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Sensor of phospholipids in Streptomyces phospholipase D Yoshiko Uesugi, Jiro Arima, Masaki Iwabuchi and Tadashi Hatanaka Research Institute for Biological Sciences (RIBS), Okayama, Japan Phospholipase D (PLD; EC 3.1.4.4) catalyzes phos- pholipid hydrolysis and phosphatidyl transfer (Fig. 1A). This is a ubiquitous and important enzyme involved in signal transduction in mammals [1,2]. Streptomyces PLDs can be categorized into two types: one is an iron-containing enzyme, such as that from Streptomyces chromofuscus (chromofuscus PLD) for which tightly bound iron is necessary for its catalytic activity [3]; and the other is a member of the PLD superfamily whose hallmark is the possession of two catalytic HxKxxxxD (HKD) motifs [4–6]. Because enzymes of the latter type have a simple structure containing two HKD motifs, they are useful as a suitable model of mammalian PLDs. A study of the chemical modification of PLD from Streptomyces sp. PMF (PMFPLD) suggested that Lys, not His, is essential for PLD activity [7]. Iwasaki et al. [8] revealed that two HKD motifs are essential for the activity, using the N- and C-terminal halves of Strep- tomyces PLD. Furthermore, Leiros et al. [9] showed that His170 in the N-terminal HKD motif of PMFPLD acts as the initial nucleophile that attacks the phospho- rus atom of the substrate, on the basis of the crystal structures of PMFPLD. Previously, using two Strep- tomyces PLDs in repeat-length independent and broad spectrum (RIBS) in vivo DNA shuffling, we constructed a random chimera library to investigate the recognition of phospholipids by Streptomyces PLD. We revealed that the N-terminal HKD motif contains the nucleo- phile, using an inactive chimera and surface plasmon resonance (SPR) analysis [10]. To date, the functions of the HKD motifs in cata- lytic mechanisms have been extensively studied [11–13]. At present, PLD-catalyzed reactions are con- sidered to consist of two steps: first, the formation of a covalently linked phosphatidyl enzyme interme- diate via the His residue of the N-terminus HKD motif; and second, the hydrolysis or transphosphati- dylation of the intermediate by a water or alcohol molecule (Fig. 1A). As mentioned above, previous experimental studies have focused on the relationship between HKD motifs Keywords phospholipase D; phospholipid; substrate recognition; SPR; Streptomyces Correspondence T. Hatanaka, Research Institute for Biological Sciences (RIBS), Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan Fax: +81 866 56 9454 Tel: +81 866 56 9452 E-mail: hatanaka@bio-ribs.com (Received 12 January 2007, revised 14 March 2007, accepted 22 March 2007) doi:10.1111/j.1742-4658.2007.05802.x Recently, we identified Ala426 and Lys438 of phospholipase D from Strep- tomyces septatus TH-2 (TH-2PLD) as important residues for activity, sta- bility and selectivity in transphosphatidylation. These residues are located in a C-terminal flexible loop separate from two catalytic HxKxxxxD motifs. To study the role of these residues in substrate recognition, we eval- uated the affinities of inactive mutants, in which these residues were substi- tuted with Phe and His, toward several phospholipids by SPR analysis. By substituting Ala426 and Lys438 with Phe and His, respectively, the inactive mutant showed a much stronger interaction with phosphatidylcholine and a weaker interaction with phosphatidylglycerol than the inactive TH-2PLD mutant. We demonstrated that Ala426 and Lys438 of TH-2PLD play a role in sensing the head group of phospholipids. Abbreviations PA, phosphatidic acid; PC, phosphatidylcholine; PG, phosphatidylglycerol; PLD, phospholipase D; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho- L-serine]; PpNP, phosphatidyl-p-nitrophenol; RU, resonance unit; SPR, surface plasmon resonance; SUV, small unilamellar vesicle. 