Insights into the Membrane Interacting Properties of the C-Terminal

Article pubs.acs.org/biochemistry

Insights into the Membrane Interacting Properties of the C‑Terminal Domain of the Monotopic Glycosyltransferase DGD2 in Arabidopsis thaliana Scarlett Szpryngiel and Lena Mal̈ er* Department of Biochemistry and Biophysics, Center for Biomembrane Research, The Arrhenius Laboratory, Stockholm University, 10691 Stockholm, Sweden ABSTRACT: Glycosyltransferases (GTs) are responsible for regulating the membrane composition of plants. The synthesis of one of the main lipids in the membrane, the galactolipid digalactosyldiacylglycerol, is regulated by the enzyme digalactosyldiacylglycerol synthase 2 (atDGD2) under starving conditions, such as phosphate shortage. The enzyme belongs to the GT-B fold, characterized by two β/α/β Rossmann domains that are connected by a flexible linker. atDGD2 has previously been shown to attach to lipid membranes by the N-terminal domain via interactions with negatively charged lipids. The role of the C-terminal domain in the membrane interaction is, however, not known. Here we have used a combination of in silico prediction methods and biophysical experimental techniques to shed light on the membrane interacting properties of the C-terminal domain. Our results demonstrate that there is an amphipathic sequence, corresponding to residues V240−E258, that interacts with lipids in a charge-dependent way. A second sequence was identified as being potentially important, with a high charge density, but no amphipathic character. The features of the plant atDGD2 observed here are similar in prokaryotic glycosyltransferases. On the basis of our results, and by analogy to other glycosyltransferases, we propose that atDGD2 interacts with the membrane through the N-terminus and with parts of the C-terminus acting as a switch, allowing for a dynamic interaction with the membrane.

A

atDGD2 important for the viability of the plants. atDGD2 is found in the outer membrane of the chloroplast and belongs to the GT4 family, with a GT-B fold characterized by two β/α/β Rossmann domains that are connected by a flexible linker. The active site is harbored between the two domains. GTs catalyze the formation of glycosidic bonds between a donor part (e.g., the sugar moiety of an activated donor, for example, nucleotide sugars) and an acceptor molecule (e.g., other sugars, proteins, small molecules, or lipids). The acceptor molecule in the mechanism of the DGDG synthases is monogalactosyldiacylglycerol (MGDG), and the donor molecule is UDP-galactose. The MGDG lipids that are used by atDGD2 specifically (i.e., regulated by phosphate shortage) are produced by two MGDG synthases (atMGD2 and atMGD3) in the outer membrane of the chloroplast, in contrast to under normal growth conditions where MGDG is provided by atMGD1 in the inner envelope.4 The presence of several GTs that regulate and fine-tune glycolipid synthesis in such ways stresses the importance of a well-functioning equilibrium between all constituents in the biomembrane. Hence, it is very important to understand how the biomolecules involved respond to different stimuli and what interaction partners they have in vivo. One of the key questions

ll plants must have protective measures toward many kinds of environmental stress factors. The stress may be due to factors that disturb their regular ecology, physiology, and/or morphology, e.g., drought, cold, wind, pathogens, or nutrient shortage. Nutrient shortage may prompt responses that alter the metabolic pathways and general biochemistry. Phosphorus is essential for the plant synthetic pathways of crucial biomolecules such as ATP, DNA, and phospholipids, molecules directly related to the very most basal functions of the cells. If there is a shortage of phosphate, the plant has to compensate for this. Examples of phosphate rescue mechanisms include extended roots structures and/or an adapted expression of genes that ultimately may redirect the phosphate bound to biomolecules. One way of doing so is to exchange phospholipids in the biomembranes for other lipids. An enzyme that has been shown to be important for the phosphate rescue mechanism in Arabidopsis thaliana is a glycosyltransferase (GT), digalactosyldiacylglycerol synthase 2 (atDGD2).1−3 atDGD2 is one of two isoforms of a GT involved in the synthesis of one of the main lipids found in the chloroplast, the galactolipid digalactosyldiacylglycerol (DGDG). Under normal growth conditions, the bulk of the DGDG lipids are produced by the isoform atDGD1 whereas the gene encoding atDGD2 is upregulated by the plant only upon phosphate shortage.1−3 The phospholipids in the bilayer are then replaced by the DGDG produced by atDGD2, and the phosphate is recycled for more acute purposes, making © XXXX American Chemical Society

Received: June 2, 2016 Revised: November 10, 2016 Published: November 10, 2016 A

DOI: 10.1021/acs.biochem.6b00559 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

