Structure and Dynamics of the Phospholipase C-
1 Pleckstrin Homology Domain Located at the Lipid Bilayer Surface*
Satoru Tuzi
,
Naoko Uekama,
Masashi Okada,
Satoru Yamaguchi,
Hazime Saitô and
Hitoshi Yagisawa
From the
Department of Life Science, Himeji Institute of Technology, Harima
Science Garden City, Kouto 3-chome, Kamigori, Hyogo 678-1297, Japan
Received for publication, January 6, 2003
, and in revised form, April 28, 2003.
 |
ABSTRACT
|
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Despite the importance of signal transduction pathways at membrane
surfaces, there have been few means of investigating their molecular
mechanisms based on the structural information of membrane-bound proteins. We
applied solid state NMR as a novel method to obtain structural information
about the phospholipase C-
1 (PLC-
1) pleckstrin homology (PH)
domain at the lipid bilayer surface. NMR spectra of the alanine residues in
the vicinity of the
5/
6 loop in the PH domain revealed changes in
local conformations due to the membrane localization of the protein. We
propose that these conformational changes originate from a hydrophobic
interaction between the amphipathic
-helix located in the
5/
6 loop and the hydrophobic layer of the membrane and contribute
to the membrane binding affinity, interdomain interactions and intermolecular
interactions of PLC-
1.
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INTRODUCTION
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Pleckstrin homology
(PH)1 domains are well
defined structural modules of about 120 amino acid residues
(1,
2) mainly found in proteins
involved in cellular signaling and cytoskeletal functions
(36).
It has been proposed that these domains function as mediators of
intermolecular interactions analogous to many other structural modules
involved in cellular signaling (e.g. SH2 and SH3 domains). Many kinds
of inositol lipids and inositol phosphates have been identified as important
ligands of PH domains
(36),
and, in some cases, the PH domains also interact with other proteins and
mediate protein-protein interactions
(5).
The PH domain of phospholipase C-
1 (PLC-
1) is one of the most
extensively studied PH domains. It has been proposed that it regulates the
membrane localization of PLC-
1
(7,
8) through its high affinity
specific interaction with phosphatidylinositol 4,5-bisphosphate
(PtdIns(4,5)P2), a PLC-
1 ligand
(9), and
D-myo-inositol 1,4,5-trisphosphate
(Ins(1,4,5)P3)
(10), a product of
PtdIns(4,5)P2 hydrolysis by PLC-
1. Despite the rather low
sequence similarity among the PH domain families, the secondary and tertiary
structural motifs of the PH domain are highly conserved
(36).
A high resolution structural model of the rat PLC-
1 PH domain forming a
complex with Ins(1,4,5)P3 has been determined by x-ray diffraction
study at 1.9-Å resolution
(11). The model consists of a
seven-stranded
sandwich formed by two orthogonal anti-parallel
-sheets and a C-terminal amphipathic
-helix. These are conserved
structural motifs among the PH domains whose structures have been determined
by x-ray diffraction and NMR studies. The loops between the
strands,
particularly the
1/
2,
3/
4, and
6/
7 loops,
differ greatly among the PH domains, and, in the case of the PLC-
1 PH
domain, the
1/
2 and
3/
4 loops mainly interact with
Ins(1,4,5)P3. The
5/
6 loop of the PLC-
1 PH
domain includes a characteristic short amphipathic
-helix
(
2-helix) that is not found in other PH domain model structures studied
so far.
Because functionally important intermolecular interactions of PLC-
1
with its ligand, PtdIns(4,5)P2, or other proteins included in the
signal transduction pathways (e.g. transglutaminase II
(G
h)) take place at the membrane surface
(1214),
structural information of PLC-
1 at the membrane surface is
indispensable for understanding the molecular mechanism underlying the
functions of PLC-
1. The conformation and dynamics of peripheral
membrane proteins at the lipid bilayer surface are expected to be different
from those in solution, due to intermolecular interactions between the protein
and lipids, changes in pH and ionic strength induced by surface charges of the
membrane, and drastic changes in the dielectric constant at the lipid bilayer
surface.
