiments were performed, with three replicates for each experiment. 2.2.6 In surfo crystallization The in surfo crystallization of aaPlsY was carried out by depositing 300 nL of protein solution onto the wells of 2-well sitting-drop plates, followed by covering the protein solution with 300 nL of precipitant solution taken from the 40-L reservoir. Plates were sealed with transparent tape (Hampton Research, Cat. HR4-506), and placed in a Formulatrix RockImager-1000 at 20 °C. The crystallization process was monitored on day-0, 1, 3, 5, 7, 14 and 30. 2.2.7 In meso crystallization LCP was made by mixing PlsY at 20-22 mg mL-1 with lipids at the volume ratio of 2:3 (for 9.9 MAG, 7.9 MAG, and 8.8 MAG) or 1:1 (for 7.7 MAG and 7.8 MAG) in a coupled syringe device.22 In the case of acylP co-crystallization, the acylP powder was added into the syringe that was to be loaded with aaPlsY. In the case of G3P co-crystallization, G3P was added to the precipitant solution at 20-100 mM. To set up crystallization, 50 nL of LCP was deposited onto 96-well glass sandwich plates with 6-mm-diameter wells, before being covered with 800 nL of precipitant solution using a Gryphon LCP robot (Art Robbins, Sunnyvale, CA). The plates were sealed and placed in RockImager-1000 either at 20 °C or 4 °C for crystal growth. The crystallization process was typically monitored on day-0, 1, 3, 5, 7, 14 and 30. For the trials with acylP where crystals tend to dissolve over time, the process was monitored at 12-hour intervals. 2.2.8 Crystal harvesting Desired wells were cut open using a pointy glass cutter under a microscope (Model Olympus SZX16), as described.60 The detached cover glass was lifted open using fine tweezers (Cat. 01035/90-PO, Dumont). Exposed crystals were harvested using MiTeGen loops (Cat. M2-L18SP-30; M2-L18SP-50; M2L18SP-75; M2-L18SP-100) that approximately match the crystal size. The crystals were rapidly plunged into liquid nitrogen and stored till X-ray diffraction experiments. 2.2.9 Data collection, and structure determination X-ray diffraction data were collected at the BL18U1 or BL19U1 beamlines at the National Center for Protein Sciences Shanghai (NCPSS), and BL17U1 at Shanghai Synchrotron Radiation Facility (SSRF).61 Crystals were mostly invisible at beamlines. At BL18U1 and BL19U1, crystals were centered by manual rastering guided by X-ray diffraction collected on a Pilatus 6M detector. At BL17U1, crystals were centered by automatic rastering guided by diffraction data collected on an ADSC Q315r CCD detector. The beam sizes were 20 × 20 and 50 × 50 microns at BL18U1 and



BL19U1, respectively, and was in the adjustable range between 10 × 10 to 50 × 50 microns at BL17U1. Detector-to-crystal distances were set appropriately according to the diffraction quality of crystals. Oscillation of 0.2 s was set for the Pilatus detector, and 1 s for the CCD detector. Diffraction data were processed with XDS,62 and scaled and merged with Aimless.63 Phenix Autosol, Autobuild, and Refinement64 modules were used to solve, build and refine the structures, respectively. The interactive model building was carried out using Coot65 as guided by improved maps generated by Phenix.Refine. Structure presentations were made using Pymol. 3. Results 3.1 ecPlsY purification PlsY was identified as a G3P acyltransferase from Streptococcus pneumonia (spPlsY) by Rock and coworkers.56 PlsY orthologs were found ubiquitously in bacteria.56 The E. coli ortholog (ecPlsY), which is 34.7% identical and 51.8% similar with spPlsY, was initially chosen for structural study. PlsY proteins are composed of ~200 residues and spPlsY was reported to cross membrane five times with a cytoplasmic C-terminus.55 This C-in topology allows convenient monitoring of recombinant overexpression using a C-terminal GFP tag.66 Accordingly, we overexpressed the fusion protein, from N- to C-terminal, as the ecPlsY, 3C protease site (LEVLFQ^GP), GFP, and 8 × His tag. The expression level was quantified as 10 mg per litre of culture, which is sufficient for structural studies. The ecPlsY-GFP fusion protein was purified using Ni-NTA beads. The His-tagged GFP fusion was removed by cleavage with His-tagged 3C protease and a reverse IMAC step. Purified tagfree ecPlsY showed a monodisperse, Gaussian-shaped peak on size exclusion chromatography (Fig. 1A), suggesting a homogeneous sample. Based on the series loading on SDS-PAGE (Fig. 1A, inset), the purity was estimated to be at least 92 %. The protein was concentrated to 30 mg mL-1 as an optically clear solution, which is suitable for crystallization trials.

Fig. 1. Purification and crystallization of ecPlsY. (A) Size exclusion chromatography of ecPlsY. The void volume (Vo) and total volume (Vt) are appropriately labeled. SDS-PAGE of series loading of the purified ecPlsY is shown in the inset. The loading amounts were 1 g (lane 1), 4.4 g (lane 2), 11 g (lane 3), and 45 g (lane 4). Molecular weights are indicated (but not shown) on the right. (B, C) Initial hits from crystallization trials of ecPlsY. Images were taken under bright light (B) and with polarized light (C). An arrow indicates a typical birefringent crystal. The crystals were obtained at 20 °C in the 9.9 MAG mesophase with the precipitant solution containing 30 %(w/v) PEG 500 DME, 0.1 M NaCl, 0.1 M CaCl2 and 0.1 M Tris-HCl pH 8.0.