France). Two variants of S240−258, one in which K254 [S240−258(K254A)] was replaced with Ala and one in which K250, K254, and K257 [S240−258(K250,254,257A)] were replaced with Ala, were also synthesized. 1-Palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-snglycero-3-phospho(1′-sn-glycerol) (POPG), 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero3-phosphoglycerol (DMPG), and deuterated 1,2-dihexanoyl-snglycero-3-phosphocoline (DHPC-d22) were obtained from Avanti Polar Lipids (Alabaster, AL). 1,2-Diacyl-3-O-(β-D-galactopyranosyl)-sn-glycerol (MGDG) was purchased from Larodan Fine Chemicals AB (Malmö, Sweden). In Silico Analyses of Structural and Physicochemical Properties in atDGD2. To predict the intrinsic membrane interacting properties of atDGD2, the full amino acid sequence was subjected to sequence analysis by the AmphiphaSeek algorithm.13 The SEG algorithm14,15 was used to screen for low-complexity regions (LCRs). Prediction of phosphorylation sites in the full-length protein was achieved with NetPhos 2.0,16 and Ser, Thr, and Tyr residues were scored according to their phosphorylation potential. Normalized values above the threshold (0.5) were examined on the basis of their localization in the amino acid sequence and the structural model of atDGD2. Agadir (http://agadir.crg.es17) was used to estimate the helical content of the peptides in this study. Default settings were always used for the analyses. A structure was modeled with the Robetta full-chain protein structure prediction server (http://robetta.bakerlab.org18), and from this model, a topology diagram was constructed. The parent X-ray structures chosen by Robetta for the initial Ginzu Domain prediction were GlgA glycogen synthase from Bacillus subtilis [Protein Data Bank (PDB) entry 3FRO and UniProt entry P39125] and human glypican-1 (PDB entry 4AD7). The sequence of residues 1−404 was modeled on GlgA with a confidence of 0.68, and the C-terminal helical region of residues 404−473 was based on human glypican-1 (PDB entry 4AD7), with a confidence score of 0.19. Five models were obtained, and model 1 was chosen for the construction of the topology diagram. The topology of atDGD2 was compared to the structures of two membraneanchored GTs of the same family: PimA (PDB entries 4NC9 and 4N9W)19,20 and WaaG (PDB entry 21W1).21 Helical wheels for S240−258 and S269−287 were prepared with the aid of the HeliQuest algorithm (http://heliquest.ipmc. cnrs.fr/22) and were used to visualize the helical propensity of the sequence23 and to calculate the hydrophobic moments of the peptides.24 The sequence of S240−258 was scanned through the database of annotated helical fragments via HeliQuest to detect proteins with similar helical patterns and to elucidate their roles in vivo. Preparation of Vesicles for CD and Fluorescence Spectroscopy. Large unilamellar vesicles (LUVs) were used as membrane mimetics in fluorescence and CD spectroscopy experiments. POPC-containing vesicles and vesicles containing increasing amounts of negative headgroup charge were produced by the extruder technique.25,26 Negative headgroup charge was introduced by substituting 10, 20, 30, 40, or 50 mol % POPC for POPG. To produce galactolipid-containing LUVs, POPC was exchanged for either 20% MGDG, 20% MGDG, and 20% POPG, or 40% MGDG and 20% POPG. The lipids were dissolved together in chloroform and dried under nitrogen gas for 2 h, until a lipid film was obtained. The lipid film was then dissolved in 50 mM phosphate buffer (pH 5.7). Stock solutions with a lipid concentration of 1 or 5 mM were prepared. In the

involves understanding how the enzyme senses the outer membrane bilayer, which is necessary for catalysis to occur. The chloroplast membranes in A. thaliana are composed of around 55 mol % MGDG and 20 mol % DGDG. In addition to these two main lipid types, phosphatidylcholine (PC), phosphatidylglycerol (PG), and sulfoquinovosyldiacylglycerol (SQDG) are present.5 Some important differences in the biophysical characteristics of these lipids concern their propensity to form bilayers (DGDG is bilayer-forming, whereas MGDG is non-bilayer-forming), and also headgroup charge.6 SQDG is a glycolipid commonly found in photosynthetic organelles, and both PG and SQDG are anionic lipids, giving a total content of anionic lipids of roughly 15 mol %. It has previously been shown that PG, as well as other negatively charged lipids such as phosphatidylserine (PS) and phosphatidic acid (PA), may increase the activity of atDGD2 in vitro.7 The reason for this is mostly unknown, but previously, several short segments in atDGD2 were identified for which the affinity for lipid bilayers is regulated by the extent of anionic charge in the lipid headgroups. This suggests that some regions in atDGD2 have a much stronger propensity to interact with bilayers.8 Similar observations have also been made for other monotopic, i.e., peripherally anchored, GTs.9,10 In contrast to the prokaryotic GTs alMGS and WaaG, it was proposed that atDGD2 connects to one leaflet of the bilayer by a general hydrophobic interaction with parts of the N-terminal domain, but also that positively charged residues located mainly in the C-terminal domain were important.8 In the study of the N-terminal domain of atDGD2, a multivariate analysis of several GTs was used to discriminate between lipid-binding and soluble proteins and to identify putative lipid-binding regions in the protein. In this way, several potential lipid-interacting sequences were identified.8 Here, the lipid interacting properties of sequences in the C-terminal domain of atDGD2 have been investigated. GTs are highly conserved in their three-dimensional structure but display a very low degree of sequence homology. Moreover, they utilize a diverse array of substrate and donor molecules (as reviewed by Albesa-Jové et al.11 and Hu and Walker12). These two observations highlight the importance of examining the structural characteristics and lipid interacting properties of GTs. On the basis of atDGD2 sequence properties, two regions have been identified as candidates for interacting with lipids in this study. These two segments, V240−E258 (denoted S240− 258) and G269−T287 (denoted S269−287), were investigated with regard to their membrane interacting properties, based on a combination of spectroscopic methods (CD, fluorescence, and NMR spectroscopy) and amino acid sequence analyses. Both segments have characteristics that are likely to govern function and/or interactions rather than the structure and/or fold. The results demonstrate that S240−258 forms an amphipathic helix and interacts with lipids in a charge-dependent way and may function as a means for the enzyme to be located at the bilayer surface. On the basis of the findings presented here, and a comparison with membrane interacting properties of other GT4 family GTs, a refined model for the atDGD2 interaction with the bilayer is proposed.

