Phosphatidylserine induces functional and structural alterations of the membrane-associated pleckstrin homology domain of phospholipase C-d1 Naoko Uekama, Takio Sugita, Masashi Okada, Hitoshi Yagisawa and Satoru Tuzi Graduate School of Life Science, University of Hyogo, Harima Science Garden City, Kamigori, Hyogo, Japan A number of processes contributing to important cellu- lar functions such as cellular signal transduction, cyto- skeletal organization, and membrane trafficking are known to be localized at membrane surfaces of the plasma membrane and organelles. For instance, extra- cellular signals are often transmitted from activated receptors to peripheral membrane proteins, such heterotrimeric G-proteins, and subsequently amplified and ⁄ or passed to successors by a number of peripheral membrane proteins located at the membrane surface. Because the interface between the aqueous phase and the hydrophobic interior of the membrane provides a unique molecular environment completely different from the homogeneous solution phase environment [1,2], one would expect induction of unique structure– function relationships for the peripheral membrane Keywords cellular signal transduction; phosphatidylserine; phospholipase C-d1; pleckstrin homology domain Correspondence S. Tuzi, Graduate School of Life Science, University of Hyogo, Harima Science Garden City, Kouto 3-chome, Kamigori, Hyogo 678- 1297, Japan Fax: +81 791 580182 Tel: +81 791 580180 E-mail: tuzi@sci.u-hyogo.ac.jp (Received 4 August 2006, revised 1 Novem- ber 2006, accepted 6 November 2006) doi:10.1111/j.1742-4658.2006.05574.x The membrane binding affinity of the pleckstrin homology (PH) domain of phospholipase C (PLC)-d1 was investigated using a vesicle coprecipitation assay and the structure of the membrane-associated PH domain was probed using solid-state 13 C NMR spectroscopy. Twenty per cent phos- phatidylserine (PS) in the membrane caused a moderate but significant reduction of the membrane binding affinity of the PH domain despite the predicted electrostatic attraction between the PH domain and the head groups of PS. Solid-state NMR spectra of the PH domain bound to the phosphatidylcholine (PC)⁄ PS⁄ phosphatidylinositol 4,5-bisphosphate (PIP 2 ) (75 : 20 : 5) vesicle indicated loss of the interaction between the amphi- pathic a2-helix of the PH domain and the interface region of the mem- brane which was previously reported for the PH domain bound to PC ⁄ PIP 2 (95 : 5) vesicles. Characteristic local conformations in the vicinity of Ala88 and Ala112 induced by the hydrophobic interaction between the a2-helix and the membrane interface were lost in the structure of the PH domain at the surface of the PC⁄ PS⁄ PIP 2 vesicle, and consequently the structure becomes identical to the solution structure of the PH domain bound to d-myo-inositol 1,4,5-trisphosphate. These local structural changes reduce the membrane binding affinity of the PH domain. The effects of PS on the PH domain were reversed by NaCl and MgCl 2 , suggesting that the effects are caused by electrostatic interaction between the protein and PS. These results generally suggest that the structure and function relationships among PLCs and other peripheral membrane proteins that have similar PH domains would be affected by the local lipid composition of mem- branes. Abbreviations DD-MAS, single pulse excitation dipolar decoupled-magic angle spinning; GST, glutathione-S-transferase; IP 3 , D-myo-inositol 1,4,5-trisphosphate; PC, phosphatidylcholine; PH domain, pleckstrin homology domain; PIP 2 , phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; POPC, 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine; PS, phosphatidylserine; SUV, small unilamellar vesicle. FEBS Journal 274 (2007) 177–187 ª 2006 The Authors Journal compilation ª 2006 FEBS 177 proteins adapted to the interfacial environment. How- ever, the structural characteristics of the membrane- associated state of peripheral membrane proteins remain unclear, mainly due to the lack of an appropri- ate technique for investigating the structures of peri- pheral membrane proteins bound to lipid bilayers. Well-established techniques for determination of pro- tein structures, such as X-ray diffraction and solution NMR spectroscopy, are not suitable for characterizing structures of peripheral membrane proteins in the membrane-associated state. As X-ray diffraction requires the highly periodic structure of a protein crystal to obtain structural information, its use is inappropriate for characterizing peripheral membrane protein-membrane complexes characterized by dynamic and nonperiodic structures. Furthermore, the massive net molecular weights of membrane protein-membrane complexes present a significant obstacle for structure determination by solution-state NMR spectroscopy because of increases in the line-width of NMR signals. In previous work, we have applied solid-state NMR spectroscopy to gain insights into the structure of the pleckstrin homology (PH) domain of phospholi- pase C-d1 (PLC-d1; EC 3.1.4.11), a peripheral mem- brane protein. As a result, we proposed a model for the conformational change of the domain induced dur- ing its membrane association based on the structure and dynamics of individual alanine residues in the domain [3]. PLC-d1 is comprised of five domains; PH, EF-hand, X, Y, and C2 [4–6]. The PH domain is located at the N-terminus and is known to dominate membrane localization of PLC-d1 by anchoring the protein through a specific high affinity binding interaction with the head group of phosphatidylinositol 4,5-bisphos- phate (PIP 2 ) [7–10]. This PIP 2 -dependent membrane localization of the PH domain has been shown to pro- vide an indirect regulatory mechanism of the phos- pholipase activity of PLC-d1 through regulation of the frequency of encounter between the PLC-d1 active site (X and Y domain) and its substrate, PIP 2 [11,12]. A study using chimeric proteins of PLC- d1 and PLC-b1 led to the proposal that the interactions between the PH domain and the active site domains also provide a direct means of regulating hydrolytic activity [13]. The above-mentioned conformational change of the PLC-d1 PH domain induced at the membrane surface may also contribute to direct and ⁄ or indirect regula- tion of PLC-d1 hydrolysis. Furthermore, structure– function relationships of the PLC-d1 PH domain may