Insights into the structure of plant a-type phospholipase D Susanne Stumpe, Stephan Konig and Renate Ulbrich-Hofmann ă Martin-Luther University Halle-Wittenberg, Institute of Biochemistry and Biotechnology, Halle (Saale), Germany Keywords calcium binding; phospholipase D; smallangle X-ray scattering; stability; structure Correspondence R Ulbrich-Hofmann, Martin-Luther University Halle-Wittenberg, Institute of Biochemistry and Biotechnology, Kurt-Mothes-Str 3, 06120 Halle (Saale), Germany Fax: +49 345 5527303 Tel: +49 345 5524864 E-mail: Renate.Ulbrich-Hofmann@ biochemtech.uni-halle.de (Received 29 December 2006, revised 23 February 2007, accepted 20 March 2007) doi:10.1111/j.1742-4658.2007.05798.x Phospholipases D play an important role in the regulation of cellular processes in plants and mammals Moreover, they are an essential tool in the synthesis of phospholipids and phospholipid analogs Knowledge of phospholipase D structures, however, is widely restricted to sequence data The only known tertiary structure of a microbial phospholipase D cannot be generalized to eukaryotic phospholipases D In this study, the isoenzyme form of phospholipase D from white cabbage (PLDa2), which is the most widely used plant phospholipase D in biocatalytic applications, has been characterized by small-angle X-ray scattering, UV-absorption, CD and fluorescence spectroscopy to yield the first insights into its secondary and tertiary structure The structural model derived from small-angle X-ray scattering measurements reveals a barrel-shaped monomer with loosely structured tops The far-UV CD-spectroscopic data indicate the presence of a-helical as well as b-structural elements, with the latter being dominant The fluorescence and near-UV CD spectra point to tight packing of the aromatic residues in the core of the protein From the near-UV CD signals and activity data as a function of the calcium ion concentration, two binding events characterized by dissociation constants in the ranges of 0.1 mm and 10–20 mm can be confirmed The stability of PLDa2 proved to be substantially reduced in the presence of calcium ions, with salt-induced aggregation being the main reason for irreversible inactivation Phospholipase D (PLD; EC 3.1.4.4), which hydrolyzes phospholipids to phosphatidic acid and the head group alcohol, occurs in plants, microorganisms and animals In plants, PLD isoenzymes play multiple regulatory roles in diverse processes, including abscisic acid signaling, programmed cell death, root hair patterning, root growth, freezing tolerance, and other stress responses [1] There is growing evidence for phosphatidic acid being an intracellular lipid messenger in plants as well as in mammals [2,3] Most PLDs also catalyze the transphosphatidylation reaction in which the phosphatidyl moiety is transferred to a suitable alcohol In biotechnology, this phospholipid-transforming reaction has been exploited on a laboratory and industrial scale [4] In plants, multiple PLD genes encoding isoforms with distinct regulatory and catalytic properties have been identified [5,6] The most prevalent isoenzyme, which is responsible for the common PLD activity observed in leaves or seeds of plants, is the a-type PLD (PLDa) The conventional PLDa does not require phosphoinositides for activity when assayed at millimolar levels of calcium ions It exhibits optimum activity at pH values between and 6, and nonphysiologic high calcium concentrations between 30 and 100 mm [3,7,8] In contrast, the PLD isoenzymes of the b, c, d and e types from Arabidopsis show highest activity at micromolar calcium concentrations, and require the presence of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) [5] The activity Abbreviations PLD, phospholipase D; PLDa, a-type phospholipase D; PLDa2, isoenzyme form of phospholipase D from white cabbage; PpNp, phosphatidyl-p-nitrophenol; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; SAXS, small-angle X-ray scattering 2630 FEBS Journal 274 (2007) 2630–2640 ª 2007 The Authors Journal compilation ª 2007 FEBS S Stumpe et al Fig Scheme of the primary structure of PLDs (A) PLDa2 from cabbage (P55939) (B) PLD1 from human (Q13393) (C) PLD from Streptomyces sp (1V0YA) The N-terminal C2 domain (A), the PX and PH domains (B) and the four PLD specific conserved regions (I–IV) are marked Regions II and IV represent the two HKD motifs forming the active site In (A), the positions of the 30 tyrosine and 15 tryptophan residues are marked by stars