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EXPERIMENTAL PROCEDURES Materials. Peptides with the sequences VWSKGYKELLKLLEKHQKE, S240−258, and GDGEDSEEIKEAARKLDLT, S269−287, in full-length atDGD2 together with sequences corresponding to S11−29, S46−64, S130−148, and S227−2458 were purchased from PolyPeptide Laboratories (Strasbourg, B


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where F0 and F are the fluorescence intensities without and with the quencher, respectively, KSV is the Stern−Volmer constant, and [Q] is the concentration of the quencher.32 To quantify the interaction between S240−258 and different vesicles, dissociation constants for binding of S240−258 to 100% POPC, 20% POPG, 40% POPG, and 20% POPG/20% MGDG LUVs were measured. A stock solution of LUVs (1 mM lipids in buffer) was titrated into a cuvette containing 1.46 μM peptide, until saturation of the Trp signal was obtained. The incubating time between addition of vesicles and the measurement was 2 min, and three spectra were recorded and averaged for each sample. Background experiments with vesicle solutions were collected and subtracted from the peptide spectra. The change in the maximal emission wavelength (λmax) was plotted against the lipid:protein ratio (to compensate for changes in the peptide concentration). The dissociation constant, KD, was estimated from the obtained saturation binding curve (assuming one-site binding) via

latter case, the sample was diluted with buffer prior to the measurements. The dissolved lipids were vortexed extensively using a tabletop vortex mixer, and the resulting large multilamellar vesicles (LMVs) were subjected to a freeze−thaw cycle in liquid nitrogen and a 40 °C water bath. This was repeated five times to reduce the lamellarity and obtain large unilamellar vesicles. The solution was extruded through double 100 nm polycarbonate Nucleopore membranes 21 times to achieve a uniform size of the vesicles.25,26 The vesicles were stored at 5 °C between measurements and used within 2 weeks of preparation. The size and homogeneity of the vesicles were controlled by dynamic light scattering (DLS) experiments, conducted on a CGS-3 Compact Goniometer from ALV-GmbH. The hydrodynamic radii were estimated to be 64−76 nm, the POPG and galactolipid-containing vesicles being somewhat larger than zwitterionic vesicles. Preparation of Bicelles for NMR Measurements. For the 1H translational diffusion and 31P one-dimensional (1D) NMR experiments, isotropic bicelles were used instead of vesicles, because their smaller size is more suitable for solution NMR spectroscopy. Bicelles with 100% DMPC, 90% DMPC, and 10% DMPG or 60% DMPC, 10% DMPG, and 30% MGDG were prepared by mixing the lipids with buffer [50 mM phosphate buffer (pH 5.7)] followed by vortexing and centrifugation, as described previously.26−30 The resulting lipid slurry of lipids was then mixed with a 1 M solution of DHPC-d22 in D2O and subsequently vortexed until the opaque liquid turned transparent, to obtain a final bicelle solution with a q ratio (q = [lipids]/[DHPC]) of 0.5 and a total concentration of lipid and DHPC of 300 mM. CD Spectroscopy. CD measurements were taken on a Chirascan circular dichroism spectrometer from Applied Photophysics. The temperature was adjusted to 25 °C with a Quantum Northwest TC 125 temperature controller. The signal was detected between 190 and 260 nm using a 0.5 nm step resolution. A 0.2 cm quartz cell was used, and five spectra were collected, averaged, solvent subtracted, and converted to mean residue ellipticity (MRE) with the aid of the software within the Chirascan interface. The samples were prepared by adding the appropriate amount of peptide to ready-made vesicle solutions or buffer [50 mM phosphate buffer (pH 5.7)] to a final peptide concentration of 50 μM and a total lipid concentration of 1 mM. The secondary structure composition was estimated with the web-based tool BeStSel.31 Fluorescence Spectroscopy. Intrinsic Trp fluorescence was used to study the local environment of the Trp in S240− 258 as a function of lipid vesicle environment (S269−287 does not contain a Trp or Tyr). The fluorescence measurements were performed on a FluoroLog spectrometer (HORIBA Jobin Yvon). The excitation wavelength was 295 nm, and the emission was observed between 300 and 400 nm. The same sample conditions (peptide concentration, temperature, and buffer) as in the CD experiments were used. Samples for quenching measurements were obtained by adding aliquots of 1 M acrylamide directly to the cuvette containing a peptide/LUV solution followed by gentle stirring with the pipet tip. The incubation time between measurements was at least 3 min to allow for complete diffusion and solubilization of the quencher. The peptide concentration was initially 50 μM, and the quencher concentration in the samples was 6.5−80 mM. To calculate the quenching constant, the Stern−Volmer equation was used: F0 = 1 + KSV[Q] F

λmax = Bmax

[L:P] KD + [L:P]

(2)

where λmax is the change in the Trp maximal emission wavelength, Bmax is the maximal level of binding, and [L:P] is the lipid:peptide ratio from which the lipid concentration at the dissociation midpoint was estimated. NMR Spectroscopy. NMR spectroscopy was used to investigate the lipid binding properties of atDGD2. The samples in the NMR experiments contained 0.5 mM peptide in buffer or in bicelle solutions. The pH was adjusted with 50 mM phosphate buffer to 5.7, and 10% D2O was added for field/frequency lock. Measurements were taken for S227−245, S240−258, and S269−287 derived from the C-terminal domain in atDGD2 and for S11−29, S46−64, and S130−148 in the N-terminal domain. All experiments were conducted at 25 °C. Processing of the NMR data was performed with TopSpin 3.1 (Bruker Biospin). 1H pulsed field gradient diffusion NMR experiments were conducted on a Bruker Avance NMR spectrometer operating at a 600 MHz 1H frequency. The translational diffusion experiments were performed using a modified Stejskal−Tanner spin−echo pulse sequence33−35 with a fixed diffusion time to measure the translational diffusion constants for the peptides and lipids in the bicelle solutions. The gradient strength was increased linearly from 5 to 95% in 32 steps. Three experiments were performed for samples with and without peptide. The gradient pulse length, δ, and diffusion delays, Δ, were optimized to appropriate values for bicelle samples and for peptide in buffer separately. Typical values for the peptide experiments were 3−4 and 150−200 ms, respectively, and for lipid experiments 5−6 and 300−350 ms, respectively. To account for viscosity differences in the sample, reference measurements of water diffusion were performed,36 and in these, δ and Δ were 2 and 90 ms, respectively. The attenuation of signal intensity, I/I0, for each peak was fitted to the Stejskal−Tanner equation as a function gradient strength, and the calculated diffusion constants for each sample were then averaged over the three measurements. Gradient calibration was performed with a standard sample of 0.01% H2O in D2O and 1 mg/mL GdCl3 at 25 °C. Diffusion rates for the DMPC bicelles were estimated from the methyl group peak at 0.78 ppm, assuming that all of the DMPC molecules participate in bicelle formation.37 In bicelles containing DMPG and/or MGDG, diffusion was also measured for these lipids using

