Phosphatidylserine induces functional and ... - Wiley Online Library

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

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 6781297, Japan Fax: +81 791 580182 Tel: +81 791 580180 E-mail: [email protected] (Received 4 August 2006, revised 1 November 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 13C NMR spectroscopy. Twenty per cent phosphatidylserine (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 (PIP2) (75 : 20 : 5) vesicle indicated loss of the interaction between the amphipathic a2-helix of the PH domain and the interface region of the membrane which was previously reported for the PH domain bound to PC ⁄ PIP2 (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 ⁄ PIP2 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 MgCl2, 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 membranes.

A number of processes contributing to important cellular functions such as cellular signal transduction, cytoskeletal organization, and membrane trafficking are known to be localized at membrane surfaces of the plasma membrane and organelles. For instance, extracellular 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

Abbreviations DD-MAS, single pulse excitation dipolar decoupled-magic angle spinning; GST, glutathione-S-transferase; IP3, D-myo-inositol 1,4,5-trisphosphate; PC, phosphatidylcholine; PH domain, pleckstrin homology domain; PIP2, 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

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proteins adapted to the interfacial environment. However, the structural characteristics of the membraneassociated state of peripheral membrane proteins remain unclear, mainly due to the lack of an appropriate technique for investigating the structures of peripheral membrane proteins bound to lipid bilayers. Well-established techniques for determination of protein 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 phospholipase C-d1 (PLC-d1; EC 3.1.4.11), a peripheral membrane protein. As a result, we proposed a model for the conformational change of the domain induced during 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-bisphosphate (PIP2) [7–10]. This PIP2-dependent membrane localization of the PH domain has been shown to provide an indirect regulatory mechanism of the phospholipase activity of PLC-d1 through regulation of the frequency of encounter between the PLC-d1 active site (X and Y domain) and its substrate, PIP2 [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 regulation of PLC-d1 hydrolysis. Furthermore, structure– function relationships of the PLC-d1 PH domain may be modified by alteration of the nature of the membrane. Since PLC-d1 is reported to shuttle between the plasma membrane and the cytoplasm, and also 178

between the cytoplasm and the nucleus [14,15], variations in lipid composition in the different membranes of the cell [16] might cause location-dependent structure–function variations for PLC-d1. Alteration of the asymmetric distribution of lipids between the inner and outer leaflets of the membrane and a lateral segregation of lipid components in the membrane may also cause fluctuations of the lipid composition in the vicinity of PLC-d1. In this study, we investigated the effects of phosphatidylserine (PS) on the structure and function of the PLC-d1 PH domain on an artificial membrane surface. 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 electrostatic 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 erythrocytes 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 alterations of phospholipid asymmetry [23,24]. Loss of asymmetric 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 membrane-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 predicted 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 coprecipitation assays of PLC-d1 PH domain performed for the


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Table 1. Membrane binding affinities of PLC-d1 PH domain

Vesicle POPC ⁄ PIP2d POPC ⁄ PIP2d POPC ⁄ PS ⁄ PIP2e POPC ⁄ PS ⁄ PIP2e POPC ⁄ PS ⁄ PIP2e POPC ⁄ PS ⁄ PIP2e

[NaCl] (mM)

PH domain bound to vesicle (%)

0 25 0 25 50 100

81.0 80.9 73.6 83.2 84.5 82.7

± ± ± ± ± ±

a,c

2.40 1.33 2.72 3.28 3.52 3.44

Kdb,c (lM) 1.62 1.63 2.77 1.36 1.21 1.41

± ± ± ± ± ±

0.33 0.17 0.48 0.40 0.40 0.40

a Estimated from ratios of the PH domain collected with the centrifuged pellets by the vesicle coprecipitation assay. bKd values were calculated as shown in the text. [PH]0 and [PIP2]0 used for the calculations were 11.2 lM and 15.9 lM, respectively. cValues are mean ± SD for more than five different experiments (P < 0.05). d Molar ratio of POPC–PIP2–biotinylated PE ¼ 93 : 5 : 2. eMolar ratio of POPC–PS–PIP2–biotinylated PE ¼ 73 : 20 : 5 : 2.