2672 FEBS Journal 274 (2007) 2672–2681 ª 2007 The Authors Journal compilation ª 2007 FEBS and activity. Recently, we demonstrated that four amino acid residues, Gly188, Asp191, Ala426 and Lys438, of PLD from Streptomyces septatus TH-2 (TH-2PLD) are associated with PLD activity and sub- strate recognition [10,14] (Fig. 2A). Substituting Ala426 and Lys438 with Phe and His, respectively, led to improvements in PLD activity, thermostability, and organic solvent tolerance and to a change in the selectiv- ity of transphosphatidylation activity compared with that in the original chimera [14]. This suggests that Ala426 and Lys438 are involved in substrate recognition. Hughes et al. [15] demonstrated that human PLD1b interacts with fluorescence-labeled phospha- tidylcholine (PC), whereas PLD1 does not interact with fluorescence-labeled phosphatidylethanolamine. This finding shows that the PLD–phospholipid inter- action correlates with PLD activity, because PLD1 has no catalytic activity toward phosphatidylethanol- amine. Recently, SPR analysis has been used to investigate the effects of the rat PLD1 Phox homo- logy (PX) domain on membrane binding properties [16], and the specific association of PLD1b with its regulator proteins, PKCa, Rac1 and ARF6 [17]. In addition, because of the interaction of inactive PLDs and PC retaining a covalent phosphatidyl-enzyme intermediate determined by SPR analysis, the N-ter- minal HKD motif was found to act as a catalytic nucleophile [10]. In this study, to investigate the roles of Ala426 and Lys438 of TH-2PLD in substrate recognition in more detail, we analyzed the association of inactive mutants of TH-2PLD, in which these residues were substituted with Phe and Ala, respectively, conco- mitantly with the substitution of His443 of the C-terminal HKD motif with Ala, with three phos- pholipid substrates (Fig. 1) by SPR analysis. Results Preparation of inactive mutants of TH-2PLD In a previous study, we used two homologous Streptomyces PLDs, TH-2PLD and PLD from Strep- tomyces sp. (PLDP), as parental enzymes by RIBS shuffling [10]. PLDP had Phe and His corresponding to Ala426 and Lys438 of TH-2PLD, respectively. Thus, we substituted these residues of TH-2PLD with Phe and His in this study. In addition, to evaluate the effect of the residues on phospholipid recognition by SPR analysis, we constructed inactive mutants of TH-2PLD, in which His443 of an HKD motif was substituted with Ala, as shown in Fig. 2B. We then expressed the resultant genes and purified the proteins they encode. All of the purified mutants mostly showed a single band with the same molecu- lar mass ( 57 kDa) as that of wild-type TH-2PLD on SDS ⁄ PAGE (Fig. 2C). Furthermore, using west- ern blot analysis with anti-(wild-type TH-2PLD) serum, these mutants were found to have similar uniformities and purities (Fig. 2D). All the mutants had low activities toward phosphatidyl-p-nitrophenol (PpNP) (< 0.7 lmolÆmin )1 Æmg )1 ), whereas wild-type TH-2PLD had high activity (59 lmolÆmin )1 Æmg )1 ). To confirm the folding of the inactive mutants of TH-2PLD, their CD spectra were measured. As shown in Fig. 3, the CD spectra showed that the inactive mutants folded with a secondary struc- ture similar to wild-type TH-2PLD. These results A B Fig. 1. Reactions catalyzed by PLD (A) and structure of phospholipid head groups (B). Y. Uesugi et al. Sensor of phospholipids in PLD FEBS Journal 274 (2007) 2672–2681 ª 2007 The Authors Journal compilation ª 2007 FEBS 2673 suggest that we successfully prepared inactive mutant enzymes. Association of inactive mutants with phospholipid vesicles