(1) C


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Figure 1. (A) Amino acid sequence, (B) modeled structure, and (C) topology diagram of atDGD2. The segments investigated by the segment-based approach in this work and in previous work8 are highlighted according to the following color scheme: orange for S11−29, purple for S46−64, green for S130−148, red for S227−245, yellow for S240−258, and pink for S269−287. The sequences studied in this work are indicated by their sequences (S227−245, S240−258, and S269−287). Also shown is an additional segment S169−187 (blue) that is strongly hydrophobic and has lytic properties on vesicles, hence believed to be important for insertion of protein into the bilayer. The helices in panel C are named on the basis of the preceding β-strand (α8 indicated). The color coding is the same as in panel B. Key residues and motifs (conserved and/or involved in membrane interaction) are indicated. 31

resonances at 3.77 ppm (DMPG) and 2.7, 1.95, and 0.86 ppm (MGDG). Diffusion rates for the peptides were estimated from peaks with sufficient intensity and gave a reasonably good fit, most often peaks corresponding to aromatic ring protons (6.5− 7.4) or, for S269−287, nonoverlapping methyl group protons. When we assumed a two-state behavior between the bound and unbound peptides, the percentage of bound peptide was calculated from D bound = xDlipid + (1 − x)Dfree

P 1D spectra were recorded for bicelles in the absence and presence of the C-terminal sequences S240−258 or S269−287 and also for bicelles with or without the previously studied N-terminal sequences S11−29, S46−64, S130−148, and S227− 245.8

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RESULTS In Silico Sequence Analyses. To be able to visualize and validate the experimental results, the Robetta structure server was used to obtain a hypothetical model structure of atDGD2 (Figure 1), and from this model, a topology diagram was generated. The predicted double Rossmann fold is evident from this model, and the segments studied experimentally are mostly located in helical or loop regions connecting the β-strands. The full-length amino acid sequence of atDGD2 was subjected to several analyses by primary structure analysis tools, commonly used to characterize proteins in silico. The theoretical pI of the full-length protein is estimated to be 7.7 according to ProtParam, i.e., quite neutral. Significant differences between

(3)

where Dlipid is the diffusion constant of DMPC, Dfree is the diffusion constant of the peptide in buffer solution, Dbound is the diffusion constant of the peptide in the presence of bicelles, and x is the fraction of bound peptide. Because of limited solubility, it was not possible to study S11−29, S46−64, and S130−148 in buffer and the fraction of peptide associated with bicelles was therefore not calculated. The diffusion rates of S11−29, S46−64, and S130−148 in buffer are, however, expected to be similar to those for S227−245, S240−258, and S269−287 (Table 2). D


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Figure 2. Results from the analysis of the atDGD2 amino acid sequence. (A) Amphipathicity and secondary structure propensity of segments S240− 258 and S269−287 illustrated with helical wheels. Anionic and cationic residues are colored red and blue, respectively. Hydrophobic residues are colored yellow, and polar residues are colored purple. His is colored pink and small, and neutral amino acid residues are colored gray. The hydrophobic moment for each segment is indicated by an arrow, showing both the direction and degree of hydropathy. The N- and C-termini are colored red. (B) Results from the SEG analysis indicating LCRs shown as gray spheres (KLEQQKLQ) and NetPhos results indicating possible phosphorylation sites shown as lime spheres (S274 and T287) on the modeled structure of atDGD2. The sequences studied in this work are colored yellow (S240−258) and pink (S269−287), and the amino acid residues that have been shown to be important for enzymatic activity7 are shown as red sticks (V25, K243, and W241). A predicted in-plane membrane anchor (by AmphiphaSeek, E247−E253) is shown as purple spheres.

while S269−286 is acidic (net charge of −4) with a pI value of 4.2 and is mainly composed of hydrophilic residues. Agadir predicted segments S240−258 and S269−287 to be 20.8 and 21.4% helical, respectively. HeliQuest was used to determine the hydrophobicity and the hydrophobic moment (⟨μH⟩) of the segments. The results demonstrated a strong hydrophobic moment in S240−258 (⟨μH⟩ of 0.644 and hydrophobicity of 0.2711), indicative of an amphipathic helix. S269−287, on the other hand, had a very hydrophilic character and a hydrophobic moment (⟨μH⟩ of 0.221 and hydrophobicity of −0.116) weaker than that of S240−258. With the segments drawn as helical wheel projections, it was observed that S240−258 has a clear amphipathic character, with all positively charged residues flanked by negatively charged residues (Figure 2A), and hydrophobic residues on the opposite side of the helix. The amino acid sequence of S269−287 contains many anionic residues but did not display the same amphipathic pattern in a helical wheel as seen for S240−258. Structure of S240−258 and S269−287. Far-UV CD spectra (190−250 nm) were measured to monitor how different lipids affected the secondary structure content of peptides

the N-terminal (1−215) and C-terminal (216−473) domains were, however, obvious (pI values of 8.8 and 6.4, respectively). The same observation, a basic N-terminal domain and a slightly more acidic C-terminal domain, was previously demonstrated for another monotopic GT, alMGS.38 The AmphipaSeek algorithm predicted one distinct region (E247LLKLLE253) to be a so-called in-plane amphipathic membrane anchor (Figure 2B). The SEG algorithm proposed two low-complexity regions, K218LEQQKLQEQ227 and K243GYKELLKLLEKHQKELAELE263. The first LCR is topologically located between the two domains, while the second contains the predicted amphipathic helix (Figure 2B). On the basis of the model structure and the predictions, two sequences were selected for further biophysical studies: V 240 WSKGYKELLKLLEKHQKE 258 , S240−258, and G269DGEDSEEIKEAARKLDLT286, S269−286 (Figure 1A). They are close to each other in primary structure and are both located in the C-terminal domain of atDGD2 (Figure 1B). They do, however, have very different physical characteristics regarding, for instance, pI values and hydrophobicities. S240−258 is basic (net charge of +3) with a theoretical pI of 9.3, but with a high degree of hydrophobic residues, E