Structure of plant phospholipase D A B C of plant PLDf is independent of calcium ions, but the enzyme requires PtdInsP2 to selectively hydrolyze phosphatidylcholine With the exception of PLDf, which contains a Phox (PX) and a Pleckstrin (PH) homology domain in the N-terminal region, all the other PLD isoenzymes possess a calcium- and phospholipid-binding domain (C2 domain) at the N-terminus [5] From white cabbage, the most traditional source of PLD for biocatalytic phospholipid transformations, two PLDs of the a type (PLD1 and PLD2) were expressed in Escherichia coli and purified [8] The amino acid sequence of PLD2, called PLDa2 in the following, is identical with that of PLD isolated from cabbage leaves [9] Figure 1A shows a schematic picture of the primary structure of PLDa2 with the C2 domain and four conserved regions (I–IV) specific for the PLD superfamily, where regions II and IV are two copies of the HxKx4Dx6GSxN (HKD) motif [10,11] The H, K and D residues of theses motifs are absolutely conserved [11] As derived from the only crystal structure of a PLD, the prokaryotic PLD from Streptomyces sp strain PMF, the two HKD motifs form one catalytic site [12] For comparison, Fig 1B,C shows schemes of the primary structures for a mammalian and a microbial PLD, demonstrating that the PLD-specific regions I–IV are present in all members of the PLD superfamily However, there is low similarity in the remaining parts of the molecules Most mammalian PLDs contain a PX and a PH domain instead of the C2 domain in plants, whereas microbial PLDs lack such probably regulatory domains Despite the increasing amount of primary structural data for PLDs, there is almost no information on the secondary and tertiary structures of plant PLDs Although Abergel et al reported the crystallization of the cowpea PLD [13], no crystal structure has been published so far Even reports on purified plant PLDs are extremely rare Obviously, the biochemical and structural characterization of plant PLD enzymes has been hindered by their instability during and after purification from plant tissues [14,15] or the low yields in recombinant protein production [16], respectively These difficulties were recently overcome by using Ca2+-mediated hydrophobic chromatography [17] combined with recombinant PLD production in E coli [8] This purification method was also successfully applied to PLDa enzymes from soybean [18], cowpea [19], and poppy [20,21] In the present study, the first insights into the secondary and tertiary structure of PLDa2 from cabbage are given The surface structure of the enzyme was investigated by small-angle X-ray scattering (SAXS) Further structural information was obtained by the spectroscopic characterization of PLDa2 in the native state The high instability of purified plant PLDs, as reported above, was shown to result from salt effects that can be eliminated in low-buffered solutions at room temperature From spectroscopic and activity measurements, two classes of calcium-binding events, with dissociation constants varying by at least two orders of magnitude, were derived Results PLDa2 preparation PLDa2 was produced according to Schaffner et al ă [8] with slight modifications as described in Experimental procedures, which improved the yield of active enzyme The amount of homogeneous PLDa2 obtained was 3.5–5.0 mg per liter of cell culture The specific activity towards the synthetic substrate phosphatidyl-p-nitrophenol (PpNp) was 10.2–14.7 mL)1, which is consistent with the activity of PLDa2 in previous preparations [8] FEBS Journal 274 (2007) 2630–2640 ª 2007 The Authors Journal compilation ª 2007 FEBS 2631 Structure of plant phospholipase D S Stumpe et al Structural information from SAXS Scattering measurements with synchrotron radiation were performed at pH 7.0 with and 4.7 mgỈmL)1 PLDa2 A typical SAXS profile of PLDa2 is shown for 4.7 mgỈmL)1 in Fig 2A From scattering intensities I(0), a molecular mass of 97 kDa was calculated (using BSA as standard), which is in accordance with a monomer of PLDa2 Correspondingly, no aggregates were detected at a PLDa2 concentration of 0.05–0.2 mgỈmL)1 in size exclusion chromatography The molecular mass deduced from the amino acid sequence is 91.9 kDa, whereas the molecular mass determined by size exclusion chromatography was 85.8 kDa (data not shown) The radius of gyration Rg (2.87 ± 0.02 nm) extracted by gnom [22] from the scattering data was in good agreement with the Rg value estimated by