To investigate the association of key C-terminal resi- dues with the head group of phospholipid substrates, we analyzed the binding profiles of TH-2(H443A), TH-2-F(H443A) and TH-2-FH(H443A) for several phospholipid vesicles with a covalent phosphatidyl- PLD intermediate using SPR analysis. Sensorgrams obtained by SPR analysis showed real-time biomole- cular interaction. Overlaid sensorgrams were obtained when TH-2(H443A), TH-2-F(H443A) and TH-2- FH(H443A) were passed at different concentrations over immobilized 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC; Fig. 4A–C), 1-palmitoyl-2- oleoyl-sn-glycero-3-[phospho-l-serine] (POPS; Fig. 4D–F) or 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1- glycerol)] (POPG; Fig. 4G–I) vesicles. As shown in Fig. 4A–F, TH-2-F(H443A) and TH-2-FH(H443A) exhibited significantly higher binding abilities for POPC and POPS vesicles than TH-2(H443A). By con- trast, sensorgrams of TH-2-F(H443A) were similar to those of TH-2(H443A) for POPG vesicles, and their interactions were stronger than those of TH-2- FH(H443A) (Fig. 4G–I). These differences in interac- tion were not caused by the heterogeneity of the mutants. If they were, each mutant would have shown the same association and dissociation curves for all the phospholipids; however, the results did not show such A B CD Fig. 2. (A) 3D structure around identified key residues (i.e. residues 188, 191, 426 and 438 of TH-2PLD) associated with activ- ity. The overall structure of TH-2PLD is sho- wn using the Swiss-PDB viewer and is based on the crystal structure of PMFPLD. The identified key residues are indicated in red. The N-terminal and C-terminal HKD motifs are shown in light blue and purple, respectively. (B) Primary structures of wild- type TH-2PLD and its inactive mutants. The gray box indicates the His residue of the C-terminal HKD motif mutated to Ala. The identified residues related to the PLD reaction are shown in black boxes. (C) SDS ⁄ PAGE results of purified PLDs. Lanes 1–4 contained 2 lg of TH-2PLD, TH- 2(H443A), TH-2-F(H443A) and TH-2-FH- (H443A), respectively. Lane M indicates SDS ⁄ PAGE standard proteins (molecular masses: 100 000, 80 000, 60 000, 50 000, 40 000, 30 000 and 20 000 Da). Samples were loaded on a 10% acrylamide gel. (D) Western blot analysis of purified PLDs using anti-(wild-type TH-2PLD) serum. Lanes 1–3 contained 2 lg of TH-2(H443A), TH-2- F(H443A) and TH-2-FH(H443A), respectively. Lane M indicates prestained SDS ⁄ PAGE standards (molecular masses: 111 000, 93 000, 53 500, 36 100 and 29 500 Da). The samples were loaded on a 10% acrylamide gel. The arrowhead indicates the position of the purified PLDs. Sensor of phospholipids in PLD Y. Uesugi et al. 2674 FEBS Journal 274 (2007) 2672–2681 ª 2007 The Authors Journal compilation ª 2007 FEBS curves. Therefore, these results suggest that substitu- tion of Ala426 with Phe led to considerably stronger interactions with POPC and POPS. It should be noted that the double mutant showed a decrease in the strength of its interaction with POPG vesicles, although the interactions of the mutant with POPC and POPS vesicles remained strong. The kinetic constant was calculated from each sensor- gram using bia evaluation 4.1 analysis software according to the global fitting of 1:1 binding with a mass transfer model. Affinity constants (K D ) for POPC vesi- cles were 5.3 ± 0.9, 5.5 ± 1.4 and 4.8 ± 0.7 nm for TH-2(H443A), TH-2-F(H443A) and TH-2-FH(H443A), respectively. There was no significant difference in K D value between these proteins; however, the maximal responses of these proteins differ markedly. In the case of POPG and POPS vesicles, K D could not be calculated because their response curves did not fit the evaluation curves. In