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CD spectrum of either of the peptides, indicating that the presence of MGDG does not alter the structure of the peptides. Trp Fluorescence and Lipid Interaction of S240−258. To directly study the bilayer interacting properties of samples with S240−258 peptide and different LUVs, different fluorescence spectroscopy measurements were performed. The emission maxima, λmax, for intrinsic Trp (W241) fluorescence together with Stern−Volmer quenching constants, KSV, are listed in Table 1. Wavelength shifts in λmax reveal information

derived from the C-terminal domain of atDGD2. Spectra were recorded for S240−258 and S269−287 peptides in buffer and in different lipid mixtures (Figure 3). In buffer solution, both of

Table 1. Trp Fluorescence Data for S240−258 in Different Environmentsa sample conditions

λmax (nm)

KSV (M−1)

buffer 100:0 POPC 90:10 POPC:POPG 80:20 POPC:POPG 70:30 POPC:POPG 60:40 POPC:POPG 50:50 POPC:POPG 80:20 POPC:MGDG 60:20:20 POPC:MGDG:POPG

361 357.5 354.5 349.5 343.0 343.5 342.5 360.5 351.0

9.8 9.4 not available 4.8 not available 3.8 not available 9.4 5.2

a The peptide concentration in these experiments was 50 μM, and the lipid concentration was 1 mM.

about the accessibility of the fluorophore (Trp) to the solvent, and a clear blue shift of the emission maximum as a function of increasing anionic lipid content was observed (Table 1). MGDG (20%) was added to LUVs composed of 100% PC or 80% PC and 20% PG to investigate the effect on the peptides when substrate lipids were present in the bilayer. In agreement with the CD results, no influence of MGDG on the maximum could be detected. Hence, we conclude that the structure induction revealed by CD is correlated with an increased level of membrane association of the peptide. In an effort to quantify the interaction strength of the peptide and the LUVs, KD values were calculated on the basis of the blue-shifted Trp signal upon binding. Figure 4A shows the changes in λmax as a function of L:P ratio. From these curves, estimates of KD values were obtained. The KD values for binding of S240−258 to 20% PG and to 20% PG/20% MGDG LUVs were in this way found to be approximately 70 μM, irrespective of the presence of MGDG. The corresponding value for the interaction with 40% PG LUVs was 10 μM. No interaction was detected with zwitterionic vesicles. Shifts in wavelength were also compared to previously measured data for N-terminal segments (Figure 5). The comparison clearly shows that the blue-shift in fluorescence is largest for the C-terminal S240−258 segment, followed by the N-terminal S130−148 segment. We conclude that these regions are likely to be the main points of bilayer interaction in the DGD2 enzyme. Fluorescence quenching was performed with the watersoluble quencher acrylamide (Table 1 and Figure 4B). The data demonstrated a large difference in the accessibility of the Trp to solvent when a sufficient amount of PG was added to the LUVs. In the presence of 20% PG, the quenching constants dropped significantly, and increasing the level of PG to 40% shielded the Trp from water even further. As in the other experiments, no effect of adding MGDG was observed. Effect of Lys Residues on S240−258 Structure and Lipid Interaction. To examine the contribution of Lys

Figure 3. Far-UV CD spectra of (A) S240−258 and (B) S269−287 in the presence of vesicles containing increasing amounts of POPG and/ or MGDG (see the inset). The lipid:peptide ratio was 20.

the peptides were mainly unstructured. Small α-helical contributions to the spectra were observed for both peptides, revealed by the slight shift in the minimum from 197 nm (typical for random coil structure) to 200 or 203 nm, and also by the general appearance of the signal between 210 and 240 nm. This partly helical structure was most pronounced in the CD spectrum for S269−287 (Figure 3B). In the presence of zwitterionic LUVs, there was no change in the CD signals of either of the peptides, i.e., there was no interaction, or the interaction was of a nature that did not alter the secondary structure of the segments. For S240−258, it was possible to detect a slight change toward the α-helix in the secondary structure when more than 20% of the lipids was exchanged for PG (Figure 3A). At higher PG contents, this effect was even more pronounced. No further change in the secondary structure of S240−258 at a PG content higher than 40−50% was observed. A quantification for S240−258 by the BeStSel circular dichroism structure analysis tool31 showed that the helical content increased from 6 to 23% with an increasing amount of PG. S269−287 was also exposed to the same LUVs, but no significant change in the overall appearance of the spectrum was observed, irrespective of the lipid composition (Figure 3B). We can therefore conclude that the secondary structure of S240−258 depends on bilayer composition, whereas no structure induction in S269−280 could be detected. The effect of MGDG was examined by substituting 20% POPC in POPC LUVs and in POPC/POPG (80:20) LUVs for MGDG (Figure 3). Addition of MGDG did not have an influence on the F


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Figure 4. Fluorescence emission of W258 in the S240−258 peptide. (A) Binding curves based on blue-shifts in λmax (in absolute value) as a function of L:P ratio. (B) Stern−Volmer plots for the quenching of fluorescence (F0/F) as a function of acrylamide concentration ([Q]). The lipid:peptide ratio was 20.