primus [23] from the Guinier plots (2.89 ± 0.01 nm) and is in the typical range for a spherical particle The bell-shaped distance distribution function illustrates the quality of the experimental data (Fig 2B) Reconstruction of the overall shape of the PLDa2 from the X-ray scattering data was achieved by the ab initio modeling program dammin [24] The superposition of the 10 calculated models (Fig 3) shows a longish molecule with loosely structured apical and basal regions The lid-like region (Fig 3, left) can be construed as a separate domain, but was not explicitly visible in all individual models The Porod volume (volume of the hydrated solute particle) of the lowresolution models averages 126.7 ± 3.5 nm3 A B Fig SAXS profile (A) and distance distribution function (B) of PLDa2 (A) Experimental data are indicated by open circles The solid line shows the scattering curve fitted by the program GNOM [22] The PLDa2 concentration was 4.7 mgỈmL)1 Structural information from spectroscopy UV absorption spectra (Fig 4A) show a maximum at 280 nm, with a slight shoulder at 295 nm assigned to the tryptophan residues The absence of absorption in the UV region > 310 nm, characteristic for light scattering of high molecular mass species, provides no Fig Ab initio low-resolution structure model of PLDa2 calculated from the SAXS pattern The balls represent the dummy atoms used in the simulated annealing procedure of program DAMMIN [24] to restore the models The model resulting from superposition of 10 individual models is shown in three different views obtained by 90° rotation around the y-axis (middle) and the z-axis (right) 2632 FEBS Journal 274 (2007) 2630–2640 ª 2007 The Authors Journal compilation ª 2007 FEBS S Stumpe et al Structure of plant phospholipase D A C Fig Spectra of native PLDa2 (A) UV absorbance spectrum (B) Fluorescence emission spectra with excitation at 278 nm (black) and 295 nm (gray) (C) Far-UV CD spectrum (D) Near-UV CD spectra in the absence (black) and the presence of 100 mM CaCl2 (gray) All spectra were obtained in 50 mM sodium acetate buffer (pH 5.5) at 20 °C The PLDa2 concentrations were 253 lgỈmL)1 (A), 25 lgỈmL)1 (B), and mgỈmL)1 (C, D), respectively evidence for aggregates, and reconfirms the size exclusion chromatography results The fluorescence spectra have an emission wavelength maximum at 334 nm and refer to a hydrophobic environment for most or all tryptophan side chains (Fig 4B) The similar shape and fluorescence emission maxima of the spectra at both excitation wavelengths (278 and 295 nm) indicate a complete resonance energy transfer from the tyrosine to the tryptophan residues The secondary structure of PLDa2 was investigated by far-UV CD spectroscopy (Fig 4C) The far-UV CD spectrum of PLDa2 exhibits: (a) a sharp maximum at 192 nm; and (b) a wide minimum between 208 and 220 nm The a-helix and b-strand contents of the protein were calculated with the online programs dichroweb [25] and k2d [26], respectively With the program k2d, the b-sheet content (0.45) was higher than the a-helix content (0.08), whereas the program dichroweb computed secondary structure contents with 0.20 a-helix, 0.28 b-sheet, and 0.21 turn conformation Both methods agreed in indicating more b-sheet than a-helix content The near-UV CD spectrum of native PLDa2 (250– 300 nm, Fig 4D), describing the chiral environment of the aromatic amino acid side chains, has a defined structure that presents two sharp minima at 288 and 295 nm, and two maxima at 274–283 nm (wide) and 290 nm (sharp) Storage stability and aggregation propensity The instability of the purified enzyme described in the literature [14,15] prompted us to analyze the inactiva- B D tion of PLDa2 under selected conditions Storage at lower temperatures (0–10 °C) resulted in faster inactivation of the enzyme than at room temperature (23 °C) Under physiologic conditions (pH 7.0 and an ionic strength of 150 mm, adjusted with sodium chloride), as well as under conditions where the enzyme is most active (pH 5.5 and 40–100 mm calcium chloride), PLDa2 was more rapidly inactivated than in the absence of higher salt concentrations In Table 1, the rate constants of irreversible inactivation, which followed first-order kinetics, in the absence and presence of NaCl and CaCl2 at pH 5.5 and 23 °C are compared The results show that the instability of PLDa2 is Table Influence of salts on the observed first-order rate constants (kobs) of PLDa2 inactivation and fluorescence decrease PLDa2 