Despite the importance of the PLC-
1 structure at the lipid bilayer
surface, there is virtually no structural information about the peripheral
membrane proteins at the membrane surface, due to a lack of means of
investigating the molecular structure of proteins at the lipid bilayer surface
at atomic resolution. In this study, we applied solid state NMR as a novel
method of gaining insights into atomic level structural information of a
peripheral membrane protein at the membrane surface under conditions similar
to those of natural membranes. Solid state NMR is a highly suitable technique
for this purpose, because it can provide information about the conformation
and dynamics of individual amino acid residues in an intact protein under a
wide variety of conditions, including those in a protein-lipid vesicle complex
suspended in buffer at ambient temperature. By metabolic introduction of
carbon-13-labeled alanine residues into a protein as NMR probes, the local
conformation and dynamics of selectively labeled amino acid residues are
readily analyzed
(1519).
Here, we applied this "site-directed" high resolution solid state
13C NMR technique to the PH domain of PLC-
1 as the first
trial of a high resolution solid state NMR investigation of peripheral
membrane proteins involved in lipid signal transduction pathways and obtained
evidence for the conformational change of the PH domain at the membrane
surface.
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EXPERIMENTAL PROCEDURES
|
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MaterialsPhosphatidylcholine (PtdCho) from bovine liver was
purchased from Avanti Polar Lipids (Birmingham, AL). PtdIns(4,5)P2
from bovine brain and Ins(1,4,5)P3 were from Sigma (St. Louis, MO).
L-[3-13C]Alanine was from CIL (Andover, MA). All
reagents were used without further purification.
Expression Vector and Site-directed MutagenesisThe cDNA
encoding rat aortic PLC-
1 fragment (1140) was subcloned into a
pGEX-2T-based bacterial expression vector (pGEX-2T from Amersham Biosciences),
designated pGST3. Individual point mutations were introduced into the plasmid
pGST3-PLC-
1
(141768) encoding the PH domain of the wild
type enzyme by T4 DNA polymerase-based mutagenesis using a TransformerTM
site-directed mutagenesis kit (Clontech). The selection primer was
(5'-GGTTTCTTAGTCGACAGGTGGCAC-3'), which converts the
AatII site (35023525) of pGST/PLC-
1
(141768) into a SalI site, and the mutagenic primers
were A21L, 5'-ACCCGGACCTTCAGCTCCTTCTGAAGGGCA-3'; A88G,
5'-TGGAGAAGTTTGGCCGAGACATCCCCGAG-3'; A112G,
5'-ACCCTAGACCTCATTGGCCCATCACCAGCTGA-3'; A116L,
5'-TTGCCCCATCACCACTTGACGCTCAGCACT-3'; and A118G,
5'-ATCACCAGCTGACGGTCAGCACTGGGTG-3'. The desired point mutation and
the sequence flanking the mutagenic primer-annealing site were confirmed by
DNA sequence analysis.
Protein Expression and PurificationThe wild-type and
mutated PLC-
1 PH domains (1140) were expressed as glutathione
S-transferase (GST) fusion proteins in Escherichia coli
(PR745) (20). Cells were grown
in M9 medium (1), which
contains 100 mg of each of 20 amino acids but with L-alanine
replaced by L-[3-13C]alanine and were incubated in the
presence of 0.1 mM
isopropyl-1-thio-
-D-galactopyranoside (5 h at 37 °C).
After centrifugation, the resulting cell pellets were resuspended in a buffer
containing a mixture of protease inhibitors (20 mM Tris-HCl, pH
7.5, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 50 units/ml aprotinin, 2 µg/ml leupeptin, 2
µg/ml pepstatin A, 0.1 mM banzamidine) and subjected to
sonication using a Vibra cell (Sonics and materials). The cell debris was
removed by centrifugation (15,000 x g for 15 min). The
[3-13C]Ala-labeled PLC-
1 PH domain-GST fusion proteins were
purified using glutathione-Sepharose 4B (Amersham Biosciences) affinity
chromatography, and the [3-13C]Ala-labeled PLC-
1 PH domain
was consequently obtained by cleavage of the link between the PH domain and
GST using thrombin (Sigma). The amino acid sequence of the wild type
PLC-
1PH domain is shown in Fig.
1. Final preparations of the wild type and mutant PH domains
included additional amino acid residues, GSRST- and -ELGPRPNWPTS, at the N and
C termini of the natural amino acid sequence, respectively.