particular, sensorgrams toward POPG showed an increase and a decrease in interactions during the association process at a low mutant concentration (Fig. 4G–I). The results suggest that the mutants have more than two binding sites, with different affinities for POPG vesicles. Unfortunately, there are no evaluation models for determining the interaction between a ligand and an analyte involving more than two binding sites. Thus, the sensorgrams of mutants toward POPG could not be analyzed appropriately. To compare the differences in affinity for phospho- lipids among TH-2(H443A), TH-2-F(H443A) and TH-2-FH(H443A), the maximal responses measured for these inactive mutant associations with POPC, POPS and POPG vesicles are shown in Fig. 5. Injection of 532 nm TH-2(H443A) resulted in a binding signal of 1409 resonance units (RU) for POPC vesicles, which was similar to that of 1204 RU for POPG vesicles (t-test; P > 0.05), whereas the association with POPS vesicles was significantly weaker (477 RU; P < 0.001). As shown in Fig. 4A,G, the sensorgram of TH- 2(H443A) increased more sharply in the association phase when POPG vesicles rather than POPC vesicles were used. In contrast, TH-2-FH(H443A) bound to POPG vesicles slowly (Fig. 4I), and the degree of bind- ing response for POPG vesicles was 5.1-fold lower than that for POPC vesicles. TH-2-F(H443A) also showed a degree of binding response for POPG vesicles 2.6-fold lower than that for POPC vesicles. Interestingly, the interactions of each inactive mutant with POPS vesicles were similar and low in degree compared with those with POPC vesicles, although each mutant exhibited different degrees of interaction with POPG vesicles. From these results, it is suggested that residues 426 and 438 of TH-2PLD play a role in sensing the head group of phospholipid vesicles. Conformational change of inactive mutants induced by phospholipids To analyze changes in the tertiary and secondary struc- tures of inactive mutants induced by phospholipids, we further measured the fluorescence and CD spectra of inactive mutants in the absence and presence of POPC and POPG vesicles [18]. TH-2PLD has 11 Trp residues that contribute to its fluorescence emission spectrum. As shown in Fig. 6A–C, the emission maxima, around 340 nm, were the same for all the inactive mutants. With inactive mutant alone, TH-2-F(H443A) showed a similar fluorescence emission spectrum to TH- 2(H443A), and TH-2-FH(H443A) had a higher fluores- cence emission intensity than TH-2(H443A). For the mutant TH-2-FH(H443A), the local environment around Trp434 is probably changed by substituting His for Lys438, because Lys438 is located adjacent to Trp434 ( 4A ˚ ). The fluorescence emission intensities of TH-2-F(H443A) and TH-2-FH(H443A) increased at 340 nm with the addition of POPC vesicles, and the degrees of increase were 0.186 and 0.083 relative to those without POPC, respectively. Interestingly, the fluorescence emission intensity of TH-2-FH(H443A) with POPG vesicles decreased to that without POPG at 0.11 degrees. However, the fluorescence emission intensity of TH-2(H443A) did not change with or without phospholipids. In contrast, the CD spectra of all the inactive mutants were similar with or without phospholipids (Fig. 6D–F). From these results, it sug- gests that TH-2-F(H443A) and TH-2-FH(H443A) were Fig. 3. CD spectra of PLDs. The spectrum of each PLD (0.1 mgÆmL )1 )in10mM potassium phosphate buffer (pH 7.0) was measured at 25 °C. Y. Uesugi et al. Sensor of phospholipids in PLD FEBS Journal 274 (2007) 2672–2681 ª 2007 The Authors Journal compilation ª 2007 FEBS 2675 induced conformational changes in their tertiary struc- tures with phospholipid introduction, although their secondary structures remain