was incubated in 50 mM sodium acetate buffer (pH 5.5) at 23 °C To follow the inactivation, PLDa2 (280 lgỈmL)1) was incubated for to 36 days, and the residual activity was measured after dilution to 9.3 lgỈmL)1 as described in Experimental procedures The fluorescence intensity of PLDa2 (25 lgỈmL)1) was measured at an excitation wavelength of 278 nm and an emission wavelength of 335 nm NM, not measurable kobs (10)7Ỉs)1) Additive Inactivation Fluorescence decrease – 10 mM EDTA 120 mM NaCl 300 mM NaCl 40 mM CaCl2 100 mM CaCl2 2.5 NM 6.5 21 6.5 29 21 NM 31 120 33 120 FEBS Journal 274 (2007) 2630–2640 ª 2007 The Authors Journal compilation ª 2007 FEBS 2633 Structure of plant phospholipase D S Stumpe et al A B Fig Kinetics of PLDa2 precipitation followed by SDS ⁄ PAGE PLDa2 (280 lgỈmL)1) was incubated in 50 mM sodium acetate buffer (pH 5.5) in the absence (A) and the presence of 300 mM NaCl (B) at 23 °C After min, min, 30 min, h, h, h, h, day, days, days and days (lanes 1–11), samples were analyzed by SDS ⁄ PAGE Lane 12 shows the protein band in the precipitate after week of incubation caused by high ionic strength rather than by specific ionic effects At comparable ionic strengths of sodium and calcium chloride, the observed kinetic constants were nearly identical Interestingly, however, the inactivation could be further decreased by the addition of EDTA; inactivation constants were no longer measurable At room temperature, the loss of activity in slightly buffered solutions (10 mm Pipes, pH 7.0, or 50 mm sodium acetate, pH 5.5) with 10 mm EDTA amounted to only 17% after months When we looked for spectroscopic alterations in the course of inactivation, neither sodium chloride nor calcium chloride was found to induce spectroscopic changes in the absorption, fluorescence or far-UV CD spectra (data not shown) Interestingly, near-UV CD spectroscopy revealed changes in the presence of calcium chloride (Fig 4D), but not in the presence of sodium chloride Therefore, the spectroscopic alterations could stem from specific calcium binding rather than from nonspecific salt effects (see next paragraph) Although no shift of the fluorescence emission maxima was measurable, the fluorescence intensity decreased with increasing incubation time (data not shown) Also, the decrease of the fluorescence signal followed a first-order reaction, and allowed us to determine rate constants (Table 1) These constants, determined at about 10-fold lower protein concentration than the measurements of inactivation, show the same trends with respect to the effects of NaCl, CaCl2 and EDTA as the inactivation constants As shown by SDS ⁄ PAGE (Fig 5), the decrease in fluorescence intensity was caused by precipitation of PLDa2, which is faster in the presence of salts Calcium binding Whereas calcium ions did not change the absorption, fluorescence and far-UV CD spectra of native PLDa2, the near-UV CD signal in the range 258–292 nm was 2634 increased in the presence of calcium chloride, with a maximum at 280 nm (Fig 4D) The increase in nearUV CD intensity was specific for calcium ions, as neither sodium nor magnesium ions showed any comparable effect The increase in the near-UV CD signal at 280 nm was dependent on the concentration of calcium ions in a hyperbolic fashion (Fig 6A) The data could be fitted well using a double hyperbolic function yielding two dissociation constants, KD1 ¼ 10.24 mm and KD2 ¼ 0.123 mm Two binding events are also evident in a Scatchard-like plot [27], where the relative change in the near-UV CD signal (DF) represents the amount of the Ca2+–PLDa2 complexes (Fig 6B) Calcium ions are crucial for PLDa2 activity, and could not be replaced by other ions such as magnesium ions At concentrations of calcium ions < mm, no significant activity could be detected Up to 100 mm, PLDa2 activity increased with increasing concentration of calcium ions, independent of the counterion, chloride or acetate (Fig 7A) At calcium ion concentrations above 100 mm, the PLDa2 activity dropped again, and the decrease was steeper with calcium acetate than with calcium chloride Interestingly, the activity data in the range 0.01–100 mm CaCl2 could be fitted in similar way as the near-UV CD data, using a double hyperbolic function (Fig 7B) The resulting dissociation constants were KD1 ¼ 17.38 mm and KD2 ¼ 0.073 mm A modified Scatchard plot of these data, with DA being the change in relative activity (Fig 7C), shows two linear parts, indicating that at least two