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Fig. 2. Purification and crystallization of aaPlsY. (A) Size exclusion chromatography of aaPlsY. The void volume (Vo) and total volume (Vt) are appropriately labeled. (B) SDS-PAGE of series loading of the purified aaPlsY. The loading amounts were 0.63 g (lane 1), 1.25 g (lane 2), 2.5 g (lane 3), 5 g (lane 4), 10 g (lane 5), and 20 g (lane 6). Molecular weights are labeled on the left. (C, D) Small triangular pyramid crystals growing in 9.9 MAG at 20 °C. The precipitant solution contained 28 %(v/v) TEG, 0.1 M (NH4)2SO4, and 0.1 M glycine pH 3.8. Images were taken on day-2 under bright light (C) and with polarized light (D). An arrow indicates a typical birefringent crystal.

3.2 In meso crystallization of ecPlsY The initial in meso crystallization screening of ecPlsY yielded crystals in a precipitant condition containing polyethylene glycol 500 dimethyl ether (PEG500 DME) within three days (Fig. 1B, 1C). The crystals were small (5-8 m in the longest dimension) and birefringent, resembling the initial hits of several membrane protein crystallization projects such as DgkA,50 mPGES1,48 and OprB.49 We reasoned that the crystals were of ecPlsY because control experiments with proteinfree buffer produced no such crystals. Extensive optimization, including temperature (4 °C and 20 °C), protein concentration (10-30 mg mL-1), precipitant concentration (7-51 %), salt concentration (0.01-0.3 M), additive salts (the Hampton Research StockOption kit), pH (5.0-8.0), was carried out. But little improvement was made, as judged by crystal size.



3.3 aaPlsY purification and in surfo crystallization Stability and homogeneity (both in conformation and composition) are probably the two most important (and sometimes they are related) determining factors for successful protein crystallization.67 For intrinsically unstable proteins, stable variants are generally obtained by protein engineering68 and ortholog screening,69 especially those from thermophiles. Accordingly, we overexpressed PlsY from A. aeolicus (growth temperature of 85-95 °C),70 which shares 35.4 % identity and 50.2 % similarity with ecPlsY. The expression level of aaPlsY was 8 mg per liter of culture based on calibrated GFP fluorescence. The acyltransferase was purified to apparent homogeneity (Fig. 2A, 2B) and concentrated to 22 mg mL-1 without noticeable precipitation. Sitting-drop in surfo crystallization of aaPlsY in two short-chain detergents, namely DM and OG, did not produce any crystals. At the time, we had demonstrated that aaPlsY reconstituted into the LCP lipid bilayer was active,54 and in meso microcrystals had been obtained for ecPlsY (Section 3.2). Thus, aaPlsY was subjected to in meso crystallization.

Fig. 3. Image and diffraction of optimized aaPlsY crystals. (A, B) Crystals growing in 9.9 MAG at 20C with optimized precipitants containing 0.1 M (NH4)2SO4, 38 %(v/v) TEG, and 0.1 M Glycine pH 3.8. Images were taken with bright light (A) and between cross-polarizers (B). The crystals relate to the Form 1 in Table 1. (C) Diffraction pattern of a crystal from (A). Data collection details are in Methods and Table 1. (D) An expanded view of the boxed area in (C).

3.4 Diffraction-quality aaPlsY crystals growing in the 9.9 MAG mesophase 3.4.1 Initial trials in 9.9 MAG Interestingly, aaPlsY did not crystallize in the precipitant conditions for ecPlsY which contained PEG 500 DME. Instead, a new hit was identified from a broad screening using the commercial


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kits listed in Section 2.1. This condition was coincidently the same as the initial hit for OprB,49 which contained 28 %(v/v) triethylene glycol (TEG), 0.1 M (NH4)2SO4, and 0.1 M glycine pH 3.8. The crystals were of triangular pyramid shape and strongly birefringent (Fig. 2C, 2D). 3.4.2 Optimization in 9.9 MAG For optimization, a grid screen was made as follows. The concentrations of TEG and (NH4)2SO4 were varied in the range of 14-50 %(v/v) and 0-0.2 M, respectively. In addition, the pH was screened between 3.5 to 8.5 using glycine and sodium acetate buffer. This simple round of optimization produced larger crystals (50-90 m) that had the same triangular pyramid shape as the initial hit (Fig. 3A, 3B). aaPlsY crystals clustered at high TEG concentrations. A high-resolution dataset at 1.62 Å was obtained (Fig. 3C, 3D, Table 1) using a single crystal growing in low TEG concentrations. The crystals belonged to space group of C2221, with unit dimension of a = 71.87, b = 79.72, and c = 89.14 Å. A Mathews coefficient analysis suggested one molecule per asymmetric unit. 3.5 7.8 MAG was critical for SeMet-aaPlsY crystallization 3.5.1 Crystallization of SeMet-aaPlsY in 9.9 MAG To solve the aaPlsY structure, experimental phasing was needed because no homologous structures were available for molecular replacement. SeMet labeling is very popular for heavy atom incorporation and phasing because the protocol with the E. coli system is relatively straightforward and rarely perturbs protein folding. In addition, the covalent nature of the labeling ensures high crystallographic occupancies of Se in ordered regions.57 aaPlsY contains three methionine residues (including the N-terminal methionine). Generally, one SeMet per fifty residues are desirable for single-wavelength anomalous dispersion (SAD) phasing; however, successful cases with one SeMet per 100 residues have also been reported.71 The required abundance might be further lowered with highly redundant data collected on detectors with high signal-noise ratios such as the shutter-free Pilatus used in this study. Therefore, we did SeMet labeling without introducing more methionines. Interestingly, SeMet labeling using the E. coli methionine auxotroph B834 strain was unsuccessful because the cells stopped growing after the addition of SeMet and IPTG, which, when combined, probably imposed too much stress on the host cells. We then switched to the methionine inhibition protocol57 and obtained 3.5-4.0 mg of SeMet-aaPlsY per liter of culture. Crystallization of SeMet-aaPlsY in 9.9 MAG was carried out the same as the native protein using the TEG-based precipitant solutions. Interestingly, the resulting crystals differed from the native crystals both in size and shape (Fig. 3A, Fig. 4A). In literature, SeMet derivatives normally produce the same crystal form as the native protein but changed forms are also frequently reported. The crystals only diffracted to 4.3 Å. The differences in both crystal shape and diffraction quality between the native and the SeMet-aaPlsY suggested that SeMet was successfully incorporated. In anticipation of stronger anomalous signals at higher resolutions, we decided to optimize crystallization with the current protein form, instead of engineering more methionines for labeling.