Figure 6. Far-UV CD spectra of (A) wtS240−258, (B) S240− 258(K254A), and (C) S240−258(K250,254,257A) in the presence of buffer (), POPC (···), and 40:60 POPG/POPC (---) LUVs. The lipid:peptide ratio was 20.

Figure 5. Trp-containing segments derived from both domains of atDGD2 display different degrees of lipid interaction, as shown here by the difference in Trp fluorescence emission wavelength maxima (Δλmax) as a function of the mole percent of PG. The lipid:peptide ratio was 20.

helix by removing one or several Lys residues, most likely related to a change in hydrophobicity. As discussed previously, wtS240−258 does not appear to undergo a significant change in its structure upon addition of POPC LUVs (Figure 6A) but becomes more clearly helical in 40:60 POPG/POPC LUVs. The same is true for S240−258(K254A), although an even larger increase in helical content is observed (Figure 6B). Contrary to this, the structure of the variant in which three Lys residues were substituted appeared to be less affected by either PC or PC/PG LUVs compared to the one in which only one Lys was removed (Figure 6C).

residues to the bilayer association of S240−258, we mutated three of the Lys residues in the segment (K250, K254, and K257) to Ala. Two constructs were examined, one in which only K254 was substituted with Ala and a second in which all three Lys residues were exchanged for Ala. CD spectra for these sequences are shown in Figure 6. All three peptides are somewhat structured already in buffer, but a shift in the CD signal at around 200 nm toward more helical-like values demonstrates that the intrinsic structure is altered toward containing more G


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Biochemistry These results were further supported by examining fluorescence spectral properties in LUVs. The fluorescence emission maximum, λmax, for intrinsic Trp (W241) fluorescence did not change as dramatically for S240−258(K250,254,257A) in the presence of either PC or 60% PC/40% PG LUVs (259 nm in buffer, 258 nm in POPC, and 248 nm in 40:60 POPG/ POPC LUVs). For S240−258(K254A), on the other hand, shifts similar to those observed for the wild type were seen (361, 359, and 343 nm in the three solvents, respectively). The difference in shifts as a function of the total number of Lys residues in the sequence is shown in Figure 7. These results

The diffusion constant of the DMPC/DHPC bicelles without peptide (Table 2) was found to be in the range previously measured under similar sample conditions (5.2 × 10−11 m2 s−1).39 Upon addition of 10% DMPG, the diffusion rates were marginally slower; i.e., the apparent size of the bicelles appeared to be somewhat altered compared to that of the zwitterionic bicelles (4.9 × 10−11 and 5.0 × 10−11 m2 s−1 for DMPC and DMPG, respectively). The diffusion coefficient of bicelles containing both 10% DMPG and 30% MGDG was 2.6 × 10−11 m2 s−1 (2.7 × 10−11, 3.0 × 10−11, and 2.6 × 10−11 m2 s−1 for DMPC, DMPG, and MGDG, respectively), indicating that MGDG made the bicelles even larger, in agreement with previous results.39 Moreover, most likely, the nonbilayer prone MGDG distorts the packing of the bilayer and thereby alters the size. Hence, DMPG had the effect of slowing the diffusion of bicelles both with and without MGDG. It is also worth noting that the bicelles containing both DMPG and MGDG were difficult to produce, because the lipids were more difficult to dissolve. However, on the basis of the fact that all lipids diffuse with the same rate within each type of bicelle, we conclude that there is no phase separation of the lipids and that the lipids used here do form bicelles.39 Three peptides derived from the C-terminal domain of atDGD2, S227−245, S240−258, and S269−287, were added to the different bicelles, and the diffusion coefficients were measured (Table 2). Via comparison of the diffusion rates for the bicelles and the peptides, it was demonstrated that S227−245 and S240−258 clearly interacted with all types of bicelles. Using eq 3, the extent of binding was calculated, and both sequences showed almost complete (96−99%) binding to all bicelles. In contrast, S269−287 showed only weak interaction, and the ∼30% binding observed may have been due to an unspecific interaction aided by the short-chain lipid DHPC. It was not possible to measure the diffusion of the N-terminal sequences in buffer, and therefore, the extent of binding was not calculated. One may note, however, that all N-terminal sequences appear to interact with all types of bicelles (Table 2). It has previously been demonstrated that bicelles provide qualitatively different bilayer environments for short peptides.40,41 A larger fraction of peptides generally associate with bicelles and on average become more structured, as compared with, e.g., LUVs at the same lipid composition. Although the peptides interact with PC bicelles, it has also been demonstrated that the qualitative differences associated with introducing charged lipids

Figure 7. Trp fluorescence emission maxima for wtS240−258, S240− 258(K254A), and S240−258(K250,254,257A) (containing five, four, and two Lys residues in total, respectively) in the presence of 40:60 POPG/POPC LUVs. The lipid:peptide ratio was 20.

together indicate that the Lys residues are responsible for a charge-dependent interaction, and removing them causes a dramatic change in the bilayer interacting properties in S240−258. Interaction with Bicelles. Translational diffusion rates for the C-terminal peptides and bicelles were measured via 1H PFG NMR spectroscopy. The normalized diffusion rates for S240− 258 and S269−287 in buffer were (17.78 ± 0.01) and (18.89 ± 0.06) × 10−11 m2 s−1, respectively, and the diffusion for S227− 245 has previously been measured to be 19.9 × 10−11 m2 s−1.8 The lower value for S240−258 may indicate that the peptide dimerizes (or forms other oligomeric states) to a certain degree in solution. On the other hand, only very little contribution of secondary structure was observed in CD spectra for both peptides (Figure 4), indicating that they mainly occur as unstructured monomers in solution.