Solid State 13C NMR Sample PreparationThe
[3-13C]Ala-labeled PLC-
1 PH
domain-PtdCho/PtdIns(4,5)P2 vesicle complexes were prepared as
follows: PtdCho and PtdIns(4,5)P2 dissolved in chloroform (molar
ratio of PtdCho and PtdIns(4,5)P2 was 20:1) were cast on glass to
form a thin film. After evaporation of the chloroform in vacuo for 1
day, the lipids were suspended in 20 mM potassium Pi
buffer (pH 6.5) containing 1 mM dithiothreitol and 0.025%
NaN3 followed by three freeze-thaw cycles. The process for the
preparation of whole vesicles was performed under N2 atmosphere to
prevent oxidization of the phospholipids. The phospholipid vesicle suspensions
were mixed with the purified wild-type and mutant
[3-13C]Ala-labeled PLC-
1 PH domains in 20 mM
potassium Pi buffer (pH 6.5) containing 1 mM
dithiothreitol and 0.025% NaN3 (molar ratio of PtdCho, PtdIns, and
PH domain was 40:2:1) and incubated for 20 min at 4 °C to allow complex
formation. The protein-vesicle complexes were concentrated by
ultra-centrifugation (541,000 x g for 6 h at 4 °C). The
[3-13C]Ala-labeled PLC-
1 PH domain-Ins(1,4,5)P3
complex was prepared by mixing the PH domain and Ins(1,4,5)P3
solutions (10 mM MES buffer (pH 6.5) containing 25 mM
NaCl, 10% glycerol, and 0.025% NaN3; the molar ratio of the PH
domain and Ins(1,4,5)P3 was 1:1.1). The PH
domain-Ins(1,4,5)P3 complex solution was concentrated by
ultrafiltration using Microcon YM-3 (Amicon). The
[3-13C]Ala-labeled PH domain-PtdCho/PtdIns(4,5)P2
vesicle complexes and the [3-13C]Ala-labeled PH
domain-Ins(1,4,5)P3 complex were placed in a 5-mm outer diameter
zirconia pencil-type solid state NMR sample rotor and sealed with epoxy resin
to prevent evaporation of water.
Measurement of Solid State 13C NMR SpectraHigh
resolution solid state 13C NMR spectra were recorded on a Varian
Infinity 400 spectrometer (13C: 100.6 MHz), using cross
polarization-magic angle spinning (CP-MAS) and single pulse excitation dipolar
decoupled-magic angle spinning (DD-MAS) methods. The spectral width,
acquisition time, and repetition time for CP-MAS and DD-MAS experiments were
40 kHz, 50 ms, and 4 s, respectively. The contact time for the CP-MAS
experiment was 1 ms. Free induction decays were acquired with 2,048 data
points and Fourier-transformed as 32,768 data points after 30,720 data points
were zero-filled. The
/2 pulses for carbon and protons were 5.0 µs, and
the spinning rates were 4 kHz. The dipolar decoupling field strength was 55
kHz unless indicated otherwise in the text. Transients were accumulated
20,00040,000 times until a reasonable signal-to-noise ratio was
achieved. The 13C chemical shifts were referenced to the carboxyl
signal of glycine (176.03 ppm from tetramethylsilane (TMS)) and then expressed
as relative shifts from the TMS value.
Measurement of Dynamic Light ScatteringDynamic light
scattering of the wild-type PH domain-Ins(1,4,5)P3 complex in 10
mM MES buffer (pH 6.5) containing 25 mM NaCl, 10%
glycerol, and 0.025% NaN3 was measured at 20 °C using a Dyna
Pro dynamic light scattering/molecular sizing instrument (Protein Solutions).
The molecular weights of the monomeric or oligomeric PLC-
1 PH domain
particles were estimated from the particle sizes by assuming the particles had
a spherical shape.
 |
RESULTS
|
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NMR Spectra of [3-13C]Ala Residues in the
PLC-
1 PH Domain-Ins(1,4,5)P3
ComplexFig. 2 (A
and B) shows NMR spectra of the
[3-13C]Ala-labeled PH domain-Ins(1,4,5)P3 complex in
solution measured using the single pulse excitation dipolar decoupled-magic
angle spinning (DD-MAS) method using different amplitudes of magnetic fields
for dipolar decoupling: 19 and 55 kHz. Five narrow peaks resonating at 14.55,
15.35, 15.77, 17.49, and 18.46 ppm in Fig.