unchanged. Discussion Recently, Sato et al. [19] reported that the phosphati- dic acid (PA) contents produced by a side reaction of several Streptomyces PLDs differ markedly during transphosphatidylation from PC to phosphatidylgly- cerol (PG). Among the PLDs used, TH-2PLD showed the lowest selectivity in transphosphatidylation, and the amount of hydrolyzed PA increased with reaction time. This phenomenon was considered to be the result of synthesized PG being hydrolyzed to PA during transphosphatidylation. We speculated that TH-2PLD recognizes synthesized PG as well as PC; therefore, the amount of hydrolyzed PA increases. Recently, we showed that the C-terminal flexible loop in Strepto- myces PLD (residues 425–442) is separate from the ABC DEF GHI Fig. 4. Sensorgrams at different concentrations of inactive mutants of TH-2PLD. As substrate, POPC (A–C), POPS (D–F) and POPG (G–I) vesicles were immobilized on an L1 sensor chip, as described in Experimental procedures. The SPR sensorgrams were obtained when TH-2(H443A) (A,D,G), TH-2-F(H443A) (B,E,H) and TH-2-FH(H443A) (C,F,I) were passed over the phospholipid vesicles at 532, 355, 236 and 158 n M, respectively (from top to bottom), at a flow rate of 20 lLÆmin )1 for 5 min at 25 °C, followed by a buffer at the same flow rate for 10 min. The affinities of the mutants to the phospholipid vesicles were determined by fitting these SPR sensorgrams using BIA EVALUATION 4.1 analysis software. Sensor of phospholipids in PLD Y. Uesugi et al. 2676 FEBS Journal 274 (2007) 2672–2681 ª 2007 The Authors Journal compilation ª 2007 FEBS two highly conserved catalytic HKD motifs, and that it is formed at the entrance of the active-site well and has multiple functional roles. A mutant PLD with Ala426 and Lys438 substituted with Phe and His, respectively, improved its selectivity in transphosphati- dylation from PC to PG [14]. Thus, we plan to investi- gate the relationship between the recognition of several phospholipids, such as PC, PG and phosphatidylserine (PS), and the residues identified by SPR analysis. These results agree well with the present findings that TH-2(H443A) has comparable interactions with POPC and POPG, and that TH-2-FH(H443A) has a much stronger interaction with POPC and a weaker interaction with POPG than TH-2(H443A) (Fig. 5). In addition, by determining the corresponding active mutants in terms of their activity toward POPC vesi- cles by the method using choline oxidase and peroxi- dase [14], the activities of TH-2-F (3.3 lmolÆmin )1 Æ mg )1 ) and TH-2-FH (3.3 lmolÆmin )1 Æmg )1 ) were found to be twofold higher than that of TH-2PLD (1.7 lmolÆmin )1 Æmg )1 ) toward POPC (data not shown). These results suggest that the activities of active mutants correlate well with the binding data of inac- tive mutants. These findings suggest that TH-2PLD recognizes PC and PG similarly, and that residues 426 and 438 of TH-2PLD play an important role in phospholipid recognition. To date, nine sequences of Streptomyces PLDs have been determined. All Strep- tomyces PLDs except TH-2PLD have a Phe residue corresponding to Ala426 of TH-2PLD. By contrast, at Lys438 of TH-2PLD, four of them have a Lys residue and the rest have a His residue. Thus, the main cause of the low selectivity of TH-2PLD in transphosphati- dylation seems to be Ala426. In this study, we con- firmed that these residues are associated with the interaction between TH-2PLD and its substrate. SPR analysis revealed that Streptomyces PLD inter- acts to a much higher degree with zwitterionic phos- pholipid vesicles (i.e. PC) than with anionic phospholipid vesicles (i.e. PG and PS) (Fig. 5). PLDs from mammals and poppy seedlings hydrolyze PC most efficiently among several phospholipids [20,21]. That is, Streptomyces PLD containing HKD motifs seems to prefer zwitterionic phospholipid vesicles, such as POPC, to anionic phospholipid vesicles POPS and POPG, similarly to other PLDs. Previous SPR analysis indicated that the PLD1 PX domain has a high phosphoinositide specificity, that is, the K D value for POPC ⁄ POPE ⁄ phosphatidylinositol 3,4,5-trisphosphate (77 : 20 : 3) is 18 ± 4 nm [16]. Powner et al. [17] showed K D values between PLD1b and regulator proteins, PKCa, Rac1 and ARF6 (i.e. 42 ± 1 5, 143 ± 28 and 660 ± 63 nm, respectively) [17]. In this study, K D values for POPC vesicles were found to be 5.3 ± 0.9, 5.5 ± 1.4 and 4.8 ± 0.7 nm for TH-2(H443A), TH-2-F(H443A) and TH-2-FH(H443A), respectively. These results suggest that the affinities of these mutants for POPC vesicles are stronger than those of the specific association between the PLD1 PX domain and phosphatidylinositol 3,4,5-trisphosphate, or PLD1b and its regulators. We speculate that the difference in the degree of interaction with POPC among TH-2PLD mutants is a result of the influence of two flexible loops, i.e. resi- dues 188–203 and 425–442, of TH-2PLD on each other. Iwasaki et al. [8] and Xie et al. [22] showed that PLD activity is restored when the N- and C-terminal fragments of Streptomyces PLD and PLD1 coexist, although these fragments have only negligible activities in isolation. From these findings, we speculate that PLD changes its conformation markedly before and after binding to the substrate. In fact, SPR analysis (Figs 4,5) and fluorescence spectroscopy (Fig. 6) showed that the inactive mutants, in which Ala426 and Lys438 were substituted with Phe and Ala, showed ter- tiary structural changes with phospholipid binding. Combined with the results of SPR analysis and fluores- cence spectroscopy, it seems that the interaction between Streptomyces PLD and POPG differs from that between the PLD and POPC, although this phe- nomenon cannot be explained experimentally at pre- sent. These two flexible loops may play a role as a Fig. 5. Interaction of PLDs and phospholipid vesicles. The maximal responses of TH-2(H443A), TH-2-F(H443A) and TH-2-FH(H443A) were measured for their specific associations with phospholipids vesicles. Each PLD (532 n M) was injected at a flow rate of 20 lLÆmin )1 . Each value represents the mean ± SD from three inde- pendent experiments. Y. Uesugi et al. Sensor of phospholipids in PLD FEBS Journal 274 (2007) 2672–2681 ª 2007 The Authors Journal compilation ª 2007 FEBS 2677 trigger of conformational change when PLDs bind to the substrate. Therefore, residues 426 and 438 located in the C-terminal loop could affect the interaction of PLDs with substrate. TH-2(H443A) and TH-2-F(H443A) exhibited stron- ger interactions with POPG vesicles than TH- 2-FH(H443A). Sensorgrams of these mutants showed an increase and a decrease in the degree of interactions during the association process at a low concentration of mutants (Fig. 4G–I). These results suggest that there are more than two binding sites that have differ- ent affinities to PG vesicles in the mutants. Using com- puter analysis with the automated docking program autodock, Aikens et al. [23] showed that the glycerol group of PG is bound to a region composed of Ser453, Lys454, Asn455, Tyr457, Ser459 and Leu461 of PMFPLD. These residues correspond to Ser458, Lys459, Asn460, Tyr462, Ser464 and Leu466, respect- ively, in the C-terminal region of TH-2PLD. PMFPLD and TH-2PLD are 85% homologous in primary struc- ture. Hence, we surmise that another PG-recognizing site is present in the C-terminal region. Ala426 and Lys438 of TH-2PLD are located in a C- terminal flexible loop separate from two catalytic HKD motifs. The loop, in coordination with