be modified by alteration of the nature of the mem- brane. Since PLC-d1 is reported to shuttle between the plasma membrane and the cytoplasm, and also between the cytoplasm and the nucleus [14,15], varia- tions in lipid composition in the different membranes of the cell [16] might cause location-dependent struc- ture–function variations for PLC-d1. Alteration of the asymmetric distribution of lipids between the inner and outer leaflets of the membrane and a lateral segre- gation of lipid components in the membrane may also cause fluctuations of the lipid composition in the vicin- ity of PLC-d1. In this study, we investigated the effects of phos- phatidylserine (PS) on the structure and function of the PLC-d1 PH domain on an artificial membrane sur- face. The negatively charged head group of PS is expected to affect the PLC-d1 PH domain, which is localized at the membrane interface as a result of elec- trostatic interactions. The asymmetric distribution of PS between the outer and inner leaflet of the biological membranes has been reported to be altered via various physiological processes including, for example, cellular activation and apoptosis. Since most of the PS in the plasma membrane (over 90% for the human erythro- cytes and platelets) is reported to be localized at the inner leaflet [17–19], an alteration of the asymmetric distribution of PS in the membrane causes changes in the extent of exposure of PS to the intracellular surface of the plasma membrane which is accessible by PLC-d1. For instance, an increase in PS in the outer leaflet of the plasma membrane occurs during apoptosis [20–22]. Surface exposure of PS has also been reported for stimulated nonapoptotic cells as a result of altera- tions of phospholipid asymmetry [23,24]. Loss of asym- metric distribution and possible segregation of acidic lipids such as PS in the plasma membrane [25–27] should result in alterations of the local concentration of PS in the vicinity of the membrane-associated protein. In this work, we evaluate the effects of PS on mem- brane-binding affinities using a vesicle coprecipitation assay. We also evaluate the structures of the PLC-d1 PH domain using solid-state NMR spectroscopy. PS is found to induce a moderate reduction of the membrane binding affinity despite the predicted electrostatic attraction between the PH domain and the head groups of PS [28]. PS also causes conformational changes of the PH domain at the membrane surface which are pre- dicted to contribute to alteration of the affinity. Results PS reduces the membrane binding affinity of the PLC-d1 PH domain Table 1 shows the results of the vesicle coprecipita- tion assays of PLC- d1 PH domain performed for the Function and structure of PLC-d1 PH domain N. Uekama et al. 178 FEBS Journal 274 (2007) 177–187 ª 2006 The Authors Journal compilation ª 2006 FEBS 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) small unilamellar vesicles (SUVs) containing 5% PIP 2 and 0 or 20% PS. The PS concentration of the latter is selected as a possible local concentration of PS in an inner leaflet of the plasma membrane. Nine per cent of the total phospholipid content of the rat liver plasma membrane is reported to be PS and >90% of PS is located at the inner leaflet [17–19]. The ratio of the PH domain partitioned into supernatant and pellet was determined from the densities of the SDS ⁄ PAGE bands and is shown in Table 1 as the percentage of PH in the pellets. The vesicle-associated PH domain collected in the pellets decreases significantly in the presence of 20% PS. This reveals that an increase in PS content in the membrane lowers the membrane binding affinity of the PH domain. Dissociation constants (K d ) for the PH domain–PIP 2 interaction were calculated for SUVs with different lipid compositions from the ratios of the PH domain partitioned into the supernatants and the preci- pitates corresponding to the free PH domain and PH domain–PIP 2 complex, respectively. Affinities of the PLC-d1 PH domain for the head groups of phosphati- dylcholine (PC) or PS are negligible compared with the high affinity for PIP 2 [8]. The K d values were calculated from following equations: K d ¼ Rf½PIP 2  0 À½PH 0 =ðR þ 1Þg where R ¼ [PH] ⁄ [PHÆPIP 2 ], [PH] is the concentration of the free PH domain and [PHÆPIP 2 ] is the concentra- tion of the PH domain forming a complex with PIP 2 at equilibrium. [PH] 0 and [PIP 2 ] 0 are total concentra- tions of PH domain and PIP 2 accessible by the PH domain, respectively. We assumed that 70% of the total lipids of the SUVs are exposed to the outer sur- face of the SUVs and that PIP 2 is evenly distributed within the SUVs. The surface areas of the inner and outer leaflets were calculated based on the thicknesses and the surface areas per lipid head groups of inner and outer leaflets reported for egg phosphatidylcholine vesicles and the average radius of SUVs determined by dynamic light scattering [29]. The K d obtained for the PC ⁄ PIP 2 vesicle without PS was very close to the pre- viously reported value determined by isothermal titra- tion calorimetry (1.66 ± 0.80 lm) [10]. As shown in Table 1, a moderate but significant increase in K d was found in the presence of 20% PS. The reduced affinity for binding to the membrane containing 20% PS was restored in the presence of 25 mm NaCl. Further addi- tion of NaCl up to 100 mm did not induce a further increase in K d . The differences in K d values for PC ⁄ PIP 2 vesicles and PC ⁄ PS ⁄ PIP 2 vesicles containing 25–100 mm NaCl was found to be insignificant. In the presence of 25 mm NaCl, the K d is close to that for the PC ⁄ PIP 2 membrane without PS, indicating that NaCl abolishes the effect of PS. The similar K d values for the PC ⁄ PIP 2 membrane with 0 and 25 mm NaCl eliminate the possibility that NaCl affects the affinity for binding to the PC ⁄ PIP 2 membrane in the absence of PS. These suggest that the lowering effect of PS on the membrane binding affinity of the PH domain is mediated by the electrostatic interaction between the negatively charged head group of PS and the charged protein surface, which would be masked by Na + and ⁄ or Cl – . Although the association of the PH domain with the membrane is thought to be domin- ated by the highly specific association to PIP 2 via its ligand binding site, the affinity-reducing effect of PS on the PH domain suggests that a