2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) small unilamellar vesicles (SUVs) containing 5% PIP2 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 (Kd) for the PH domain–PIP2 interaction were calculated for SUVs with different lipid compositions from the ratios of the PH domain partitioned into the supernatants and the precipitates corresponding to the free PH domain and PH domain–PIP2 complex, respectively. Affinities of the PLC-d1 PH domain for the head groups of phosphatidylcholine (PC) or PS are negligible compared with the high affinity for PIP2 [8]. The Kd values were calculated from following equations: Kd ¼ Rf½PIP2 0 ½PH0 =ðR þ 1Þg where R ¼ [PH] ⁄ [PHÆPIP2], [PH] is the concentration of the free PH domain and [PHÆPIP2] is the concentration of the PH domain forming a complex with PIP2 at equilibrium. [PH]0 and [PIP2]0 are total concentrations of PH domain and PIP2 accessible by the PH domain, respectively. We assumed that 70% of the total lipids of the SUVs are exposed to the outer surface of the SUVs and that PIP2 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 Kd obtained for the PC ⁄ PIP2 vesicle without PS was very close to the previously reported value determined by isothermal titration calorimetry (1.66 ± 0.80 lm) [10]. As shown in Table 1, a moderate but significant increase in Kd 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 addition of NaCl up to 100 mm did not induce a further increase in Kd. The differences in Kd values for PC ⁄ PIP2 vesicles and PC ⁄ PS ⁄ PIP2 vesicles containing 25–100 mm NaCl was found to be insignificant. In the presence of 25 mm NaCl, the Kd is close to that for the PC ⁄ PIP2 membrane without PS, indicating that NaCl abolishes the effect of PS. The similar Kd values for the PC ⁄ PIP2 membrane with 0 and 25 mm NaCl eliminate the possibility that NaCl affects the affinity for binding to the PC ⁄ PIP2 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 dominated by the highly specific association to PIP2 via its ligand binding site, the affinity-reducing effect of PS on the PH domain suggests that a mechanism independent of PIP2 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 spectra of 13C-labeled methyl carbons in [3-13C]Ala-labeled PH domain bound to PC vesicles containing 5% PIP2 (PC ⁄ PIP2 vesicle) and PC ⁄ PIP2 vesicle containing 20% PS (PC ⁄ PS ⁄ PIP2 vesicle), respectively, suspended in 20 mm Mops buffer (pH 6.5) containing 1 mm dithiothreitol and 0.025% NaN3. The assignments of the spectral signals to each of the Ala residues determined for the PH domain bound to PC ⁄ PIP2 vesicles were made by using a series of site-directed replacements of Ala residues [3] indicated at the top of Fig. 1. Chemical shifts of Ala residues in the PH domain bound to d-myo-inositol 1,4,5-trisphosphate (IP3) in solution are shown as vertical bars at the bottom of the spectra. These chemical shift values correspond to the structure


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Fig. 1. DD-MAS NMR spectra of the [3-13C]Ala-labeled PLC-d1 PH domain bound to PC ⁄ PIP2 or PC ⁄ PS ⁄ PIP2 vesicles. (A) PH domain bound to a PC ⁄ PIP2 vesicle at 20 C, (B) PH domain bound to PC ⁄ PS ⁄ PIP2 vesicle at 20 C, and (C) PH domain bound to a PC ⁄ PS ⁄ PIP2 vesicle at 4 C. The PH domain–vesicle complexes were suspended in 20 mM Mops buffer (pH 6.5) containing 1 mM dithiothreitol and 0.025% NaN3. 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 IP3 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.

of the PH domain in solution in the absence of membrane. This structure is expected to be similar to the three dimensional structure determined for the PLC-d1 PH domain–IP3 complex by X-ray diffraction. As shown with dotted lines, the chemical shifts of the signals 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 180