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Fig. 4. Crystallization of SeMet-aaPlsY and the anomalous map. (A) Crystals growing in 9.9 MAG at 20 C. An arrow indicates a typical crystal. The precipitant solution contained 34 %(v/v) TEG, 0.3 M (NH4)2SO4, 0.1 M glycine pH 3.8. (B) Crystals growing in 7.9 MAG at 20 C. The precipitant solution contained 34 %(v/v) TEG, 0.2 M (NH4)2SO4, 0.1 M glycine pH 3.8. (C) Crystals growing in 7.8 MAG at 20 C. The precipitant solution contained 28 %(v/v) TEG, 0.2 M (NH4)2SO4, 0.1 M glycine pH 3.8. The crystals relate to Form 2 in Table 1. (D) The anomalous map (magenta) contoured at 3.0 . The two strong density blobs were from residues 94 and 166, and the weak density was from the N-terminal formyl SeMet. The structure has been described elsewhere.54 Membrane boundary defined by the OPM server72 is marked with two lines.

3.5.2 Crystallization of SeMet-aaPlsY in 7.8 MAG Protein constructs (with less disordered regions and higher stability), precipitant type and concentration, and host lipids are common variables for optimizing in meso crystals.12 aaPlsY contains very little extra-membrane domains or loops for trimming of flexible regions. In addition, its ultra-high stability (half-life of 30 min at 90C)54 suggests no need for further stabilization by protein engineering. Regarding the precipitant solution, the crystals in figure 4A were obtained in an already-optimized, TEG-based condition. Further optimization of precipitant conditions would involve another broad screening experiment. This requires a significant amount of protein which was undesirable for the SeMet-aaPlsY because of the relatively low yield. We therefore turned our attention to the host lipids. Host lipid screening has proven to be very useful in optimization of in meso crystals, notably, of proteins that have similar sizes as PlsY.48,50 In particular, five non-monoolein lipids have been more regularly reported in literature than others. They are 7.7 MAG, 7.8 MAG, 7.9 MAG, 8.8 MAG and 9.7 MAG.5,12-15,48-51,53,73,74 Accordingly, we performed in meso crystallization trials in all of the MAGs except for 9.7 MAG which ran out in the laboratory at the time. For precipitant conditions, we used a TEG-based screen with extended concentration ranges instead of a new broad screening to save costly lipids and protein.


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The host lipid screening was successful. Rod-shaped crystals (10 ×120 m) were obtained within one day, in both 7.9 MAG and 7.8 MAG (Fig. 4B, 4C). Importantly, they both diffracted to 2.0 Å at the synchrotron, with the I222 space group (Form 2, Table 1) which was different from that in 9.9 MAG (Form 1, Table 1). The structure was solved using the Se-SAD data collected with the crystals from 7.8 MAG. Three Se sites, one of which was later attributed to the N-terminal formyl SeMet (Fig. 4D), were found using SHELX.75 An initial monomer model containing 187 residues was built using the Phenix.autobuild module with Rfree/work of 0.2715/0.2431.

Fig. 5. The problem of lamellar phase and citrate. (A, B) Images of a typical 7.8 MAG lamellar phase drop taken under bright field (A) and polarized light (B). (C) A crystal (indicated by an arrow) growing in 7.8 MAG under citrate-containing conditions at 4 °C. The precipitant solution contained 24 %(v/v) PEG 400, 0.4 M ammonium acetate, sodium citrate pH 6.0. AcylP was included at 1 mol% (in relation to 7.8 MAG). The crystal relates to Form 3 in Table 1. (D) The structure solved using crystals in (C). Two citric acid molecules were observed in the V-shaped54 active site. (E) An expanded view of the Fo-Fc simulated annealing omit map at 3.0 σ level for the two citric acid molecules. (F-H) The same with that for (C-E) except that the crystals were obtained in 9.9 MAG at 20 C with the precipitant solution containing 47 %(v/v) PEG 400, 0.1 M NiCl2 and sodium citrate pH 6.0. Crystals in (F) relate to Form 4 in Table 1.

3.6 Crystallization of aaPlsY-acylP – 7.8 MAG and low temperature were important 3.6.1 Overcoming the problem of detergent-induced lamellar phase The next aim of the project was to determine the PlsY structure with its substrates, acylP and G3P, in order to reveal its active site architecture and catalytic mechanism. AcylP is a detergent-like molecule. Detergents are known to flip LCP to lamellar phase at high enough concentrations.20 For instance, the 9.9 MAG LCP can tolerate approximately 10 %(w/v) of DDM.19,20 At 20 mg