mechanism inde- pendent of PIP 2 binding also exists. PS induces a conformational change of the PH domain at the membrane surface Figure 1A,B shows single pulse excitation dipolar decoupled-magic angle spinning (DD-MAS) NMR spec- tra of 13 C-labeled methyl carbons in [3- 13 C]Ala-labeled PH domain bound to PC vesicles containing 5% PIP 2 (PC ⁄ PIP 2 vesicle) and PC ⁄ PIP 2 vesicle containing 20% PS (PC ⁄ PS ⁄ PIP 2 vesicle), respectively, suspended in 20 mm Mops buffer (pH 6.5) containing 1 mm dithio- threitol and 0.025% NaN 3 . The assignments of the spectral signals to each of the Ala residues determined for the PH domain bound to PC ⁄ PIP 2 vesicles were made by using a series of site-directed replacements of Ala residues [3] indicated at the top of Fig. 1. Chem- ical shifts of Ala residues in the PH domain bound to d-myo-inositol 1,4,5-trisphosphate (IP 3 ) in solution are shown as vertical bars at the bottom of the spectra. These chemical shift values correspond to the structure Table 1. Membrane binding affinities of PLC-d1 PH domain Vesicle [NaCl] (m M) PH domain bound a,c to vesicle (%) K d b,c (lM) POPC ⁄ PIP 2 d 0 81.0 ± 2.40 1.62 ± 0.33 POPC ⁄ PIP 2 d 25 80.9 ± 1.33 1.63 ± 0.17 POPC ⁄ PS ⁄ PIP 2 e 0 73.6 ± 2.72 2.77 ± 0.48 POPC ⁄ PS ⁄ PIP 2 e 25 83.2 ± 3.28 1.36 ± 0.40 POPC ⁄ PS ⁄ PIP 2 e 50 84.5 ± 3.52 1.21 ± 0.40 POPC ⁄ PS ⁄ PIP 2 e 100 82.7 ± 3.44 1.41 ± 0.40 a Estimated from ratios of the PH domain collected with the centri- fuged pellets by the vesicle coprecipitation assay. b K d values were calculated as shown in the text. [PH] 0 and [PIP 2 ] 0 used for the cal- culations were 11.2 l M and 15.9 lM, respectively. c Values are mean ± SD for more than five different experiments (P<0.05). d Molar ratio of POPC–PIP 2 –biotinylated PE ¼ 93:5:2. e Molar ratio of POPC–PS–PIP 2 –biotinylated PE ¼ 73 : 20 : 5 : 2. N. Uekama et al. Function and structure of PLC-d1 PH domain FEBS Journal 274 (2007) 177–187 ª 2006 The Authors Journal compilation ª 2006 FEBS 179 of the PH domain in solution in the absence of mem- brane. This structure is expected to be similar to the three dimensional structure determined for the PLC-d1 PH domain–IP 3 complex by X-ray diffraction. As shown with dotted lines, the chemical shifts of the sig- nals of Ala116 and Ala118 are not affected by addition of PS, indicating that conformations of Ala116 and Ala118 within the C-terminal a3-helix of the PH domain are unchanged. These signals are indifferent to the presence of the membranes, probably because the positions of Ala116 and Ala118 are located far from the membrane surface [3]. The peak at 16.76 p.p.m. in Fig. 1B is assigned to Ala88 taking the same confor- mation as Ala88 in the PH domain bound to PC ⁄ PIP 2 vesicles that resonates at 16.81 p.p.m. (Fig. 1A), although the intensity of the signal is reduced. In con- trast, a peak at 17.64 p.p.m. in the spectrum of the PH domain bound to PC ⁄ PS ⁄ PIP 2 vesicles does not have a corresponding peak at 17.6 p.p.m. in the spectrum of the PH domain bound to PC ⁄ PIP 2 vesicles. The inten- sity of this peak at 17.64 p.p.m. decreases at lower temperatures or at a higher salt concentration, as shown in Figs 1C and 2B, respectively, accompanied Fig. 1. DD-MAS NMR spectra of the [3- 13 C]Ala-labeled PLC-d1PH domain bound to PC ⁄ PIP 2 or PC ⁄ PS ⁄ PIP 2 vesicles. (A) PH domain bound to a PC ⁄ PIP 2 vesicle at 20 °C, (B) PH domain bound to PC ⁄ PS ⁄ PIP 2 vesicle at 20 °C, and (C) PH domain bound to a PC ⁄ PS ⁄ PIP 2 vesicle at 4 °C. The PH domain–vesicle complexes were suspended in 20 m M Mops buffer (pH 6.5) containing 1 mM dithiothreitol and 0.025% NaN 3 . Peaks assigned to lipid molecules are indicated with asterisks. The chemical shifts of the Ala residues obtained for the PLC-d1 PH domain bound to IP 3 in solution are shown as vertical bars at the bottom of the spectra. Assignments of the signals to alanine residues are shown at the top and bottom of the spectra in parentheses. Fig. 2. DD-MAS NMR spectra of the [3- 13 C]Ala-labeled PLC-d1PH domain bound to PC ⁄ PS ⁄ PIP 2 vesicle in the presence of different concentrations of salts at 20 °C. Vesicles with PH domain were suspended in (A) 20 m M Mops buffer containing 1 mM dithiothreitol and 0.025% NaN 3 , (B) 20 mM Mops buffer containing 1 mM dithio- threitol, 0.025% NaN 3 and 25 mM NaCl, and (C) 20 mM Mops buf- fer containing 1 m M dithiothreitol, 0.025% NaN 3 ,25mM NaCl and 10 m M MgCl 2 . Peaks assigned to lipid molecules are indicated with asterisks. Function and structure of PLC-d1 PH domain N. Uekama et al. 180 FEBS Journal 274 (2007) 177–187 ª 2006 The Authors Journal compilation ª 2006 FEBS by increases in the intensity of Ala88 peaks at 16.7– 16.8 p.p.m. This reveals that Ala88 of the PH domain at the surface of PC ⁄ PS ⁄ PIP 2 vesicles takes two differ- ent conformations, one that takes the same conforma- tion as that at the surface of PC ⁄ PIP 2 vesicles, resonating at 16.81 p.p.m., and the other, which is characteristic for the PH domain bound to PC ⁄ PS ⁄ PIP 2 vesicles, resonating at 17.64 p.p.m. The chemical shift of Ala88 of the latter conformation is close to that of Ala88 of the PH domain bound to IP 3 in solution. The signal of Ala112 is obtained as a sharp peak at 18.44 p.p.m. for the PH domain bound to PC ⁄ PS ⁄ PIP 2 vesicles, in contrast to a broad peak for the PH domain bound to PC ⁄ PIP 2 vesicles centered at 18.8 p.p.m. The greater line width of the latter in Fig. 1A reflects conformational heterogeneity of Ala112 in the PH domain bound to PC ⁄ PIP 2 vesicles. The chemical shift and the line shape of the 18.44 p.p.m. peak are similar to the chemical shift and line shape of the peak representing Ala112 in the PH domain bound to IP 3 in solution. The matching of chemical shifts of Ala88 and Ala112 with those of the PH domain–IP 3 complex in solution indicates that a fraction of the total PH domain content bound to PC ⁄ PS ⁄ PIP 2 vesicles containing 20% PS adopts a con- formation similar to that of the PH domain bound to IP 3 in solution. A slight downfield displacement of the Ala88 peak at 17.64 p.p.m. for the PH domain bound to PC ⁄ PS ⁄ PIP 2 vesicles relative to that of the PH domain bound to IP 3 indicates that the structure of the PH domain bound to PC ⁄ PS ⁄ PIP 2 vesicles is not completely identical to that of the PH domain bound to IP 3 , presumably due to the membrane surface