Fig. 2. DD-MAS NMR spectra of the [3-13C]Ala-labeled PLC-d1 PH domain bound to PC ⁄ PS ⁄ PIP2 vesicle in the presence of different concentrations of salts at 20 C. Vesicles with PH domain were suspended in (A) 20 mM Mops buffer containing 1 mM dithiothreitol and 0.025% NaN3, (B) 20 mM Mops buffer containing 1 mM dithiothreitol, 0.025% NaN3 and 25 mM NaCl, and (C) 20 mM Mops buffer containing 1 mM dithiothreitol, 0.025% NaN3, 25 mM NaCl and 10 mM MgCl2. Peaks assigned to lipid molecules are indicated with asterisks.

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 conformation as Ala88 in the PH domain bound to PC ⁄ PIP2 vesicles that resonates at 16.81 p.p.m. (Fig. 1A), although the intensity of the signal is reduced. In contrast, a peak at 17.64 p.p.m. in the spectrum of the PH domain bound to PC ⁄ PS ⁄ PIP2 vesicles does not have a corresponding peak at 17.6 p.p.m. in the spectrum of the PH domain bound to PC ⁄ PIP2 vesicles. The intensity 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


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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 ⁄ PIP2 vesicles takes two different conformations, one that takes the same conformation as that at the surface of PC ⁄ PIP2 vesicles, resonating at 16.81 p.p.m., and the other, which is characteristic for the PH domain bound to PC ⁄ PS ⁄ PIP2 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 IP3 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 ⁄ PIP2 vesicles, in contrast to a broad peak for the PH domain bound to PC ⁄ PIP2 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 ⁄ PIP2 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 IP3 in solution. The matching of chemical shifts of Ala88 and Ala112 with those of the PH domain–IP3 complex in solution indicates that a fraction of the total PH domain content bound to PC ⁄ PS ⁄ PIP2 vesicles containing 20% PS adopts a conformation similar to that of the PH domain bound to IP3 in solution. A slight downfield displacement of the Ala88 peak at 17.64 p.p.m. for the PH domain bound to PC ⁄ PS ⁄ PIP2 vesicles relative to that of the PH domain bound to IP3 indicates that the structure of the PH domain bound to PC ⁄ PS ⁄ PIP2 vesicles is not completely identical to that of the PH domain bound to IP3, presumably due to the membrane surface properties. Electrolytes suppress the effects of PS on the PH domain As shown in Fig. 2B, the addition of 25 mm NaCl causes 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 ⁄ PIP2 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. accompanied 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 ⁄ PIP2 vesicles and simultaneous destruction of the structure which resembles the PH domain bound to IP3 at the surface of the PC ⁄ PS ⁄ PIP2 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 MgCl2 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 Mg2+ being the most common [30]. In the presence of 10 mm MgCl2, the Ala88 signal at 17.5–6 p.p.m. originating from the structure similar to that of the PH domain bound to IP3 disappears, while the Ala88 peak at 16.86 p.p.m. remains as shown in Fig. 2C. MgCl2 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 ⁄ PIP2 complex (Fig. 2C) in the presence of MgCl2 is virtually identical to that of the PH domain bound to PC ⁄ PIP2 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-d1 PH domain shows that positively charged residues are concentrated around the PIP2-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 membrane association of the PH domain through electrostatic 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


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PS is that PS lowers the stability of the PH domain– lipid bilayer complex after association of the membrane with the PH domain. The solid-state NMR spectra 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 alanine residues observed by solid-state NMR are shown in a schematic representation of the secondary and tertiary structures of the PLC-d1 PH domain bound to IP3 based on the structure determined by the X-ray diffraction study [5]. In our previous study, we proposed 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 ⁄ PIP2 vesicle as an auxiliary membrane interacting site in addition to the PIP2 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 hydrophobic inner layer. This interaction causes the conformational 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 conformational change of Ala112 which appears during the

Fig. 3. Positions of 13C-labeled alanine residues in the PLC-d1 PH domain shown in a schematic representation of the three-dimensional structure based on the model proposed by the X-ray diffraction study for the PH domain–IP3 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 13C NMR spectra due to an overlap of the signal at 14.4 p.p.m. with an intense peak arising from the lipids. IP3, located at the PIP2-specific lipid binding site, is indicated by a hexagon.