separate calcium-binding events affect PLDa2 activity A B Fig Calcium binding measured by near-UV CD spectroscopy (A) Direct plot The solid line shows the double hyperbolic fit (B) Modified Scatchard plot The CD signals, measured at different concentrations of CaCl2, were taken at 280 nm and averaged over The PLDa2 concentration was mgỈmL)1 DF corresponds to the change in the CD signal [Q]MRW (degỈcm2Ỉdmol)1) at 280 nm FEBS Journal 274 (2007) 2630–2640 ª 2007 The Authors Journal compilation ª 2007 FEBS S Stumpe et al Structure of plant phospholipase D A B C Fig Influence of the calcium concentration on PLDa2 activity (A) Relative activity as function of calcium chloride (black) and calcium acetate (gray) concentration, respectively (B) Double hyperbolic plot of the data from (A) in the range 0–100 mM Ca2+ (C) Modified Scatchard plot of the data from (B) DA corresponds to the change in relative activity The PLDa2 activity was measured against PpNp in sodium acetate buffer (pH 5.5) at 30 °C Discussion PLDa2 is a barrel-shaped monomer with loosely structured tops Despite the great number of plant PLDs identified at the DNA or cDNA level, only a few of these enzymes have been characterized in purified, isolated form Correspondingly, there is almost no information on the secondary and tertiary structures of plant PLDs Similarly, structural information on other eukaryotic PLDs is rare The two members of the PLD superfamily whose crystal structures have been elucidated [28,29] are much smaller in size, so that knowledge of their structures cannot be generalized to plant PLDs In this article, the first information on the spatial structure of a plant a-type PLD has been obtained on the basis of recombinantly produced PLDa2 from cabbage SAXS analysis (Fig 2) and analytic size exclusion chromatography indicate unequivocally that native PLDa2 from cabbage is a monomeric protein The surface structure of PLDa2 indicates a longish molecule with loosely structured regions at the two ends of the molecule (Fig 3) An assignment of these relaxed structures to sequence regions of PLDa2 has not yet been possible According to the volume estimation on the basis of the partial specific volume of a protein (0.73 mgỈg)1) [30], the C2 domain comprising residues A2 to E153 should occupy 16% of the molecular volume, and therefore seems too large to represent the lid-like structure at the top of the left view in Fig We speculate that the loosely structured region on the opposite side belongs to the C2 domain (141 amino acids) with its extended loops, whereas the lid-like region at the top represents another flexible part of the structure Assuming that the core protein containing the two essential HKD motifs (Fig 1) is similar in PLDa2 from cabbage (812 amino acids) and in PLD from Streptomyces sp (506 amino acids) with the known crystal structure [29], the C-terminal part of the plant enzyme is longer by approximately 60 amino acids and might form this part A PtdInsP2-binding domain, which has recently been desribed as a separate folding unit conserved in eukaryotic PLDs and comprises about 50 amino acids between the two HKD motifs [31], would also fit this lid-like region The loosely structured parts of the molecule and the deviation from a spherical shape may be the reason why the experimentally determined Porod volume of PLDa2 (126.7 ± 3.5 nm3) is slightly larger than the molecular volume (111.45 nm3) calculated from the partial specific volume of a protein [30] This result is in accordance with the smaller volume (99 nm3) deduced from the radius of gyration (Rg: 2.9 nm), which considers the distances between scattering masses of the molecule Spectroscopic properties of PLDa2 reflect an ordered tertiary structure with a high content of b-structure The first UV-absorption (Fig 4A), fluorescence (Fig 4B) and CD spectra (Fig 4C,D) of a plant PLD reflect the properties of a common protein Obviously, tyrosine, and particularly tryptophan, residues (Fig 1) dominate the absorption spectra of PLDa2 from cabbage, with a maximum at 280 nm (Fig 4A) The fluorescence spectra of PLDa2 with a maximum at 334 nm (Fig 4B) indicate a hydrophobic environment for most or possibly all tryptophan residues Therefore, tryptophan residues are probably mainly located in the core of the protein The tyrosine residues contribute to the fluorescence spectra by fluorescence resonance energy FEBS Journal 274 (2007) 2630–2640 ª 2007 The Authors Journal compilation ª 2007 FEBS 