prop- erties. Electrolytes suppress the effects of PS on the PH domain As shown in Fig. 2B, the addition of 25 mm NaCl cau- ses a decrease in the intensity of the Ala88 peak at 17.48 p.p.m. in the spectrum of the PH domain bound to PC ⁄ PS ⁄ PIP 2 vesicles. This decrease in intensity is accompanied by an increase in the intensity of another Ala88 peak at 16.73 p.p.m. These changes of the Ala88 signal and simultaneous downfield displacement of the Ala112 peak from 18.44 to 18.59 p.p.m. accom- panied by an increase in line width indicate that the addition of NaCl facilitates formation of the structure that resembles the PH domain bound to PC ⁄ PIP 2 vesi- cles and simultaneous destruction of the structure which resembles the PH domain bound to IP 3 at the surface of the PC ⁄ PS ⁄ PIP 2 vesicles. This change in the structure of the PH domain is probably due to a masking effect induced by Na + and ⁄ or Cl – ions on the electrostatic interaction between the negative charges of PS head groups and charged groups in the PH domain. We further examined the effect of MgCl 2 on the structure of the PH domain, considering the greater effect of divalent cations on the electrostatic interaction compared with monovalent cations. The total concentration of divalent cations in the cytoplasm is about 30 mm, with Mg 2+ being the most common [30]. In the presence of 10 mm MgCl 2 , the Ala88 signal at 17.5–6 p.p.m. originating from the structure similar to that of the PH domain bound to IP 3 disappears, while the Ala88 peak at 16.86 p.p.m. remains as shown in Fig. 2C. MgCl 2 also causes an increase in the line width and downfield displacement of the Ala112 signal to 18.77 p.p.m. Consequently, the spectrum of the PH domain bound to the PC⁄ PS⁄ PIP 2 complex (Fig. 2C) in the presence of MgCl 2 is virtually identical to that of the PH domain bound to PC ⁄ PIP 2 vesicles (Fig. 1A). This suggests that the structures of both PH domain complexes are identical. Discussion As shown in Table 1, PS in the membrane induces a significant increase in the dissociation constant of the PLC-d1 PH domain and SUV. Although the decrease in the binding affinity is moderate, it reveals that the function of the PLC-d1 PH domain could be altered by changes in the lipid composition of membranes. Suppression of this effect by an electrolyte indicates that the increase in the dissociation constant originates in the electrostatic interaction between the PH domain and the PS head groups. It is difficult, however, to ascribe this moderate inhibitory effect of PS on the membrane association of PLC-d1 PH domain to mere electrostatic repulsion between the negatively charged groups of the PH domain and the PS head groups. A three-dimensional structural model of the PLC-d1PH domain shows that positively charged residues are con- centrated around the PIP 2 -specific lipid binding site to form a positively charged surface which would be expected to attract acidic lipids [5]. Therefore, it could be expected that an increase in the negative charge of a given membrane composition could facilitate mem- brane association of the PH domain through electro- static attractions. In fact, a theoretical study of biophysical properties of the PLC-d1 PH domain with a continuum electrostatic approach predicted a remarkable reduction of electrostatic free energy for the membrane interaction of the PH domain by PS [28]. One of the possible explanations for the observed suppression of the membrane association induced by N. Uekama et al. Function and structure of PLC-d1 PH domain FEBS Journal 274 (2007) 177–187 ª 2006 The Authors Journal compilation ª 2006 FEBS 181 PS is that PS lowers the stability of the PH domain– lipid bilayer complex after association of the mem- brane with the PH domain. The solid-state NMR spec- tra of the PH domain (Fig. 1) indicate that PS induces a drastic change in the structure of the membrane associated PH domain. In Fig. 3, the positions of alan- ine residues observed by solid-state NMR are shown in a schematic representation of the secondary and ter- tiary structures of the PLC-d1 PH domain bound to IP 3 based on the structure determined by the X-ray diffraction study [5]. In our previous study, we pro- posed that the long loop between the b5 and b6 strands of the b-sheet core of the PH domain (b5 ⁄ b6 loop) interact with the membrane interface of the PC ⁄ PIP 2 vesicle as an auxiliary membrane interacting site in addition to the PIP 2 specific-high affinity lipid binding site consisting of the b1 ⁄ b2, b3 ⁄ b4 ⁄ and b6 ⁄ b7 loops. The short amphipathic a-helix included in the b5 ⁄ b6 loop (a2-helix) appears to be responsible for the interaction with the interface region of the membrane located between the aqueous phase and the hydropho- bic inner layer. This interaction causes the conforma- tional changes detectable by solid-state NMR as chemical shift displacements for Ala88 (located at the C-terminus of the a2-helix) and Ala112 (located at the C-terminus of the b7-strand connected with the base of the b5 ⁄ b6 loop by hydrogen bonds). The conforma- tional change of Ala112 which appears during the process of membrane association was interpreted as being related to removal and formation of the hydro- gen bond between Arg95 (NH) and Pro91 (C¼O) and the hydrogen bond between Arg95 (NH) and Ala112 (C¼O), as determined by solid-state NMR spectro- scopy [3]. The conformational change associated with the upfield shift of the Ala88 signal from 17.49 to 16.81 p.p.m. observed for the membrane-associated state could be ascribed to formation of a structure simi- lar to the typical a-helix and is expected to resonate at approximately 15–16 p.p.m. at the C-terminus of the a2-helix. These conformational changes of the indivi- dual Ala residues are consistent with the proposed conformational change of the entire PH domain, which include an opening of the b5 ⁄ b6 loop as shown in Fig. 4B caused by the interaction between the a2-helix and the membrane interface [3]. PS abolishes these conformational changes of the PH domain and induces the formation of a structure similar to that of the PH domain bound to IP 3 . The proposed structures of the PLC-d1PHdomainare as follows: (a) with IP 3 in solution (structure I); (b) with PIP 2 embedded in PC ⁄ PIP 2 vesicle (structure II); and (c) with PIP 2 embedded in PC ⁄ PS⁄ PIP 2 vesicle (struc- ture III), and