Table 2. Diffusion Coefficients for atDGD2 Segments and Bicelles at a DMPC/DMPG Concentration of 100 mM and a Peptide Concentration of 0.5 mM ([L:P] = 200) 100:1 DMPC

90:10 DMPC:DMPG

D (×10−11 m2 s−1) lipid

a

peptide

60:10:30 DMPC:DMPG:MGDG

D (×10−11 m2 s−1) b

xb (%)

no peptide

5.2 ± 0.1

−

−

S227−245 S240−258 S269−287

5.6 ± 0.1 5.4 ± 0.04 5.2 ± 0.03

5.8 ± 0.04 5.5 ± 0.05 14.7 ± 0.1

99 99 31

S11−29 S46−64 S130−148

4.6 ± 0.1 5.0 ± 0.01 5.0 ± 0.05

5.0 ± 0.02 5.4 ± 0.02 5.4 ± 0.1

lipid

a

peptide

D (×10−11 m2 s−1) b

4.8 ± 0.1 − C-Terminal Sequences 5.6 ± 0.1 5.7 ± 0.2 5.7 ± 0.03 5.8 ± 0.1 5.3 ± 0.1 14.9 ± 0.1 N-Terminal Sequences 5.3 ± 0.02 5.5 ± 0.1 5.6 ± 0.04 5.8 ± 0.03 5.4 ± 0.04 5.7 ± 0.1

xb (%) − 99 99 29

lipida 2.6 ± 0.1

peptideb −

2.7 ± 0.04 2.8 ± 0.1 N/A

3.3 ± 0.1 3.0 ± 0.1 14.2 ± 0.2

2.5 ± 0.01 2.5 ± 0.05 2.4 ± 0.01

3.2 3.1 ± 0.3 2.8 ± 0.1

xb (%) − 96 99 29c

a

Based on the methyl group resonance at 0.78 ppm. bBased on signals from the aromatic (if present) or methyl groups. cEstimate based on DMPC and DMPC/DMPG diffusion. H


Article

Biochemistry

interact with lipids. The strong anionic character, together with the very low hydrophobicity of S269−287, indicates that this region may still be important for the protein in some other way. In an effort to determine additional interaction roles for the atDGD2 segments, the galactolipid MGDG was added to the membrane mimetic systems. MGDG/POPC vesicles did not alter the Trp fluorescence signal in any peptide and did not alter the secondary structure of S240−258 or S269−287. The KD values for the interaction of S240−258 with 20% PG LUVs (70 μM, with or without MGDG present) were ∼10-fold larger than the KD value for 40% PG LUVs (10 μM), indicating a charge-dependent binding of the peptides to lipids. Moreover, substituting three of the Lys residues with Ala in S240−258 removed at least part of this bilayer interaction. Taking all results together, we can conclude that negative charge on the bilayer surface is essential for membrane interactions for both the N-terminal and C-terminal domains. Interaction with acceptor molecules is likely to take place in the N-terminal domain, and the MGDG-binding site is presumably within a pocket that originates from both domains. Here we see that our results show that MGDG does not seem to be involved in anchoring to the membrane, at least not in a way that affects Trp fluorescence, or inducing structural changes. GT4 Glycosyltransferases Share Common Structural and Lipid Interacting Features. Glycosyltransferases that utilize nucleotide sugars as substrate molecules (so-called Leloir GTs) have a very low degree of sequence homology, but still, only two main folds have been found.11,12,42 This is a remarkable trait because they perform their enzymatic activity with a vast amount of different glycosyl donor molecules (e.g., lipids, proteins, and small sugar molecules) and can be either membrane-attached or soluble. The GT-B fold enzymes generally bind the nucleotide sugar to the C-terminal domain and the accepting substrate molecule (i.e., the glycosylated lipid for atDGD2) to the N-terminal domain.42 Recent results on the membrane interacting properties of two prokaryotic GTs, WaaG (from Escherichia coli) and PimA (from Mycobacterium smegmatis), have also provided valuable information about GT4 structure and function. The N-terminal S130−158 and S168−186 segments of atDGD2 were previously suggested to be involved in a permanent, monotopic, membrane anchoring of atDGD2. S168−186 is located in a position corresponding to a membrane-interacting region of WaaG (MIR-WaaG, consisting of three helices after β5) with many Tyr and cationic residues, capable of specific interaction with anionic lipids. WaaG has been proposed to insert into the membrane in a monotopic manner via this segment.10 W177 in the atDGD2 S168−186 segment is located in an exposed fashion in the model structure of atDGD2 (Figure 2), and the aromatic nature of this amino acid residue is conserved in very many, if not all, GT4 GTs. Additionally, an exceptional lipid interacting mechanism was recently discovered in PimA, where large structural changes modulated catalysis via anionic lipids in the bilayer. Two helices in the N-terminal domain, corresponding to the location of the α5 helices in atDGD2, were shown to be involved in large secondary structure reshuffling events.20 In earlier work with atDGD2, it was demonstrated that S130−148 in the α4 position altered its structure from random coil to α-helix via a β-sheet structure upon being exposed to anionic lipids. There are, however, very few reports of membrane-bound proteins in which the tertiary structure is believed to undergo dramatic changes in contact with lipids.20,43 Nevertheless, these recent findings clearly

(e.g., structural conversion and increased level of association) are also present in bicelles.40 Hence, these results should be viewed in light of the qualitative differences in bilayer properties provided by bicelles and LUVs. One may also note that the peptide:lipid ratio was 10-fold higher in the LUVs. We therefore conclude that the sequences that display a lipid chargedependent structural conversion interact with bicelles, while S269−287 does not interact significantly with lipids at all. The peptides also had small effects on the bicelles. Overall, there were no consistent changes in bicelle size, but the N-terminal sequences had an effect on the size, or morphology, of the bicelles somewhat different than the effect of the C-terminal sequences. While the N-terminal sequences made the DMPC bicelles diffuse somewhat slower, the two C-terminal sequences that interact with DMPC bicelles had the opposite effect, but the differences were small. When PG were added to the bicelles, all peptides produced faster lipid diffusion. This may be either due to peptides interacting with the bicelle rim, acting as surfactants and thereby decreasing the effective q value (q = [lipid]/DHPC), or due to a change in bicelle morphology, possibly due to specific interaction between the negatively charged lipids and peptides. The fact that the peptides and lipids have similar diffusion coefficients indicates that no phase separation occurs, and that the lipids are still integrated in the bicelles together with the peptides. In an effort to characterize the interactions in more detail, 31P NMR spectroscopy was used to measure the effect of the peptides on lipid headgroups. No effect on signal intensity or chemical shifts in either of the lipids in bicelles could however be detected. We conclude that no effect on the lipid phosphates could be detected from either of the atDGD2 fragments.