the N- terminal loop, forms the entrance of the active well comprising two HKD motifs (Fig. 2A). It is reasonable to consider that these residues play a role in sensing the head group of phospholipids from a geometrical point of view. It might be possible to change the sub- strate preference of Streptomyces PLD by substituting these two residues with other amino acid residues. ABC DEF Fig. 6. Fluorescence emission spectra of TH-2(H443A) (A),TH-2-F(H443A) (B) and TH-2-FH(H443A) (C) in the absence and presence of phosp- holipids vesicles. The inactive mutants were excited at 290 nm and emission spectra were recorded between 300 and 380 nm. Fluores- cence measurements were carried out at 25 °C with 1.2 l M PLDs in 10 mM Tris ⁄ HCl (pH 7.0) containing 4 mM CaCl 2 . CD spectra of TH- 2(H443A) (D), TH-2-F(H443A) (E) and TH-2-FH(H443A) (F) in the absence and presence of phospholipids vesicles. The spectrum was meas- ured at 25 °C with 1.7 l M PLDs in 10 mM Tris ⁄ HCl (pH 7.0) containing 4 mM CaCl 2 . POPC or POPG of SUVs was added PLD solution at a final concentration of 1 m M, and incubated for over 1 h. All spectra were corrected by subtracting the spectra of the corresponding back- ground media without PLD. Sensor of phospholipids in PLD Y. Uesugi et al. 2678 FEBS Journal 274 (2007) 2672–2681 ª 2007 The Authors Journal compilation ª 2007 FEBS Experimental procedures Materials The plasmid pETKmS2 [24] was kindly provided by T. Yamane (Nagoya University, Japan). PpNP was pre- pared from soybean phosphatidic acid and p-nitrophenol according to the procedure of D’Arrigo et al. [25]. POPC, POPS and POPG were obtained from Avanti Polar Lipids (Alabaster, AL, USA) and used without further purification. All the other chemicals were of the highest purity available. Preparation of PLDs To construct the mutant TH-2(H443A), the mutagenic gene was amplified by PCR using the following primers: 5¢- CCCTGCGCGCGCTCGTCGGCA-3¢ (corresponding to the gene th-2pld, nucleotides 962–982) and 5¢-ACCAG CTTGTGG(TG fi GC, His fi Ala)CTGCGCGTACG-3¢ [corresponding to nucleotides 1316–1340 from th-2pld, TH-2(H443A)]. The amplified DNA fragment was cloned, sequenced and digested with BglII and Van91I. Next, the plasmid pUC19(TH-2) [26] was digested with BglII and Van91I, and the product was substituted for the corresponding region in the subcloned th-2pld. The resul- tant mutant gene was digested with NcoI and BamHI and ligated into the NcoI–BamHI gap of the vector pETKmS2(TH-2(H443A)). To prepare the mutants TH-2-F(H443A) and TH- 2-FH(H443A), a partial th-2pld was amplified by PCR using the following primers: 5¢-ACTACGTCGACACCTCCCA CC-3¢ (corresponding to nucleotides 575–595 from th-2pld) and 5¢-GAAGGTG GCTAGCTGGAGGTTG-3¢ for the silent mutation of the NheI site (underlined) (corresponding to nucleotides 1257–1278 from th-2pld). Then the amplified DNA fragments were cloned, sequenced and digested with PstI and NheI. Next, the plasmids pETKmS2(G-F) and pETKmS2(G-FH) [14] were digested with NheI and BsiWI. The two resulting fragments were ligated into the PstI– BsiWI gap of the vector pETKmS2(TH-2(H443A)) to con- struct the expression vector. The expression vectors obtained were confirmed by DNA sequencing. The recombinant TH-2PLD and inactive mutant enzymes were expressed as secreted proteins with a C-terminal His6 tag, and purified with TALON metal affinity resin (Clontech, Palo Alto, CA, USA) according to standard protocols. The purities of proteins obtained were confirmed by SDS ⁄ PAGE [10] and western blot analysis using an anti-(wild-type TH- 2PLD) serum. Assay for PLD activity using PpNP Hydrolytic activity was determined on the basis of the hydrolytic activity of PpNP. The procedure was similar one described previously [27]. One unit of PLD was defined as the amount of the enzyme that releases 1 lmol of p-nitrophenol per minute under the assay conditions. The reactions were carried out in 1.5-mL cuvettes. The 1-mL reaction mixture consisted of 0.07–0.2 lg of purified PLDs and 2 mm PpNP in 0.1 m sodium acetate buffer (pH 5.5) at 37 °C. CD spectroscopy The folding of PLDs was confirmed by CD spectroscopy using a J-720WI spectrometer (Jasco, Tokyo, Japan). Pro- teins were dissolved to a final concentration of 0.1 mgÆmL )1 in 10 mm potassium phosphate buffer (pH 7.0). Spectra were acquired at 25 °C using a 2-mm path-length cuvette. The spectra of PLDs that averaged 10 scans were corrected by subtracting the spectra of the corresponding background media without PLD. Preparation of vesicles An aliquot of phospholipids dissolved in chloroform was evaporated and further dried in vacuum for at least 3 h. The lipids were hydrated to a concentration of 10 mm in phosphate-buffered saline for SPR analysis or in 10 mm Tris ⁄ HCl (pH 7.0) for fluorescence and CD measurements. The lipid suspension was vortexed vigorously, and frozen and thawed 10 times in liquid nitrogen. To obtain small uni- lamellar vesicles (SUVs), the suspension was passed 30 times through polycarbonate membranes (50 nm pore diameter) using a Lipofast extruder (Avestin, Ottawa, Canada) [28]. SPR analysis Real-time interactions between PLD molecules and phos- pholipids were measured using a Biacore instrument (Bia- core 2000, Biacore AB, Uppsala, Sweden). Liposomes were captured on the surface of a Sensor Chip L1 (Biacore AB) as ‘ligand’. The surface of the L1 sensor chip was first cleaned with 20 mm 3-[(3-cholamidopropyl) dimethylammonio]-1- propanesulfonic acid (CHAPS) at a flow rate of 5 lLÆmin )1 followed by the injection of SUVs (60 lL, 0.5 mm phospho- lipids) at a flow rate of 2 lLÆmin )1 in buffer A (10 mm sodium acetate, pH 5.5, 4 mm CaCl 2 ). This resulted in the deposition of  5000–7000 RU. To measure the association of PLDs with phospholipids, a purified inactive mutant enzyme (105–532 nm diluted in buffer A) as ‘analyte’ was applied to the captured SUVs at a flow rate of 20 lLÆmin )1 at 25 °C. After the binding of PLDs to phospholipids, disso- ciation was observed at the same flow rate for 10 min. The evaluation of the kinetic parameters of PLD binding to phospholipids was performed by the nonlinear fitting of binding data using bia evaluation 4.1 analysis software. The apparent association (k a ) and dissociation (k d ) rate Y. Uesugi et al. Sensor of phospholipids in PLD FEBS Journal 274 (2007) 2672–2681 ª 2007 The Authors Journal compilation ª 2007 FEBS 2679 constants were evaluated from the differential binding curves (sample–control) shown in Fig. 4, assuming an A + B ¼ AB association type for protein–lipid interaction. The affinity constant K D was calculated from the equation K D ¼ k d ⁄ k a . Fluorescence spectroscopy Fluorescence spectra were obtained with an F-4500 spectro- fluorometer (Hitachi, Tokyo, Japan). All measurements were carried out at 25 °C with 1.2 lm PLDs in 10 mm Tris ⁄ HCl (pH 7.0) containing 4 mm CaCl 2 using 2-mm path-length quartz cuvette. The excitation wavelength was 290 nm, and excitation and emission slits were 5 nm. 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Sensor of phospholipids in PLD FEBS Journal 274 (2007) 2672–2681 ª 2007 The Authors Journal compilation ª 2007 FEBS 2681 . results did not show such A B CD Fig. 2. (A) 3D structure around identified key residues (i.e. residues 188, 191, 426 and 438 of TH-2PLD) associated with. structure of TH-2PLD is sho- wn using the Swiss-PDB viewer and is based on the crystal structure of PMFPLD. The identified key residues are indicated in red. The