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mL-1, aaPlsY solution contained 4.5-5.0 %(w/v) DDM. Optically clear LCP was obtained for both 9.9 MAG and 7.8 MAG under this DDM concentration. For acylP co-crystallization, we wanted to include acylP at 1-2 mol% (in relation to the host lipid), considering its instability and the Km in LCP (0.1 mol%).54 Whilst the 9.9 MAG LCP was stable under these conditions, the 7.8 MAG mesophase was converted to lamellar phase, showing characteristical3 glowing birefringence under polarized light (Fig. 5A, 5B). This was consistent with the reported observation that the tolerance of LCP to chemical stresses generally reduces as the chain length decreases.48 To avoid the lamellar phase problem for 7.8 MAG, we reduced the DDM concentration in the aaPlsY sample (20 mg mL-1) from 4.5-5.0 %(w/v) to 1.6-1.8 %(w/v) using -CD54,59 during the concentration process. Transparent and non-birefringent 7.8 MAG LCP was obtained with this sample in the presence of 2 mol% acylP. 3.6.2 Avoiding citrate in the precipitant solutions Co-crystallization of aaPlsY-acylP in several citrate-containing conditions produced cubic crystals which diffracted to high resolutions (Fig. 5C, 5F, Table 1), but the active site was frequently occupied with citric acid. Typical results in 7.8 MAG and 9.9 MAG are shown in figure 5C-H. The negatively charged citric acid probably outcompeted acylP/G3P. To avoid this problem, the citrate conditions were ignored or eliminated in subsequent rounds of screening and optimization. 3.6.3 Ambiguous electron density for acylP – crystallization in 9.9 MAG aaPlsY-acylP co-crystallization was first performed in the relatively inexpensive 9.9 MAG. Several non-citrate conditions, after optimization, produced crystals that diffracted beyond 3.0 Å, but no convincing acylP density was observed in the active site. The most promising structure, which was from crystals growing in a potassium-acetate-containing condition (Fig. 6A), showed electron density that resembled acylP when this ligand was included for map calculation. However, the unbiased simulated annealing (SA) omit map showed non-continuous density in the phosphate ester region (Fig. 6B-D) and thus did not support such a fitting. The density covering the phosphate moiety could also accommodate an acetic acid which is used in the crystallization. This ambiguity prompted us to look for alternative host lipids because the likelihood of finding new meaningful conditions with 9.9 MAG would be low after the broad screening. 3.6.4 Interpretable electron density for acylP – crystallization in 7.8 MAG at 20 °C Next, we performed PlsY-acylP co-crystallization in 7.8 MAG at 20°C. Crystals appeared shortly (within 12 h) but deteriorated within a few days, as exemplified in Figure 7A-C. Thus, the crystal growth was monitored every 12 h and the crystals were harvested as soon as they stopped growing. One of the crystals (Fig. 7D) harvested on day-2 yielded a structure with blobs of electron density in the active site. AcylP appeared to fit well with the density, especially for the phosphate head group (Fig. 7E-G). However, the deterioration of crystals and the instability of acylP raised concerns about the ligand integrity; in other words, the density could accommodate equally well for a fatty acid and an orthophosphate, which are the two products of acylP hydrolysis.


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Fig. 6. Crystals and structure of aaPlsY with ambiguous ligand electron density in the active site. (A) Crystals growing in 9.9 MAG (doped with acylP) at 20 °C. An arrow indicates a typical crystal. The crystals relate to Form 5 in Table 1.The precipitant solution contained 47 %(v/v) PEG 400, 0.1 M potassium acetate and 0.1 M MES-NaOH pH 6.5. (B) The structure solved using the data collected from crystals in (A). An acylP molecule was placed in the electron density. (C) Sigma-A weighted Fo-Fc simulated annealing (SA) omit map at 3.0 σ level. (D) 2Fo-Fc map at 1.0 σ level. Note that the density did not justify an acylP, but it was nevertheless placed for visualization purposes.

Fig. 7. Co-crystallization of aaPlsY-acylP in 7.8 MAG and a promising hit. (A) Image of a crystallization well taken within 12 h. The precipitant solution contained 24 %(v/v) PEG 400, 0.1 M KF, 0.1 M MES-NaOH pH 6.0. (B) and (C) are images of the same drop taken on day-1 and day-2, respectively. (D) Crystals growing in precipitants containing 24 %(v/v) PEG 400, 0.4 M ZnSO4, HEPES-NaOH pH 7.0. The image was taken on day-2. The crystals relate to Form 6 in Table 1. (E) Structure solved from data collected with crystals in (D). (F) Sigma-A weighted Fo-Fc SA omit map at 3.0 σ level. (G) 2Fo-Fc SA omit map at 1.0 σ level. The map supports either an acylP molecule, or a fatty acid with an inorganic phosphate as the result of acylP hydrolysis.



3.6.5 Continuous electron density for acylP – crystallization in 7.8 MAG at 4 °C A reasonable strategy to prevent or slow down the hydrolysis of the unstable acylP would be to carry out crystallization at low temperatures. Conveniently, a previous report demonstrated the feasibility of low-temperature crystallization in 7.8 MAG.50 Thus, we performed crystallization trials at 4 °C and observed continuous and unambiguous acylP density in two structures from crystals growing in two different conditions. Both conditions contained PEG 400 but differed in salts; one with magnesium formate and the other with KF. The latter (Fig. 8, Form 7 in Table 1) was refined and reported54 because of its higher resolution.