2635 Structure of plant phospholipase D S Stumpe et al transfer This energy transfer seems to be very effective in PLDa2, because no separate tyrosine fluorescence is detectable The far-UV CD spectrum of PLDa2 with the maximum at 192 nm and the broad minimum at 208– 220 nm (Fig 4C) refers to both a-helix and b-sheet conformations As the signal of a-helices is more intense [32], the spectrum suggests that b-sheet structures dominate This assumption was confirmed by the calculation of the a-helix and b-strand contents of the protein with the online programs dichroweb [25] and k2d [26], respectively The well-structured near-UV CD spectrum of PLDa2 (Fig 4C) points to a tertiary structure with tight packing of the aromatic side chains in an asymmetric environment As all eight cysteine residues of PLDa2 should be half-cysteines [33], no influence of disulfide bonds on the near-UV CD spectrum must be taken into account The relatively low signal intensity can probably be attributed to the compensation of positive and negative signals of the individual aromatic residues PLDa2 is stable at low ionic strengths The low stability of purified cabbage PLD was shown to originate from a high aggregation propensity of the enzyme under physiologic conditions Inactivation and precipitation of PLDa2, the latter being followed by SDS ⁄ PAGE or decreasing fluorescence intensity, were accelerated with growing salt concentrations (Table 1, Fig 5) In the comparison of the presence of NaCl and of CaCl2, the ionic strength was more important for inactivation or precipitation than the individual ions Hydrophobic interactions, which are known to be favored by rising salt concentrations, are probably the reason for this tendency The fact that EDTA effects a marked stabilization (Table 1), however, indicates an additional destabilizing effect by calcium ions Destabilization of PLDa2 by calcium ions was also indicated by the proteolytic sensitivity of PLDa2 [34] Calcium-induced decrease of stability and biological activity due to facilitated aggregation was also reported for a-crystallin [35] Although the incubation with salts in the inactivation experiments was performed at higher PLDa2 concentrations than in the fluorescence measurements, the observed inactivation was slower than the observed fluorescence decrease due to protein precipitation This discrepancy could be due to the different experimental conditions Whereas the fluorescence decreases were measured in situ, the remaining activities were determined after dilution, and therefore partial resolubilization of precipitates is possible We assume that the 2636 precipitation is followed by (small) structural alterations in a consecutive aggregation reaction that cause irreversible inactivation It is not clear whether the destabilizing effects of calcium ions and other salts have any physiologic relevance However, the finding that pure plant PLDs may be more stable in the absence of calcium and other ions at room temperature than in their presence and at lower temperatures is of considerable practical importance PLDa2 binds calcium ions at two affinity levels CD spectroscopy in the near UV-region proved to be the only spectroscopic method able to detect specific binding of calcium ions to PLDa2 (Fig 4D) The signal increases in this spectrum caused by calcium ions indicate stiffening of the PLDa2 molecule Binding curves obtained with this method (Fig 6A) are similar to the curves of PLDa2 activity as a function of calcium ion concentration (Fig 7B), showing that the well-known effect of calcium ions on activity closely correlates with conformational changes of the enzyme These results unambiguously show that activation of PLDa2 by calcium ions is due to the binding of calcium ions to the enzyme, and not (or not only) to better structuring of the substrate aggregates From both the CD and the activity data, two different calcium-binding events can be derived The corresponding dissociation constants differ by about two orders of magnitude Even the higher affinity (with the dissociation constant in the range of 0.1 mm) is still relatively low for biospecific interactions We assume that calcium ions are involved in enzyme activation by guiding the protein to the membrane, mediating substrate binding, or adjusting the charge distribution at the active site Unfortunately, the number of calcium ions bound to the enzyme cannot be deduced from these data The binding event with the dissociation constant