are schematically shown in Figs 4A–C, respectively. At the surface of the PC ⁄ PIP 2 membrane, structure II differs from structure I (in solution) with respect to having an altered conformation of the b5 ⁄ b6 loop. The alteration would accompany a change in orientation and opening of the loop relative to the b-sheet core due to the interaction between the amphi- pathic a2-helix and the membrane. Twenty per cent PS in the membrane causes a loss of the conformational characteristics of structure II and a decrease in the membrane binding affinity of the domain. Structure III is virtually identical to structure I, even though nearly 100% of the PH domain is expected to form complexes with PIP 2 in the membrane as indicated by NMR meas- urements. The coordinated changes in membrane binding affin- ity and the PH domain structure induced by PS imply that a transition from structure II to structure III (accompanied by the loss of the interaction of the a2-helix with membrane interface) is one of the factors that destabilize the membrane binding state of the PH domain. As shown in Table 1 and Fig. 2, addition of NaCl induced both a conformational change from structure III to structure II, as shown Fig. 4D, and a restoration of the membrane binding affinity. This sug- gests that both the structural and functional changes of the PH domain caused by PS are mediated by an electrostatic interaction that could be masked by rather low concentrations of the electrolyte. This is consistent Fig. 3. Positions of 13 C-labeled alanine residues in the PLC-d1PH domain shown in a schematic representation of the three-dimen- sional structure based on the model proposed by the X-ray diffrac- tion study for the PH domain–IP 3 complex (1MAI) [5]. b-Sheets consisting of a b-sandwich core of the domain (b1-b7) are indicated by rectangles and a-helices (a1-a3) are indicated by cylinders. The alanine residues are indicated by filled gray circles. Among the five alanines, Ala21 was not resolved in the 13 C NMR spectra due to an overlap of the signal at 14.4 p.p.m. with an intense peak arising from the lipids. IP 3 , located at the PIP 2 -specific lipid binding site, is indicated by a hexagon. Function and structure of PLC-d1 PH domain N. Uekama et al. 182 FEBS Journal 274 (2007) 177–187 ª 2006 The Authors Journal compilation ª 2006 FEBS with the above model in which the PS-dependent con- formational change of the PH domain at the mem- brane interface contributes to the change of the membrane binding affinity induced by PS. The remark- able effect of low concentrations of MgCl 2 on the for- mation of the structure II (Fig. 4D) suggests that cations play important roles on the conformational and affinity changes in a process that most probably originates from effective shielding of the negative charge of the PS head group. There is a discrepancy, however, between the effect of NaCl on the membrane binding affinity and the structure. A solution of 25 mm NaCl completely restores the membrane binding affin- ity, but the structure of the PH domain undergoes only a partial transition from structure III to structure II. Because structure II and structure III are in equilib- rium at the surface of PC ⁄ PS ⁄ PIP 2 vesicles, there might be a critical level of structure II required for the restoration of the higher membrane binding affinity. There also might be additional electrostatic interaction between the PH domain and PS which reduce the sta- bility of the membrane-associated state of the PH domain. The effect of PS demonstrated in this study might provide a regulatory mechanism of PLC-d1 in response to an alteration of the lipid composition of the mem- branes during physiological processes. PH domains of certain mammalian PLC isoforms such as PLC-d3, -d4, -c1 and -c2 contain amphipathic a2-helices in the b5 ⁄ b6 loop, as indicated by patterns of hydrophobic and hydrophilic amino acid residues. The alteration of the lipid composition therefore would also be expected to affect functions and structures of these PLCs through alterations in membrane association states of the PH domains after translocation to membrane sur- faces. It has been reported that membranes from dif- ferent organelles contain different amounts of PS, and that local concentrations of PS in those membranes would be further altered by a variety of factors such as alterations of the asymmetric distribution and lateral segregation of lipids in membranes caused by physiolo- gical processes of the cell. Fluctuations in the PS con- centrations in the membrane might regulate the function of PLCs through alterations in the conforma- tion and membrane binding affinity of the PH domain. Over 90% of PS in the plasma membrane is located in the inner leaflet due to the asymmetrical distribution of lipids between the outer and inner leaflet regulated by flippases and floppases [17–19]. It has been reported that the PS content of the outer leaflet increases during A C B D Fig. 4. Schematic representations of the probable conformational differences of the PLC-d1 PH domains bound to IP 3 in solution (structure I) (A), PIP 2 in PC ⁄ PIP 2 vesicle (structure II) (B), PIP 2 in PC ⁄ PS ⁄ PIP 2 vesicle (structure III) (C), and PIP 2 in PC ⁄ PS ⁄ PIP 2 vesicle under a masking effect of ions (D). The b-sheets are indicated by rectangles. The a-helices are indicated by cylinders, with the exception of the a2-helix which is indicated by an a-helix wheel viewed from the C-terminus to emphasize the amphipathic nature of the a2-helix and its change in orienta- tion at the membrane surface. As shown in (A), hydrophilic residues (Thr81, Glu82, Glu85, and Lys86) are indicated by red circles, and hydro- phobic residues (Leu84, Phe87 and Ala88) are indicated by blue circles. The white circle indicates Gly83. IP 3 and PIP 2 head groups are shown by blue and yellow hexagons, respectively. The head groups of PC and PS are shown by yellow and red circles, respectively. Cations and anions in (D) are shown by cyan and orange circles. The blue background indicates the aqueous phase, and the orange background indi- cates the interface and hydrophobic region of the membrane. N. Uekama et al. Function and structure of PLC-d1 PH domain FEBS Journal 274 (2007) 177–187 ª 2006 The Authors Journal compilation ª 2006 FEBS 183 apoptosis, probably as a result of collapse of the asym- metric distribution of