Fig. 8. Co-crystallization of aaPlsY-acylP in 7.8 MAG at 4 °C yielded a complex structure. (A) Crystals growing in 7.8 MAG at 4 °C. An arrow indicates a typical crystal. The precipitant solution contained 0.5 M KF, 22 %(v/v) PEG 400, 0.1 M Tris-HCl pH 8.0. AcylP was included at 1 mol%. The crystals relate to Form 7 in Table 1. (B) The structure solved using X-ray diffraction data collected from crystals in (A). (C) An expanded view of the Fo-Fc SA omit map contoured at 3.0 σ level. The map was generated using the published data.54

3.6.6 Crystallographic evidence for in-crystal degradation of acylP at 4 °C The crystals growing in the same condition as in figure 8 were left for three weeks at 4 °C. During this period, the crystals did not dissolve (Fig. 9A). We collected the diffraction data from the crystals and solved the structure (Fig. 9B). It was evident that the acylP was broken down after three weeks because the electron density supported the modeling of a palmitic acid and an orthophosphate (Fig. 9C). This result was consistent with the speculation that the acylP was hydrolyzed in crystallization trials at 20 °C (Fig. 7D-G). The in-crystal degradation of acylP raises the question of whether PlsY possesses hydrolase activity. According to our published aaPlsY-acylP structure,54 the head group of acylP was located in a hydrophilic interior deep in the membrane and interacted with four waters. One of the waters (W1, Fig. 9D) hydrogen-bonded with the phosphate moiety and was 3.2-Å away from the carbonyl carbon, suitable for the nucleophilic attack in the proposed enzyme-mediated hydrolysis. Using a coupled assay,54 we measured the hydrolysis rate of acylP in the presence and absence of aaPlsY. The results showed that aaPlsY possessed weak hydrolase activity of 2.8 ± 0.4 nmol min-1 mg-1 (n=3), which was 12,000 folds lower than its primary, acyltransferase activity. This ambiguous activity will be discussed.


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Fig. 9. Crystallographic evidence for acylP hydrolysis in the active site. (A) Crystals growing in the same condition as in figure 8A. An arrow indicates a typical crystal. The image was taken on day-19 postcrystallization. The crystals relate to Form 8 in Table 1. (B) The structure from diffraction data collected with crystals in (A). (C) An expanded view of the Fo-Fc SA omit map contoured at 3.0 σ level and fitted with a palmitic acid and a phosphate. (D) The aaPlsY-acylP structure (PDB entry 5XJ7) suggested hydrolase activity. AcylP (cyan stick-ball) was located in a hydrophilic interior of aaPlsY, with the head group interacting with four waters (light blue sphere). The water W1 interacted with the phosphate group (3.0 Å) and was located in a position suitable for nucleophilic attack (3.2 Å, red dashed line) at the carbonyl carbon. Dashed lines indicate distances within 3.4 Å and are colored differently for better visualization. The side chains of Lys104, Val106, and Ala107 are not shown.

3.7 Crystallization of the aaPlsY-G3P complex 3.7.1 No G3P captured in crystals growing in 7.8 MAG Thus far, 7.8 MAG appeared to be more suitable for PlsY crystallization than 9.9 MAG, based on the results with the SeMet crystals and the PlsY-acylP co-crystals. Therefore we decided to use 7.8 MAG as the host lipid to conduct co-crystallization of aaPlsY-G3P. Several high-resolution structures (exemplified as Form 9 in Table 1) were obtained from crystals growing in 7.8 MAG under different precipitant conditions (exemplified in Fig. 11A). However, none of the structures showed electron density for G3P in the active site. 3.7.2 Crystallization of aaPlsY-G3P in 9.9 MAG yielded a complex structure Although the PlsY-G3P structure had not been known at the time, several structures revealed a sulfate binding site near the membrane boundary which contains critical residues for G3P binding.54 Thus, this region was identified as the G3P binding site.54 A b-factor analysis of the structure from crystals growing in 7.8 MAG under TEG-based conditions revealed that the G3P binding site was more mobile than the transmembrane domain, especially for the C-terminal helix (named 3 in ref.54) where a G3P-binding residue (Asn180) resides (Fig. 10A). By contrast, the 3 region in the 9.9 MAG structure from crystals growing in similar TEG conditions had similar b-factors with the relatively inflexible transmembrane domain (Fig. 10B). We reasoned that the high mobility was unfavorable for capturing a G3P-binding conformation, and performed PlsYG3P co-crystallization in 9.9 MAG.



Fig. 10. B-factor and crystal contact analysis of structures from crystals growing in 7.8 MAG and 9.9 MAG. (A) The b-factor distribution of the structure (PDB entry 5XJ5)54 obtained from crystals growing in 7.8 MAG. (B) The b-factor distribution of the structure obtained from crystals growing in 9.9 MAG. The structure relates to Form 1 in Table 1. B-factor profile is shown as putty representation with blue lines indicating low values (low flexibility), and red tubes indicating high values. Residues that interact with the sulphate (yellow) are shown as magenta sticks. Membrane boundaries are marked with dashed lines. The helix 3, which displays very different flexibilities between the two structures, is labeled. (C,D) The involvement of 3 region in crystal packing in the 7.8 MAG structure (C) and the 9.9 MAG structure (D). The boxed areas in the overview (bottom) are expanded and shown at the top. MAGs at the crystal packing sites are shown as sticks.


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After screening a number of crystal forms, we obtained a 2.37-Å structure that had a G3P molecule in the active site (Fig. 11).54 This structure provided critical evidence for a ‘substrate-assisted catalysis’ mechanism that was completely different from the ‘Asp-His dyad’ mechanism for conventional acyltransferases.54

Fig. 11. Co-crystallization of aaPlsY-G3P. (A) Crystals growing in 7.8 MAG at 20 °C. The precipitant solution contained 40 mM G3P, 23 %(v/v) PEG 400, 1 mM Na2SO4, 0.8 M LiCl, 0.1 M Tris-HCl pH 7.8. The crystals relate to Form 9 in Table 1. (B) Crystals growing in 9.9 MAG at 20 °C. The precipitant solution contained 80 mM G3P, 0.12 M LiCl, 37 %(v/v) PEG 400, 0.1 M Tris-HCl pH 8.0. (C) Structure of the aaPlsY-G3P complex obtained with crystals in (B). (D) Sigma-A weighted Fo-Fc SA omit map contoured at 3.0 σ level. (C) and (D) were re-drawn using the structure and data published in ref.54. This structure (PDB entry 5XJ6) relates to Form 10 in Table 1.