in the range of 0.1 mm might be associated with calcium binding to the C2 domain For the separately expressed C2 domain of PLDa from Arabidopsis thaliana, one to three low-affinity calcium-binding sites with dissociation constants in the range of 0.5 mm were estimated from isothermal titration calorimetry [36] The C2 domains of other proteins showed calcium-binding affinities in the range of 1–50 lm [37,38] As the C2 domain of PLDa2 from cabbage lacks two conserved residues that are probably responsible for the calcium binding [8], we hypothesize that this binding event takes place at the C2 domain A second calcium-binding site of lower affinity at the C2 domain (corresponding to the dissociation constant in the range 10–20 mm) cannot be excluded It FEBS Journal 274 (2007) 2630–2640 ª 2007 The Authors Journal compilation ª 2007 FEBS S Stumpe et al is more likely, however, that the calcium-binding event of lower affinity is attributable to the catalytic site of PLDa2, as found for PLDb from A thaliana [39] The decrease in PLD activity at concentrations above the optimal calcium concentration (100 mm), as demonstrated in Fig 7A, is scarcely reported in the literature [7] In contrast to the activation effect of calcium ions, inactivation at high calcium ion concentrations is dependent on the counterion In contrast to the stabilizing effects of salts according to the Hofmeister series, the chloride salt is less destabilizing than the acetate salt As discussed above, the inactivation by salts is connected with aggregation and precipitation of PLDa2 Obviously, the anion is specifically involved in this process In summary, we conclude that calcium ions bind specifically at two sites of PLDa2 and activate the enzyme in two steps As discussed above, the resulting conformational changes destabilize the molecule However, at higher salt concentrations, precipitation occurs, and destabilization by calcium ions is therefore no longer specific Experimental procedures Materials Bactotryptone and yeast extract were from Difco (Detroit, MI, USA) All other chemicals were the purest ones commercially available Production of PLDa2 The E coli strain BL21 (DE3) containing plasmids pUBS520 and pld2pRSET5a [8] was grown without preculture in a 200 mL culture of · YT medium (2% bactotryptone, 1% yeast extract, and 1% NaCl, pH 7.0) with 100 lgỈmL)1 ampicillin and 50 lgỈmL)1 kanamycin at 30 °C for 15 h At an absorbance of 1–2 at 600 nm, the temperature was shifted to 15 °C and the cells were grown to maximum cell density (24– 48 h) without induction Cells were harvested by centrifugation at °C and 6000 g for 10 (Avanti J-25 centrifuge, Beckman Coulter, JA-10 rotor), and stored at ) 20 °C Purification of PLDa2 The pellet from a 1.2 L culture was suspended in 37 mL of buffer (30 mm Pipes, pH 6.2, 10 mm EDTA), and the cells were disrupted by high-pressure homogenization (APV Homogeniser; Gaulin, Lubeck, Germany) The cell debris was ă separated by centrifugation at °C and 48 000 g for 20 (Avanti J-25 centrifuge, JA-30.50 rotor) Calcium chloride was added to the supernatant (final concentration Structure of plant phospholipase D 50 mm), and after centrifugation (at °C, 4800 g, 10 min, Rotofix 32 centrifuge, Hettlich, 1620A rotor), the protein solution was loaded onto an Octylsepharose column (Amersham Biosciences, Piscataway, NJ, USA) equilibrated with 30 mm Pipes (pH 6.2) containing 50 mm CaCl2 The column was carefully washed with equilibration buffer, and then the protein was eluted with 0.1 mm EDTA in mm Pipes buffer (pH 6.2) The PLDa2-containing fractions were pooled and Tris ⁄ HCl buffer (pH 7.5) was added to a final concentration of 20 mm After filtration (0.2 lm; WiCom, Heppenheim, Germany), the solution was applied to a Source 15Q column (Amersham Biosciences) Elution was performed using a linear gradient of 50 mL of 15–35% m NaCl in Tris ⁄ HCl buffer (pH 7.5) The fractions with highest catalytic activity and homogeneity in SDS ⁄ PAGE were pooled, dialyzed twice (cut-off 20 kDa; Roth, Karlsruhe, Germany) against a 100-fold volume of 10 mm Pipes buffer (pH 7.0), and stored at ) 20 °C Determination of protein concentration The concentration of PLDa2 in the range between 75 lgỈmL)1 and mgỈmL)1 was determined spectrophotometrically, using the molar extinction coefficient e280 nm ¼ 123 720 m)1Ỉcm)1 [40] Other PLDa2 concentrations were measured by the BCA assay (Pierce, Rockford, IL, USA) with BSA as standard SDS ⁄ PAGE SDS ⁄ PAGE was performed