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DISCUSSION The C-Terminal Domain Contains a Region That Interacts with Lipids. Little is known about the substrate recognition and biomembrane interactions in GT-B glycosyltransferases. Here the structural and lipid interacting characteristics of the GT atDGD2 have been investigated using a segment-based approach, with the aim of clarifying the roles of the two domains. Earlier work proposed that regions in the N-terminal domain interact with the bilayer in a monotopic way, dependent on electrostatics via cationic amino acid residues and by an exposed hydrophobic segment that also contained many aromatic residues (e.g., W132 and W177, Y145, Y173, and Y182).8 In contrast, the C-terminal domain of atDGD2 has here been shown to contain two segments with a pronounced amphipathic character and a strong hydrophobic moment: S227−245 and S240−258. A third segment with a highly charged nature and a helical propensity (S269−287) was also identified as being potentially important. A CD spectroscopy structural analysis of S240−258 and S269−287 in large unilamellar vesicles showed that approximately 30−40% of anionic lipids in the bilayer transforms S240−258 from a largely unstructured into an α-helical conformation (Figure 3), while no change in secondary structure was observed for S269−287. This result is supported by fluorescence and NMR measurements that clearly showed a chargedependent lipid interaction of S240−258, while no significant interaction could be detected for S269−287 (Tables 1 and 2). On the basis of fluorescence results, S240−258 had the most pronounced lipid-induced changes in spectral properties, followed by the proposed N-terminal anchor S130−148. Contrary to this, all results showed that S269−287 does not appear to I


Article

Biochemistry

three isoforms of monogalactosyldiacylglycerol synthases (transferases) in A. thaliana; CD, circular dichroism; DGDG, digalactosyldiacylglycerol; DHPC, 1,2-dihexanoyl-sn-glycero-3phosphocholine; DLS, dynamic light scattering; DMPC, 1,2dimyristoyl-sn-glycero-3-phosphocholine; DMPG, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol; GT, glycosyltransferase; GT4 GT, glycosyltransferase of the GT4 family; LCR, lowcomplexity region; LUV, large unilamellar vesicle; MGDG, monogalactosyldiacylglycerol; NMR, nuclear magnetic resonance; PimA, GT involved in the phosphatidylinositol metabolism in M. smegmatis; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; POPG, 1-palmitoyl-2-oleoylphosphatidylglycerol; WaaG, GT involved in synthesis of lipopolysaccharides in E. coli.

demonstrate a potential dynamic behavior connected to the function and interactions of GT-B GTs, involving structural rearrangements. The SEG analysis revealed several so-called low-complexity regions (LCRs) in the C-terminal domain of atDGD2. LCRs are usually not involved in any critical way in the folding of the protein or in sustaining the three-dimensional structure. Instead, they may act as regions that perform some proteinspecific function or are involved in specific interactions, as LCR-containing proteins have more interaction partners44 and are believed to be part of regions that are rapidly evolving.45 This is interesting because GTs display a wide range of interaction partners and substrates. LCRs are often helical and may be enriched in regions involved in structural transitions.46 The LCRs detected in atDGD2 are located in the first helix after the linking region of the domains [KLEQQKLQ (Figure 2C)] and in the region between β7 and β8 (including segment S240−258). A comparison of different GTs may shed light on the possible role of S268−287. Interestingly, atDGD2 and PimA share a high degree of sequence identity in residues K243−285 (K202−241 in PimA), including both S240−258 and the nonlipid-interacting S268−286. S269−287 is located between β8 and β9 (Figure 2C), and in both PimA and atDGD2, there is a helix (α8) with a very low pI and many solvent-exposed Glu and Asn residues. Additionally, a recent structure of the soluble GT4 family member trehalose synthase (TreT, PDB entry 2X6Q) in the Archaea Pyrococcus horikoshii47 showed that TreT forms dimers through a helix corresponding to α8 in atDGD2. The amino acid sequence in this helix contains many charged residues in TreT but carries a net charge of zero. The presence of charged and strongly hydrophilic helices, such as S268−286 in atDGD2, may indicate a switchlike interaction with substrate molecules, biomembranes, or other interaction partners. In summary, the C-terminal domain of atDGD2 has several regions with characteristics that indicate that specificity in sensing the membrane surface and/or activation of the enzyme may be located within this domain. Together with the previously characterized S227−245 segment,8 S240−258 is a good candidate for a lipid-sensing region in atDGD2, whereas the function of the S269−287 segment is still to be discovered. On the basis of our findings that the interaction is chargedependent, we propose that S240−258 may function as a lipidsensing switch related to the location of the enzyme with respect to the bilayer, allowing for dynamic events, and that this is dependent on membrane surface charge.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +46 8 162448. Fax: +46 8 155597. ORCID

Lena Mäler: 0000-0002-9464-4311 Funding

This work was supported by the Swedish Science Research Council (Contract 621-2014-3706). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS atDGD1 and atDGD2, two isoforms of digalactosyldiacylglycerol synthases (transferases) in A. thaliana; atMGD1−atMGD3, J


Article

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Insights into the Membrane Interacting Properties of the C-Terminal

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