2A, which are unaffected by dipolar decoupling field
strength, are ascribed to the 13C-labeled side-chain methyl groups
of five alanine residues in the monomeric or oligomeric PH domain. The
dipole-dipole interactions between 1H and 13C nuclei
that cause serious increases in the line width of 13C signals are
decoupled by fast isotropic rotational motions of the small PH domain
particles. A peak at 11.41 ppm, indicated by an asterisk, is thought
to be an artifact because of the lack of reproducibility. An obvious increase
in line width of the peak at 16.79 ppm under the weaker decoupling field
(Fig. 2A) indicates
that this signal arises from larger complexes that undergo slow rotational
motions with a frequency lower than 105 Hz, that of
1H-13C dipolar interactions. Dynamic light scattering
measurement indicated that the [3-13C]Ala-labeled PH
domain-Ins(1,4,5)P3 complex forms two components with different
particle sizes in solution. A major component consists of particles with a
hydrodynamic radius of 1.55 nm and average molecular mass between 19
and 50 kDa. A minor component consists of particles with a hydrodynamic radius
greater than 10 nm and an average molecular mass higher than 5 MDa. The former
component is thought to comprise the monomeric, dimeric, and/or trimeric PH
domains, and the latter aggregated represents clusters of PH domains
consisting of a large number of molecules. These results support the
above-mentioned assignments of the signals to particles with different sizes.
Although the large aggregated clusters undergo slow rotational motions, these
motions are sufficiently fast to eliminate most of the dipole-dipole
interaction between 1H and 13C nuclei that is required
to form magnetization through cross-relaxation between 1H and
13C nuclei, because no signal was observed using the cross
polarization-magic angle spinning (CP-MAS) method (data not shown).
Studies of synthetic polypeptides, structural proteins, and membrane
proteins have shown that the chemical shift of the C
carbon
in the Ala residue in high resolution solid state 13C NMR is
primarily determined by the torsion angles (
,
) of the main chain of
the residue itself
(2123).
The narrow line widths and the symmetric line shape of the peaks from the
smaller particles in Fig.
2A reveal that the conformation of the Ala residues in
the monomeric PH domain and dimer and/or trimer, if any, observed by NMR are
identical, showing no structural variation that causes displacement of
chemical shifts. The buffer composition of the [3-13C]Ala-labeled
PH domain-Ins(1,4,5)P3 complex solution was the same as the
crystallization buffer used in the x-ray diffraction study in which the
three-dimensional structural model of the PH domain-Ins(1,4,5)P3
complex was determined (11).
Because the secondary structures of the PH domain-Ins(1,4,5)P3
complex in solution is expected to be similar to those of the complex in the
crystal, the narrow peaks observed in the 13C NMR spectra of the PH
domain-Ins(1,4,5)P3 complex
(Fig. 2A) were
assigned on the basis of the conformational dependence of chemical shift and
the three-dimensional model structure of the PH domain-Ins(1,4,5)P3
complex. The chemical shifts of the C
carbons of Ala residues
contained in the typical
-helix (14.9 ppm) and
-sheet (19.9 ppm)
of poly L-alanine and random coil (16.9 ppm) of the C terminus of
bacteriorhodopsin (16,
24) are shown by vertical
bars at the bottom of Fig.
2. The peaks resonated at 14. 55, 15.35, and 15.77 ppm in
Fig. 2A, similar to
the chemical shifts of the typical
-helix attributed to three Ala
residues, Ala-21, Ala-116, and Ala-118, contained in the
helices at
the N and C termini of the PH domain. The peak at 18.46 ppm can be ascribed to
the Ala-112 located at the C terminus of the
7 strand of the PH domain
based on the chemical shift similar to that of the
-sheet. The peak at
17.49 ppm, whose chemical shift does not correspond to any secondary
structure, is ascribed to Ala-88 contained in the loop between the
5 and
6 strands (the
5/
6 loop). These assignments of the peaks
were found to be consistent with the assignments of the NMR spectra of the PH
domain-PtdCho/PtdIns(4,5)P2 vesicle complex based on site-directed
replacements of the Ala residues as described below. The chemical shift of the
peak arising from the large aggregated particle, 16.79 ppm, agrees with the
chemical shift of the C-terminal random coil of bacteriorhodopsin. This signal
presumably comes from the mobile random coil moiety of the large denatured
aggregate.