lipids [20–22]. Consequently, PS exposed to the cytoplasm would be reduced during the course of apoptosis. Analogous alterations of the lipid asymmetry have been reported to be induced during processes of cellular stimulation unrelated to apoptosis [23,24]. PLC-d1 has been found localized at the clea- vage furrow of dividing cells [31], and PIP 2 hydrolysis is important for progression of cytokinesis. The furrow has also been known to form a region of unusual lipid composition and distribution, where PIP 2 accumulates at the inner leaflet of the membrane but PS seems to transfer from the inner to the outer leaflet of the plasma membrane. Therefore, not only the availability of PIP 2 but also the presence (or absence) of other aci- dic lipids may regulate physiological events such as cytokinesis. Lateral segregation of lipids in a mem- brane plane, such as the formation of lipid microdo- mains or rafts, should also alter local concentrations of PS or other acidic phospholipids, such as phosphati- dylinositol-polyphosphates, in the vicinity of PLC-d1. Segregation of PIP 2 induced by positively charged macromolecules such as poly(Lys) and MARCKS frag- ment have been reported for vesicle systems in vitro [25,26]. Notably, a 2 H NMR study has also suggested that poly(Lys) causes segregation of PS in lipid bilay- ers [27]. Local enrichment or exclusion of PS and other negatively charged lipids, which would generally occur in lipid microdomains or rafts, should also alter relat- ive populations of structure II and structure III of the PH domain. The cytoplasm of a typical mammalian cell contains about 150 mm monovalent cations, predominantly K + and Na + , and about 0.5 mm divalent cations, predom- inately Mg 2+ and Ca 2+ as free ions [32]. Although the total concentration of the divalent cations in the typ- ical mammalian cell is around 30 mm [30], most of the divalent cations are bound to macromolecules or stored within organelles. Figure 3 indicates that the PH domain takes the conformation of either struc- ture II or structure III at the surface of the membrane containing 20% PS in the presence of 25 mm NaCl. Structure II becomes dominant in the presence of 25 mm NaCl and 10 mm MgCl 2 . This suggests that structure II and structure III coexist at the surface of the plasma membrane, as far as effects of other mem- brane components such as phosphatidylethanolamine and cholesterol are eliminated. Structure III may rep- resent a minority fraction at the surface of cellular membranes, but changes in the local lipid composition and the composition of electrolytes in the cytoplasm would alter the equilibrium between structure II and structure III under certain physiological conditions. Local concentrations of cations in the vicinity of the membrane surface are expected to be fluctuated in con- nection with the lipid composition due to a formation of a diffuse electric double layer [33,34]. An increase in acidic lipids causes an increase in the negative charge density at the membrane surface, causing accumulation of cations on the membrane surface and consequently prevents the formation of structure III. Because elec- trostatic properties and affinities for ligands vary among PH domains of PLC isoforms, the effect of PS may be different among different isoforms. Thus, the conformational change of the PH domain mediated by the interaction between the amphipathic a2-helix and the membrane interface might provide a functional switch for the PH domain and consequently for PLC-d1. Dynamic changes in the lipid composition of the plasma membrane would be capable of provi- ding a means of regulating the properties of PLC-d1 and the PLC isoforms at the membrane-associated state through the PS-dependent structural and func- tional alterations of the PH domains. Although the a2-helix located in the b5 ⁄ b6 loop is unique to the PH domain of PLC-d1 and the PLC isoforms closely rela- ted to PLC-d1, other examples of PH domains, such as the PH domains of insulin receptor substrate-1 and spectrin, also have short amphipathic a-helices in the structures [35]. We propose that these PH domains are also likely to be susceptible to changes in the local lipid composition of the membrane. Experimental procedures Materials PC from bovine liver and PS from bovine brain were pur- chased from Avanti Polar Lipid (Birmingham, AL, USA). PIP 2 from bovine brain, POPC and streptavidin were pur- chased from Sigma (St Louis, MO, USA). N-(Biotinoyl)- 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine and N-{[6-(biotinoyl)amino]hexanoyl}-1,2-dihexadecanoyl-sn- glycero-3-phosphoethanolamine were purchased from Molecular Probes (Eugene, OR, USA). l-[3- 13 C]Alanine was obtained from CIL (Andover, MA, USA). All reagents were used without further purification. Whole experimental processes including treatments of phospholipids were car- ried out under a nitrogen or argon atmosphere to prevent oxidization of lipids. Protein expression and purification The rat PLC-d1 PH domain fragment (1–140) was sub- cloned into a pGEX-2T-based bacterial expression vector (pGEX-2T from Amersham Bioscience, Piscataway, NJ, Function and structure of PLC-d1 PH domain N. Uekama et al. 184 FEBS Journal 274 (2007) 177–187 ª 2006 The Authors Journal compilation ª 2006 FEBS USA). The resulting vector product was designated pGST3. The processes of expression and purification of the PH domain were carried out as previously described [3]. In brief, the PLC-d1 PH domain was expressed as a glutathi- one-S-transferase (GST) fusion protein in Escherichia coli (PR745) cultured with M9 medium in the presence of 20 amino acids but with l-alanine replaced by l-[3- 13 C]alanine. After induction with 0.1 mm isopropyl-1-thio-b-d-galacto- pyranoside, cells were harvested and subjected to sonication in the presence of a mixture of protein inhibitors. The [3- 13 C]Ala-labeled PLC-d1 PH domain-GST fusion protein was purified using glutathione-sepharose 4B affinity resin (Amersham Bioscience). GST was removed by cleavage of the linker connecting GST and PH domain by using throm- bin (Sigma) to obtain the [3- 13 C]Ala-labeled PLC-d1PH domain. The final preparation of the PH domain inclu- des additional amino acid residues, GSRST- and -ELGPRPNWPTS, at the N- and C-termini of the natural amino acid sequence, respectively. Vesicle coprecipitation assay Dissociation constants (K