4. Discussion 4.1. The back and forth with host lipids Here we report the successful application of the host lipid screening strategy for the structure determination of PlsY, a ubiquitous bacterial G3P acyltransferase. Encouraged by the great success of the use of non-monoolein lipids in a number of challenging in meso crystallization projects,11-13,15,48-53,73,74 we screened host lipids as soon as the crystallization of SeMet-aaPlsY in 9.9 MAG was unsatisfactory, despite that 9.9 MAG supported native crystals that diffracted to 1.62 Å. It remains possible that, with extensive optimization, crystallization of SeMet-aaPlsY in 9.9 MAG could eventually lead to a structure. However, the host lipid screening improved crystal quality with one relatively straightforward optimization experiment. Our results should encourage the more vigorous use of the short-chain lipids for in meso crystallization. Among the five host lipids tested, 7.8 MAG and 7.9 MAG performed better than others. These two lipids should be considered with high priority in host lipid screening. For availability reasons, we did not test 9.7 MAG for aaPlsY crystallization. This lipid can be obtained from Nu-Chek



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Prep at a similar price as 9.9 MAG (currently 65 US dollars per gram), which is ~30 times lower than other short-chain lipids from Avanti Polar Lipids. Although 9.7 MAG did not improve much on the crystal quality for DgkA50 and mPGES1,48 it was used in several crystallization projects with great success.12,13,73,74 Thus, 9.7 MAG should also be considered for future host lipid screening experiments. Crystallization of enzyme-substrate complexes are generally challenging due to the transient nature of the interactions between them, yet the information is crucial for mechanistic studies. For instance, the only other G3P acyltransferase structure available did not have acyl-CoA or G3P bound.76 Indeed, obtaining the PlsY-substrate structures was not straightforward. Thus, exhaustive screening of PlsY-acylP in 9.9 MAG failed to produce convincing acylP electron density in the active site. Crystallization in 7.8 MAG at 20 °C showed promises that justify further optimization and provided a rationale for low-temperature crystallization which led to the success. As for G3P, extensive screening in 7.8 MAG was carried out, but to no avail. Unlike acylP, which binds to the pocket deeply buried in the transmembrane region, G3P binds to the membrane boundary region. Membrane rigidity, especially near the surface, may be important for maintaining a stable PlsY-G3P complex that can survive the crystallization process. With a shorter fatty chain, 7.8 MAG is surely much more dynamic than 9.9 MAG, and so would be the lipid bilayer and the membrane protein within. In support of the speculation, the b-factor analysis showed that the membrane boundary region from the 7.8 MAG structure was more flexible than that from the 9.9 MAG structure, despite that both crystals were grown in TEG-based conditions. It should be noted, however, that in the two structures, the 3 region participated in crystal packing to different extents. The 3 helix in the 9.9 MAG structure was located in a tighter contact site and formed more contacts with neighboring protomers than that in the 7.8 MAG structure (Fig. 10C, 10D). Therefore, the lower b-factor observed in the 9.9 MAG structure (Fig. 10B) might be caused by either or both of the crystal-contact effects and the higher physical rigidity associated with longer hydrocarbon. With the current data, we were unable to dissect the two possible causes. But based on the different crystallization outcome of aaPlsY-G3P in the two different MAGs, we tentatively propose that membrane rigidity is favorable for ligand binding at the membrane boundary. This hypothesis needs to be further tested with more experiments. Overall, the optimization was not straightforward and we went back and forth between the two host lipids. Importantly, the process was not entirely empirical. Precipitant components, host lipids, and temperatures were chosen carefully as guided by crystallization results, and crystal qualities were improved incrementally through rounds of optimization. 4.2. Low-temperature crystallization For unstable membrane proteins/ligands, or for cases where crystal growth needs to slow down, low-temperature crystallization is an attractive and useful approach. LCP is capable of undercooling,17,77 making it possible for low-temperature crystallization. However, the undercooled LCP is metastable and thus prone to the chemical-induced phase transition. Our experiences with the 9.9 MAG LCP are detailed below.