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process of membrane association was interpreted as being related to removal and formation of the hydrogen bond between Arg95 (NH) and Pro91 (C¼O) and the hydrogen bond between Arg95 (NH) and Ala112 (C¼O), as determined by solid-state NMR spectroscopy [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 similar 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 individual 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 IP3. The proposed structures of the PLC-d1 PH domain are as follows: (a) with IP3 in solution (structure I); (b) with PIP2 embedded in PC ⁄ PIP2 vesicle (structure II); and (c) with PIP2 embedded in PC ⁄ PS ⁄ PIP2 vesicle (structure III), and are schematically shown in Figs 4A–C, respectively. At the surface of the PC ⁄ PIP2 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 amphipathic 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 PIP2 in the membrane as indicated by NMR measurements. The coordinated changes in membrane binding affinity 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 suggests 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


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A

C

B

D

Fig. 4. Schematic representations of the probable conformational differences of the PLC-d1 PH domains bound to IP3 in solution (structure I) (A), PIP2 in PC ⁄ PIP2 vesicle (structure II) (B), PIP2 in PC ⁄ PS ⁄ PIP2 vesicle (structure III) (C), and PIP2 in PC ⁄ PS ⁄ PIP2 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 orientation at the membrane surface. As shown in (A), hydrophilic residues (Thr81, Glu82, Glu85, and Lys86) are indicated by red circles, and hydrophobic residues (Leu84, Phe87 and Ala88) are indicated by blue circles. The white circle indicates Gly83. IP3 and PIP2 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 indicates the interface and hydrophobic region of the membrane.

with the above model in which the PS-dependent conformational change of the PH domain at the membrane interface contributes to the change of the membrane binding affinity induced by PS. The remarkable effect of low concentrations of MgCl2 on the formation 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 affinity, 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 equilibrium at the surface of PC ⁄ PS ⁄ PIP2 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 stability 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 membranes 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 surfaces. It has been reported that membranes from different 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 physiological processes of the cell. Fluctuations in the PS concentrations in the membrane might regulate the function of PLCs through alterations in the conformation 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


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apoptosis, probably as a result of collapse of the asymmetric 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 cleavage furrow of dividing cells [31], and PIP2 hydrolysis is important for progression of cytokinesis. The furrow has also been known to form a region of unusual lipid composition and distribution, where PIP2 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 PIP2 but also the presence (or absence) of other acidic lipids may regulate physiological events such as cytokinesis. Lateral segregation of lipids in a membrane plane, such as the formation of lipid microdomains or rafts, should also alter local concentrations of PS or other acidic phospholipids, such as phosphatidylinositol-polyphosphates, in the vicinity of PLC-d1. Segregation of PIP2 induced by positively charged macromolecules such as poly(Lys) and MARCKS fragment have been reported for vesicle systems in vitro [25,26]. Notably, a 2H NMR study has also suggested that poly(Lys) causes segregation of PS in lipid bilayers [27]. Local enrichment or exclusion of PS and other negatively charged lipids, which would generally occur in lipid microdomains or rafts, should also alter relative 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, predominately Mg2+ and Ca2+ as free ions [32]. Although the total concentration of the divalent cations in the typical 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 structure 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 MgCl2. This suggests that structure II and structure III coexist at the surface of the plasma membrane, as far as effects of other membrane components such as phosphatidylethanolamine and cholesterol are eliminated. Structure III may represent 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. 184