d ) of the PH domain and PIP 2 embedded in POPC vesicles in the presence or absence of PS were evaluated by PH domain-vesicle coprecipitation assays using SUVs [36]. Phospholipid mixtures containing 2% biotinylated PE dissolved in chloroform were cast on glass to form thin films. After evaporation of chloroform in vacuo for 1 day, the lipids were re-suspended in 20 mm Mops buffer (pH 6.5) containing different amount of salts (NaCl and MgCl 2 ) followed by sonication using a probe- type sonicator. The sonication was carried out at 40 °C for 15 min in order to prepare the SUVs. All of the phospholi- pid mixtures used in this study formed lipid bilayers either in the presence or absence of the salts. The sizes of the vesi- cles were evaluated by dynamic light scattering. Average diameters of SUVs were 30 nm, regardless of the lipid com- positions. The SUVs were mixed with PLC-d1 PH domain and incubated for 15 min at 25 ° C. Subsequently, streptavi- din was added to generate a molar ratio of biotinylated lipid and streptavidin of 8 : 1. The mixture was incubated for 30 min at 25 °C. The vesicles were pelleted by ultracen- trifugation at 43 000 g for 20 min at 20 °C by using himac CS100GXL with S100 AT4 rotor (Hitachi Koki, Ibaragi, Japan). The pellets were re-suspended in 20 mm Mops buf- fer (pH 6.5) containing different amount of salts (NaCl and MgCl 2 ) to adjust the volumes to the original volumes of the suspensions. Mops buffer is suitable for evaluating the influence of salt on the membrane binding affinity due to its low ionic strength and low capability of forming com- plexes with multivalent cations. The content of PH domain in the supernatants and the pellets was estimated from the densities of the SDS ⁄ PAGE bands stained with Coomassie brilliant blue. NIH Image was used to determine the band densities. The efficiency of precipitation of the membrane fraction was estimated by quantification of phosphorus in the pellet and supernatant. Densities of SDS ⁄ PAGE bands of streptavidin were also used as indicators of the efficiency of precipitation, since all the streptavidin was proved to form a complex with SUVs by the quantification of phos- phorus. The sedimentation efficiency of the vesicles contain- ing PS tended to be lower than that of the PC ⁄ PIP 2 vesicles, probably due to an electrostatic repulsion between the negatively charged surfaces of the PC ⁄ PIP 2 ⁄ PS vesicles. The condition of the ultracentrifugation was chosen so that at least 88% of the total phospholipid content was collected in the pellet. Precipitation of the free PH domain during ul- tracentrifugation was found to be negligible in the presence and absence of streptavidin. Solid-state 13 C NMR spectroscopy [3- 13 C]Ala-labeled PLC-d1 PH domain–phospholipid vesicle complexes were prepared as follows: PC, PS and PIP 2 were dissolved in chloroform and mixed to achieve the required molar ratio of the lipids, and subsequently cast on glass to form a thin film. After evaporation of chloroform in vacuo for 1 day, the lipids were suspended in 20 mm Mops buffer (pH 6.5) containing 1 mm dithiothreitol and 0.025% NaN 3 , followed by three freeze ⁄ thaw cycles. The phospholipid vesicle suspensions were mixed with [3- 13 C]Ala-labeled PLC-d1 PH domain dissolved in 50 mm Tris buffer (pH 7.5) containing 150 mm NaCl and 0.25 mm CaCl 2 to enable formation of the protein–vesicle complex. The pro- tein–vesicle complex suspensions were subsequently dialyzed into 20 mm Mops buffer (pH 6.5) containing 1 mm dithio- threitol and 0.025% NaN 3 at 4 °C. The protein–vesicle complexes were concentrated by ultracentrifugation [himac CS100GXL with S100AT4 rotor (Hitachi Koki) 541 000 g for 6 h at 4 °C] immediately prior to obtaining NMR spect- roscopic measurements. The concentrated suspensions were placed in a 5-mm outer diameter zirconia pencil-type solid- state NMR sample rotor and sealed with epoxy resin to prevent water evaporation. Measurement of solid-state NMR spectra High resolution solid-state 13 C NMR spectra were recorded on a Chemagnetics Infinity 400 spectrometer ( 13 C: 100.6 MHz), using single-pulse excitation DD-MAS method. The spectral width was 40 kHz, the acquisition time was 50 ms, and the repetition time was 4 s. Free induction decay profiles were acquired with 2048 data points, and Fourier transformed as 32 768 data points, after 30 720 data points were zero-filled. p ⁄ 2 pulses for car- bon and proton nuclei were 5.0 ls, and the spinning rate for magic angle spinning was 2.6 kHz. The dipolar decou- pling field strength was 55 kHz. Transients were accumu- lated 20 000–40 000 times until a reasonable signal-to-noise ratio was achieved. 13 C chemical shifts were referenced to N. Uekama et al. Function and structure of PLC-d1 PH domain FEBS Journal 274 (2007) 177–187 ª 2006 The Authors Journal compilation ª 2006 FEBS 185 the carboxyl signal of glycine (176.03 p.p.m. from tetra- methyl silane) and then expressed as relative shifts from the tetramethyl silane value. Measurement of dynamic light scattering Dynamic light scattering of SUVs suspended in 20 mm Mops buffer (pH 6.5) containing 1 mm dithiothreitol and 0.025% NaN 3 was measured at 20 °C with a Dyna Pro dynamic light scattering ⁄ molecular sizing instrument (Wyatt Technology Co., Santa Barbara, CA, USA). Acknowledgements This work was supported by Grants 17048029 and 18570184 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to ST). References 1 White SH, Ladokhin AS, Jayasinghe S & Hristova K (2001) How membranes shape protein structure. J Biol Chem 276, 32395–32398. 2 Hristova K, Wimley WC, Mishra VK, Anantharamiah GM, Segrest JP & White SH (1999) An amphipathic a-helix at a membrane interface: a structural study using a novel X-ray diffraction method. J Mol Biol 290, 99–117. 3 Tuzi S, Uekama N, Okada M, Yamaguchi S, Saito H & Yagisawa H (2003) Structure and dynamics of the phos- pholipase C-d1 pleckstrin homology domain located at the lipid bilayer surface. J Biol Chem 278, 28019–28025. 4 Hirata M, Kanematsu T, Takeuchi H & Yagisawa H (1998) Pleckstrin homology domain as an inositol com- pound binding module. Jpn J Pharmacol 76 , 255–263. 5 Ferguson KM, Lemmon MA, Schlessinger J & Sigler PB (1995) Structure of the high affinity complex of inositol trisphosphate with a phospholipase C pleckstrin homo- logy domain. Cell 83, 1037–1046. 