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For PEG 500 DME conditions, the LCP was stable in the concentration range of 31-49%(w/v) at pH 6.0 – 8.0 at 20 °C. At 4 °C, the mesophase stability depended on the pH. At pH 6.0 and 6.5, the mesophase remained LCP. However, about half of the wells at pH 7.0 and all wells at pH 7.5 and 8.0 converted to opaque and birefringent phases. For MPD conditions, previous reports have demonstrated that the 9.9 MAG mesophase was quite stable at low temperatures.48,50 Interestingly, the 9.9 MAG mesophase was quite stable at 4 °C in PEG 400 conditions, based on observations from the current study and our unpublished results with the group-I cobalt energy coupling factor transporter CbiM/N/Q/O.78 LCP displayed little visual differences in PEG 400based conditions under the two temperatures, although the cubic-to-sponge phase transition at 4 °C required slightly higher concentrations (49-51%) compared to that at 20 °C (45-47%). PEG 400 is one of the most reported precipitants for in meso crystallization. Indeed, it was used in the crystallization of almost all GPCRs with known crystal structures. The fact that LCP is stable at 4 °C in this common precipitant should therefore encourage more vigorous exploration of lowtemperature crystallization for GPCRs and other unstable membrane proteins. It is important to note a few useful lipids for low-temperature crystallization because they form stable LCP at 4-6 °C. They include the rationally designed 7.9 MAG,35 the monodihydrosterculin (a 9.9 MAG analog with a cyclopropyl ring replacing the olefin),34 and two lipids with isoprenoid acyl chain.36 These lipids should allow low-temperature in meso crystallization in a wider range of precipitant conditions compared to 9.9 MAG. 4.3. In-crystal degradation of acylP in the inflexible active site Previously, we speculated that the PlsY structure was relatively inflexible based on the observation that the structures with different substrates and products were superimposable.54 This was again supported in the current study. The in-crystal degradation of acylP which happened between day-7 and day-19 (Fig. 8, Fig. 9) did not damage the crystals. The crystals before and after acylP degradation belonged to the same space group with very similar cell dimensions (Form 7 and 8, Table 1). Presumably, the hydrolysis of acylP occurred in the active site but did not cause big enough conformational changes to destroy crystal contacts. 4.4. The hydrolase activity of PlsY Enzymes are known as specific biocatalysts, but promiscuity is also quite common.79 Prompted by the in-crystal degradation of acylP, we examined the structures and reasoned that PlsY might possess hydrolase activity against acylP (Fig. 9D). Indeed, our PlsY coupled assay revealed weak hydrolase activity with kcat of 3.9 h-1, which is ~12,000 fold slower than its primary function. We believe that the hydrolase activity is too weak to be physiologically relevant. The synthesis of acylP is an endergonic process, consuming either adenosine triphosphate80,81 or acyl-carrier protein,56 and acylP is unstable and degrades spontaneously. In addition, acylP can be enzymatically consumed by PlsX to acylate apo acyl-carrier proteins.56 Therefore, there is probably no need for the cells to develop such a mechanism to eliminate it. It should be noted that, in our assay, the hydrolase activity probably started as soon as LCP was



made, but the phosphate-releasing activity was monitored approximately 20 min later. The delay was unavoidable because acylP needed to be loaded into the LCP prior to the assay, and the soaking steps prior to the monitoring were necessary for the removal of phosphate contaminant in the system which will otherwise saturate the phosphate-biosensor. One might argue that the activity rate we measured was not the initial velocity because of the delay. However, we reasoned that the reaction rate was probably still in the linear region. In kinetic assays, catalysis slows down due to a number of factors including enzymes denaturation, substrate depletion, and product inhibition. The ultra-stable aaPlsY54 should have remained active during the course of the assay at 30 °C. As for the substrate availability, it was estimated that the enzyme consumed less than 2% of the substrate at the time of monitoring, assuming the activity was at 2.8 nmol min-1 mg-1. Therefore the effect of substrate depletion on activity was probably neglectable. We also tested the hydrolase activity at 20 min, 30 min, and 40 min post reconstitution and found that the activities were the same. This linearity (20-40 min) suggested no product inhibition. Therefore, the measured hydrolase activity was likely close to the initial velocity. 4.5. Thermostability Thermostability is probably the most important determinant of a crystallization project, especially for membrane proteins because of their inherent instability. For prokaryotic membrane proteins, orthologs from extremophiles were often used15,82-89 and will likely continue to be exploited, particularly with the ever-increasing genomics data. Desired genes can be synthesized at relative low-costs using PCR with overlapping primers, in cases where the genomic DNA is not available, or codon optimization is required. Indeed, the synthesis of the aaplsY gene cost approximately 60 US dollars in the laboratory. For long genes, overlapping PCR may be difficult; Gibson assembly90 of short (0.5 - 1 kb) fragments can be used instead. For membrane proteins with no thermophile orthologs, mutagenesis is often used to stabilize the target protein. Three main approaches exist in the literature. The first approach, known as directed evolution, screens thermostable mutants from a random library.91,92 Because of numerous mutants to be tested, this approach often relies on high-throughput and convenient assays, preferably with isolated colonies. The second approach systematically scans the target protein with alanine/leucine mutations, evaluates the stability of each mutant, and combines additive stabilizing mutations. This approach has been successfully applied to the thermostabilization of several GPCRs and transporters.64,65,87-93 The third is a more rational but less-reported approach called the consensus method,93,94 where residues are mutated to the most frequent ones (the consensus) based on sequence alignment with dozens of orthologs/homologs. Because consensus mutations are not always stabilizing,95 they might also need to be evaluated individually before being combined. Given the generally poor stability of membrane proteins and the seemingly unavoidable stability barriers, development of simple, rapid, reliable, and generally applicable methods for high-throughput stability screening will be of great importance. 4.6. Removal of excess detergents The presence of excess detergents may cause undesired phase-flipping problems for in meso crystallization, as in the case of aaPlsY-acylP co-crystallization in 7.8 MAG (Fig. 5A, 5B). The problem was solved by reducing detergent concentrations using -CD during the concentration process. In our experience, membrane protein may precipitate upon the addition of -CD depending on the stability. Therefore, it is worth running a small-scale test prior to the detergent


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removal in large-scale. First reported by Li et al.,59 this method should prove useful for other applications as well. For example, reducing detergent concentrations could help improving image contrast for cryoelectron microscope analysis of membrane proteins. The solubility of -CD is modest (~10 mM). As a result, voluminous -CD solution needs to be added for samples with high starting detergent concentrations (8-10%, for example). More soluble derivatives such as methyl -CD or 2-hydroxypropyl-β-CD might work better in this regard. 4.7. A benchmark protein for the in meso method aaPlsY is ultra-stable. It readily crystallizes in the inexpensive 9.9 MAG at 20 °C, and the crystals diffract to high resolution. Thus, aaPlsY may be used as a benchmark protein for training and demonstration of the in meso method.

5.

