Báo cáo khoa học: Phosphatidylserine induces functional and structural alterations of the membrane-associated pleckstrin homology domain of phospholipase C-d1 docx

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:
(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).
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