Local concentrations of cations in the vicinity of the membrane surface are expected to be fluctuated in connection 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 electrostatic 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 providing a means of regulating the properties of PLC-d1 and the PLC isoforms at the membrane-associated state through the PS-dependent structural and functional 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 related 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 purchased from Avanti Polar Lipid (Birmingham, AL, USA). PIP2 from bovine brain, POPC and streptavidin were purchased from Sigma (St Louis, MO, USA). N-(Biotinoyl)1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine and N-{[6-(biotinoyl)amino]hexanoyl}-1,2-dihexadecanoyl-snglycero-3-phosphoethanolamine were purchased from Molecular Probes (Eugene, OR, USA). l-[3-13C]Alanine was obtained from CIL (Andover, MA, USA). All reagents were used without further purification. Whole experimental processes including treatments of phospholipids were carried 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 subcloned into a pGEX-2T-based bacterial expression vector (pGEX-2T from Amersham Bioscience, Piscataway, NJ,


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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 glutathione-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-[3-13C]alanine. After induction with 0.1 mm isopropyl-1-thio-b-d-galactopyranoside, cells were harvested and subjected to sonication in the presence of a mixture of protein inhibitors. The [3-13C]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 thrombin (Sigma) to obtain the [3-13C]Ala-labeled PLC-d1 PH domain. The final preparation of the PH domain includes 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 (Kd) of the PH domain and PIP2 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 MgCl2) followed by sonication using a probetype sonicator. The sonication was carried out at 40 C for 15 min in order to prepare the SUVs. All of the phospholipid mixtures used in this study formed lipid bilayers either in the presence or absence of the salts. The sizes of the vesicles were evaluated by dynamic light scattering. Average diameters of SUVs were 30 nm, regardless of the lipid compositions. The SUVs were mixed with PLC-d1 PH domain and incubated for 15 min at 25 C. Subsequently, streptavidin 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 ultracentrifugation 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 buffer (pH 6.5) containing different amount of salts (NaCl and MgCl2) 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 complexes 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 phosphorus. The sedimentation efficiency of the vesicles containing PS tended to be lower than that of the PC ⁄ PIP2 vesicles, probably due to an electrostatic repulsion between the negatively charged surfaces of the PC ⁄ PIP2 ⁄ 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 ultracentrifugation was found to be negligible in the presence and absence of streptavidin.

Solid-state

13

C NMR spectroscopy

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[3- C]Ala-labeled PLC-d1 PH domain–phospholipid vesicle complexes were prepared as follows: PC, PS and PIP2 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% NaN3, followed by three freeze ⁄ thaw cycles. The phospholipid vesicle suspensions were mixed with [3-13C]Ala-labeled PLC-d1 PH domain dissolved in 50 mm Tris buffer (pH 7.5) containing 150 mm NaCl and 0.25 mm CaCl2 to enable formation of the protein–vesicle complex. The protein–vesicle complex suspensions were subsequently dialyzed into 20 mm Mops buffer (pH 6.5) containing 1 mm dithiothreitol and 0.025% NaN3 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 spectroscopic measurements. The concentrated suspensions were placed in a 5-mm outer diameter zirconia pencil-type solidstate NMR sample rotor and sealed with epoxy resin to prevent water evaporation.

Measurement of solid-state NMR spectra High resolution solid-state 13C NMR spectra were recorded on a Chemagnetics Infinity 400 spectrometer (13C: 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 carbon and proton nuclei were 5.0 ls, and the spinning rate for magic angle spinning was 2.6 kHz. The dipolar decoupling field strength was 55 kHz. Transients were accumulated 20 000–40 000 times until a reasonable signal-to-noise ratio was achieved. 13C chemical shifts were referenced to


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the carboxyl signal of glycine (176.03 p.p.m. from tetramethyl silane) and then expressed as relative shifts from the tetramethyl silane value. 10

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% NaN3 was measured at 20 C with a Dyna Pro dynamic light scattering ⁄ molecular sizing instrument (Wyatt Technology Co., Santa Barbara, CA, USA).

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Acknowledgements This work was supported by Grants 17048029 and 18570184 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to ST).

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