Conclusion

Host lipid and temperature screening were once again proved to be important for in meso crystallization with the integral membrane G3P acyltransferase PlsY. Despite the high costs (currently cost 1,900 US dollars per gram for non-9.9/9.7 MAG), host lipid screening should be considered as soon as initial 9.9 MAG hits are obtained to speed up the optimization process. Temperature is another important parameter that may get overlooked depending on the laboratory culture and set up. For membrane proteins, lowering the crystallization temperature from the usual 20 °C to 4 °C could mean a huge gain, considering how much efforts are needed to increase melting temperature by 16 °C through protein engineering.

Acknowledgment. The authors thank Dr. D. Yao for assistance and advice in X-ray data collection for the initial low-resolution SeMet data sets, Dr. H. Tao at ShanghaiTech University for useful discussions and providing lab facilities for the chemical synthesis of acylP, and the National Centre for Protein Science Shanghai for providing the protein expression, purification and crystallization facilities and technical assistance. D.L.’s lab is supported by the 1000 Young Talent Program, the Shanghai Pujiang Talent Program (15PJ1409400), the National Natural Science Foundation of China (31570748 and U1632127), the CAS-Shanghai Science Research Center (CAS-SSRC-YJ-2015-02), Key Program of CAS Frontier Science (QYZDB-SSWSMC037) and CAS Facility-based Open Research Program.

Conflict of interest. The authors declare no competing financial interests.



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Crystal Growth & Design 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

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Table 1. Crystallization conditions and data collection statistics. Form1

Form2a

Form3

Form4

Form5

Form6

Form7a

Form8

Form9

Form10a

Crystal growth conditions Protein

native

SeMet

native

native

native

native

native

native

native

native

Host lipid

9.9 MAG

7.8 MAG

7.8 MAG

9.9 MAG

9.9 MAG

7.8 MAG

7.8 MAG

7.8 MAG

7.8 MAG

9.9 MAG

Temperature (°C)

20

20

4

20

20

20

4

4

20

20

Substrate added

-

-

acylP

acylP

acylP

acylP

acylP

acylP

G3P

G3P

Substrate revealed

-

-

-

-

-

inconclusive

acylP

degraded

-

G3P

Relates to

figure 3A

figure 2C

figure 5C

figure 5F

figure 6A

figure 7D

figure 8A

figure 9A

figure 11A

figure 11B

Space group

C222

I222

C222

P4 2 2

C222

C22 2

P2 2 2

P2 2 2

I222

P22 2

1

1

2 1

1

1

1 1 1

1 1 1

1 1

Cell dimensions a, b, c (Å)

71.87,

79.72,

83.44,

65.37,

100.16,

68.84,

68.84

63.20,

78.39,

82.92,

93.12,

46.22,

65.60,

47.23,

65.06,

59.60,

86.22,

42.15,

53.77,

89.14

107.42

82.51

101.06

86.24

57.36

84.68

84.06

89.29

87.69

α, β, γ (°)

90, 90, 90

90, 90, 90

90, 90, 90

90, 90, 90

90, 90, 90

90, 90, 90

90, 90, 90

90, 90, 90

90, 90, 90

90, 90, 90

Wavelength (Å)

0.97853

0.97861

0.97776

0.97776

0.97776

0.97776

0.97854

0.97915

0.97776

0.97853

Resolution (Å)

53.38 - 1.62

47.45 - 2.04

45.62 - 1.75

48.68 - 1.64

42.73 - 2.19

46.56 - 2.59

46.22 - 1.77

47.23 - 2.10

49.03 - 1.65

45.84 - 2.37

(1.65 - 1.62)

(2.09 - 2.04)

(1.78 - 1.25)

(1.67 - 1.64)

(2.26 - 2.19)

(2.70 - 2.59)

(1.81 - 1.77)

(2.16 - 2.10)

(1.68 - 1.65)

(2.46 - 2.37)

Rmerge

0.087 (1.327)

0.173 (1.245)

0.072 (1.286)

0.109 (1.735)

0.084 (1.535)

0.143 (1.266)

0.064 (0.798)

0.115 (1.396)

0.098 (1.101)

0.173 (1.145)

Rpim

0.037 (0.515)

0.060 (0.450)

0.022 (0.375)

0.022 (0.373)

0.024 (0.442)

0.052 (0.476)

0.029 (0.429)

0.046 (0.545)

0.046 (0.511)

0.052 (0.382)

I/σI

12.0 (1.9)

10.9 (2.0)

22.3 (1.9)

19.4 (2.1)

20.4 (1.7)

11.4 (1.6)

18.0 (2.0)

13.2 (1.7)

11.5 (1.7)

11.9 (1.7)

Completeness (%)

100 (100)

99.7 (96.4)

99.9 (100)

99.7 (94.2)

99.7 (96.7)

99.6 (97.4)

98.4 (97.1)

98.9 (98.1)

99.8 (100)

98.9 (89.9)

Multiplicity

7.5 (7.5)

9.5 (8.7)

13.3 (13.5)

25.6 (20.3)

13.0 (12.6)

8.5 (7.8)

5.6 (5.2)

7.2 (7.5)

5.4 (5.5)

12.4 (9.7)

b

a

57.68,

The structure and statistics have been published.54

b

Highest resolution shell is shown in parenthesis.


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For Table of Contents Use Only In MesoCrystallization of the Integral Membrane Glycerol 3-phosphate Acyltransferase with Substrates Zhenjian Li, Yannan Tang, Dianfan Li

Table of Contents Graphic

Synopsis The use of an ultra-stable ortholog, host lipid screening, and low-temperature crystallization were critical for the in meso crystallization of the apo- and substrate-bound forms of the integral membrane glycerol 3-phosphate acyltransferase PlsY. Co-crystallization of PlsY with its lipid substrate required the use a short-chain monoacylglycerol at 4 °C. By contrast, co-crystallization with the water-soluble substrate required the longer-chain monoolein.


Cover Page Most Important Figure: Synopsis figure

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