according to the method of Laemmli [41] Gels [10% (w ⁄ v) acrylamide] were stained with Coomassie Brilliant Blue G250 and quantified by densitometric evaluation at 595 nm (CD60; Desaga, Darmstadt, Germany) Small angle X-ray solution scattering with synchrotron radiation Data were collected with the EMBL beamline X33 at the DORIS storage ring, Desy, Hamburg Measurements were performed at a camera length of m, using a Mar345 image plate detector (Marresearch, Norderstedt, Germany), ˚ at a wavelength of 1.5 A, a temperature of 12 °C, and PLDa2 concentrations between and 4.7 mgỈmL)1 in 10 mm Pipes buffer (pH 7.0) Calibration of the momentum transfer axis (s) was done using collagen or tripalmitin as standards The experimental data were normalized to the intensity of the incident beam and corrected for the detector response, the buffer scattering was substracted, and the statistical errors were calculated using the program primus [23] To obtain the forward scattering intensity I(0) and the radius of gyration (Rg), the data were processed with the program gnom [22] The molecular mass was calculated FEBS Journal 274 (2007) 2630–2640 ª 2007 The Authors Journal compilation ª 2007 FEBS 2637 Structure of plant phospholipase D S Stumpe et al from the ratio of the forward scattering intensity of the sample and that of the molecular mass standard BSA The ab initio deconvolution of the SAXS profiles to restore a low-resolution shape of PLDa2 from the experimental data was executed using the program dammin [24] The final model was computed by superposition of 10 individual models using the program damaver [42] Size exclusion chromatography A Superdex 200 HR 10 ⁄ 30 FPLC column (Amersham Biosciences) was equilibrated with 10 mm Pipes buffer (pH 7.0) and 140 mm NaCl Proteins were eluted with the same buffer at a flow rate of 0.5 mLỈmin)1 at °C, and detected by the protein absorbance at 280 nm Aldolase (150 kDa), ovalbumin (45 kDa), chymotrypsinogen A (25 kDa), and cytochrome c (12.3 kDa) (Serva, Heidelberg, Germany) were used as molecular mass standards Absorption and CD spectroscopy A Jasco V-560 spectrophotometer (Jasco, Gross-Umstadt, Germany) and a 10 mm quartz cuvette were used in UV absorption measurements CD spectra were obtained using a Jasco J-810 spectropolarimeter The protein spectra were measured in 10 mm (near-UV) and 0.1 mm (far-UV) quartz cuvettes by scanning at 20 nmỈmin)1 with a resolution of 0.1 nm, a response time of s, a bandwidth of nm, and a temperature of 20 °C An average of 10 scans was recorded and corrected by subtracting the baseline spectrum of the buffer The CD signal was converted to molar ellipticity [Q]MRW (degỈcm2Ỉdmol)1) [43] The ellipticity at 280 nm was used for monitoring calcium binding The CD signal was recorded over Then, calcium ions were added stepwise, and the CD signal was recorded again The measured signal was corrected for the protein dilution by calcium chloride addition Fluorescence spectroscopy Fluorescence experiments were performed at 20 °C with a FluoroMax-2 spectrofluorimeter (Horiba Jobin Yvon, Munich, Germany) using 10 · mm fluorescence quartz cuvettes PLDa2 was excited at 278 nm (excitement of tyrosine and tryptophan residues) or at 295 nm (excitement of tryptophan residues only) The slit width was nm for excitation and emission The signal was acquired with a response time of s, and the wavelength increment was nm The fluorescence signal of the blank buffer was substracted Enzyme assays The hydrolytic activity of PLDa2 was determined by measuring the p-nitrophenol release from PpNp (synthesized as 2638 described by D’Arrigo et al [44]) at 405 nm [8] In general, PLDa2 was incubated in 220 lL of 65 mm sodium acetate buffer (pH 5.5) containing 50 mm CaCl2 at 30 °C With the addition of the substrate solution [10 mm PpNp, 5% Triton X-100 (v ⁄ v), and mm SDS], the reaction was started After 10 of incubation, which is in the linear range of the progress curve, the reaction was stopped with 60 lL of m Tris ⁄ HCl buffer (pH 8.0) containing 0.1 m EDTA One unit (U) of PLDa2 corresponds to the release of lmol p-nitrophenolỈmin)1 Acknowledgements The authors thank Ch Kuplens and Dr R Schops for ă assistance with protein purication and for preparation of the PpNp The financial support of the Kultusministerium des Landes Sachsen-Anhalt and the Graduiertenkolleg 1026 of the Deutsche Forschungsgemeinschaft, Bonn (Germany), and the support of 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