6 Essen LO, Perisic O, Cheung R, Katan M & Williams RL (1996) Crystal structure of a mammalian phospho- inositide-specific phospholipase Cd. Nature 380, 595–602. 7 Varnai P, Lin X, Lee SB, Tuymetova G, Bondeva T, Spat A, Rhee SG, Hajnoczky G & Balla T (2002) Inosi- tol lipid binding and membrane localization of isolated pleckstrin homology (PH) domains. Studies on the PH domains of phospholipase C d1 and p130. J Biol Chem 277, 27412–27422. 8 Kavran JM, Klein DE, Lee A, Falasca M, Isakoff SJ, Skolnik EY & Lemmon MA (1998) Specificity and pro- miscuity in phosphoinositide binding by pleckstrin homology domains. J Biol Chem 273, 30497–30508. 9 Garcia P, Gupta R, Shah S, Morris AJ, Rudge SA, Scarlata S, Petrova V, McLaughlin S & Rebecchi MJ (1995) The pleckstrin homology domain of phospholi- pase C-d 1 binds with high affinity to phosphatidylinosi- tol 4,5-bisphosphate in bilayer membranes. Biochemistry 34, 16228–16234. 10 Lemmon MA, Ferguson KM, O’Brien R, Sigler PB & Schlessinger J (1995) Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homo- logy domain. Proc Natl Acad Sci USA 92, 10472–10476. 11 Yagisawa H, Sakuma K, Paterson HF, Cheung R, Allen V, Hirata H, Watanabe Y, Hirata M, Williams RL & Katan M (1998) Replacements of single basic amino acids in the pleckstrin homology domain of phospholipase C-d1 alter the ligand binding, phospho- lipase activity, and interaction with the plasma mem- brane. J Biol Chem 273, 417–424. 12 Lomasney JW, Cheng HF, Wang LP, Kuan Y, Liu S, Fesik SW & King K (1996) Phosphatidylinositol 4,5- bisphosphate binding to the pleckstrin homology domain of phospholipase C-d1 enhances enzyme activ- ity. J Biol Chem 271, 25316–25326. 13 Guo Y, Philip F & Scarlata S (2003) The Pleckstrin homology domains of phospholipases C-b and -d confer activation through a common site. J Biol Chem 278, 29995–30004. 14 Yagisawa H, Okada M, Naito Y, Sasaki K, Yamaga M & Fujii M (2006) Coordinated intracellular transloca- tion of phosphoinositide-specific phospholipase C-d with the cell cycle. Biochim Biophys Acta 1761, 522–534. 15 Yagisawa H, Yamaga M, Okada M, Sasaki K & Fujii M (2002) Regulation of the intracellular localization of phosphoinositide-specific phospholipase Cd(1). Adv Enzyme Regul 42, 261–284. 16 Daum G (1985) Lipids of mitochondria. Biochim Bio- phys Acta 822, 1–42. 17 Daleke DL (2003) Regulation of transbilayer plasma membrane phospholipid asymmetry. J Lipid Res 44, 233–242. 18 Zachowski A (1993) Phospholipids in animal eukaryotic membranes: transverse asymmetry and movement. Bio- chem J 294, 1–14. 19 Rothman JE & Lenard J (1977) Membrane asymmetry. Science 195, 743–753. 20 Kagan VE, Borisenko GG, Serinkan BF, Tyurina YY, Tyurin VA, Jiang J, Liu SX, Shvedova AA, Fabisiak JP, Uthaisang W et al. (2003) Appetizing rancidity of apop- totic cells for macrophages: oxidation, externalization, and recognition of phosphatidylserine. Am J Physiol Lung Cell Mol Physiol 285, L1–L17. 21 Bratton DL, Fadok VA, Richter DA, Kailey JM, Guth- rie LA & Henson PM (1997) Appearance of phosphati- dylserine on apoptotic cells requires calcium-mediated nonspecific flip-flop and is enhanced by loss of the amino- phospholipid translocase. J Biol Chem 272, 26159– 26165. Function and structure of PLC-d1 PH domain N. Uekama et al. 186 FEBS Journal 274 (2007) 177–187 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... sequestration of phosphatidylinositol 4,5-bisphosphate by the basic effector domain of myristoylated alanine-rich C kinase substrate is due to nonspecific electrostatic interactions J Biol Chem 277, 34401–34412 27 Franzin CM & Macdonald PM (2001) Polylysineinduced 2H NMR-observable domains in phosphatidylserine ⁄ phosphatidylcholine lipid bilayers Biophys J 81, 3346–3362 Function and structure of PLC-d1 PH domain. .. modeling of the membrane targeting of phospholipase C pleckstrin homology domains Protein Sci 12, 1934–1953 29 Huang C & Mason JT (1978) Geometric packing constraints in egg phosphatidylcholine vesicles Proc Natl Acad Sci USA 75, 308–310 30 Rawn JD (1989) Biochemistry Neil Patterson Publishers, Burlington, NC 31 Naito Y, Okada M & Yagisawa H (2006) Phospholipase C isoforms are localized at the cleavage... Biology of the Cell, 4th edn Garland Science, New York, NY 33 Israelachvili JN (1992) Intermolecular and Surface Forces, 2nd edn Academic Press, London 34 Szundi I & Stoeckenius W (1989) Surface pH controls purple-to-blue transition of bacteriorhodopsin A theoretical model of purple membrane surface Biophys J 56, 369–383 35 Lemmon MA & Ferguson KM (2000) Signal-dependent membrane targeting by pleckstrin homology. .. Signal-dependent membrane targeting by pleckstrin homology (PH) domains Biochem J 350, 1–18 36 Tortorella D, Ulbrandt ND & London E (1993) Simple centrifugation method for efficient pelleting of both small and large unilamellar vesicles that allows convenient measurement of protein binding Biochemistry 32, 9181–9188 FEBS Journal 274 (2007) 177–187 ª 2006 The Authors Journal compilation ª 2006 FEBS 187 ... Uekama et al 22 Kunzelmann-Marche C, Freyssinet JM & Martinez MC (2002) Loss of plasma membrane phospholipid asymmetry requires raft integrity Role of transient receptor potential channels and ERK pathway J Biol Chem 277, 19876–19881 23 Frasch SC, Henson PM, Nagaosa K, Fessler MB, Borregaard N & Bratton DL (2004) Phospholipid flip-flop and phospholipid scramblase 1 (PLSCR1) co-localize to uropod rafts in formylated... (2003) Caspase-independent exposure of aminophospholipids and tyrosine phosphorylation in bicarbonate responsive human sperm cells Biol Reprod 68, 2122– 2134 25 Gambhir A, Hangyas-Mihalyne G, Zaitseva I, Cafiso DS, Wang J, Murray D, Pentyala SN, Smith SO & McLaughlin S (2004) Electrostatic sequestration of PIP2 on phospholipid membranes by basic ⁄ aromatic regions of proteins Biophys J 86, 2188–2207 . properties of PLC-d1 and the PLC isoforms at the membrane-associated state through the PS-dependent structural and func- tional alterations of the PH domains. Although the a2-helix located in the b5. Phosphatidylserine induces functional and structural alterations of the membrane-associated pleckstrin homology domain of phospholipase C-d1 Naoko Uekama, Takio Sugita,. loop is unique to the PH domain of PLC-d1 and the PLC isoforms closely rela- ted to PLC-d1, other examples of PH domains, such as the PH domains of insulin receptor substrate-1 and spectrin, also