High Resolution Solid State NMR Spectra of the PH
Domain-PtdIns(4,5)P2
ComplexFig. 3 (A
and B) show DD-MAS and CP-MAS NMR spectra of the
[3-13C]Ala-labeled PH domain forming a complex with
PtdIns(4,5)P2 in the PtdCho/PtdIns(4,5)P2 liposome
suspended in buffer solution at 20 °C. The chemical shifts of the signals
from the [3-13C]Ala-labeled PH domain-Ins(1,4,5)P3
complex are shown by vertical bars at the bottom of the
spectra. The signals from the lipid molecules are indicated by
asterisks. The relative intensity of the lipid signals in the CP-MAS
spectrum (Fig. 3B) is
lower than that in the DD-MAS spectrum
(Fig. 3A) because of
the lower efficiency of magnetization formation through the cross-polarization
process due to the high mobility of the lipid molecules. Chemical shifts of
the peaks at 14.41, 15.37, and 15.83 ppm in the CP-MAS NMR spectrum
(Fig. 3B) and those at
15.40 and 15.83 ppm in the DD-MAS NMR spectrum
(Fig. 3A) are very
similar to those of the peaks observed for the PH
domain-Ins(1,4,5)P3 complex (14.55, 15.35, and 15.77 ppm;
Fig. 2A). In contrast
to these signals, the signals resonated at 17.49 and 18.46 ppm in the spectra
of the PH domain-Ins(1,4,5)P3 complex
(Fig. 2A) show an
upfield displacement to 16.99 ppm and a downfield displacement up to 19.1 ppm,
respectively. To assign these signals, mutant PH domains in which Ala residues
are replaced by Gly or Leu residues are prepared.
Fig. 4 (A and
B) show the CP-MAS NMR spectra of the
[3-13C]Ala-labeled A112G mutant PH domain in which Ala-112 is
replaced by Gly and A88G mutant PH domain in which Ala-88 is replaced by Gly
forming complexes with PtdCho/PtdIns(4,5)P2 vesicles, respectively.
The signals resonating between 18.5 and 19.1 ppm (Figs.
3B and
4A) were assigned to
Ala-112 based on the disappearance of these peaks in the spectrum of A112G.
The replacement of Ala-112 by Gly induced downfield displacement of the peak
at 16.99 ppm in the CP-MAS spectrum of the wild type PH domain
(Fig. 3B) to 17.50 ppm
(Fig. 4B). The peaks
at 15.37 and 15.83 ppm in the CP-MAS NMR spectrum of the wild type PH domain
(Fig. 3B) were also
shifted in the range of 15.51 and 16.40 ppm
(Fig. 4B).
Fig. 4C shows the
CP-MAS NMR spectrum of the [3-13C]Ala-labeled A88G mutant PH
domain. The peak at 16.99 ppm in the CP-MAS spectrum of the wild type PH
domain (Figs. 3B and
4A) was assigned to
Ala-88, because this peak disappeared in the spectrum of A88G
(Fig. 4C).
Fig. 5 (AC)
shows the DD-MAS NMR spectra of A116L, A118G, and A21L mutant PH
domain-PtdCho/PtdIns(4,5)P2 vesicle complexes, respectively. The
signals resonating at 15.83 and 15.40 ppm were ascribed to Ala-116 and Ala-118
based on the disappearances of the peaks indicated by arrows in the
spectra of A116L (Fig.
5A) and A118G (Fig.
5B), respectively. As shown by a closed triangle
in Fig. 5B, a strong
suppression of the signal of Ala-112 was induced by the replacement of Ala-118
by Gly. This suppression would be caused by decrease in an efficiency of the
dipole decoupling due to an interference between the frequency of the dipole
decoupling field (55 kHz) and a newly induced thermal motion of the Ala-112
residue at a frequency around 104-105 Hz
(25). The removal of the
side-chain methyl group of Ala-118 could facilitate such a thermal motion of
Ala-112, because the side chain of Ala-118 is in van der Waals contact with
the side chain of Ala-112 in the three-dimensional model structure.
Fig. 5C shows the
DD-MAS spectrum of A21L mutant PH domain-PtdCho/PtdIns(4,5)P2
vesicle complex. A remarkably intense signal at 16.9 ppm indicated by an
open triangle was ascribed to the random coil structure of the
denatured PH domain included in the aggregated cluster similar to that
observed for the PH domain-Ins(1,4,5)P3 complex
(Fig. 2). The replacement of
Ala-21 might reduce a stability of the PH domain. Because the peaks at 15.83
and 15.40 ppm remained intact in the spectrum of A21L, the peak resonating at
14.41 ppm in the CP-MAS spectrum of the wild type PH
domain-PtdCho/PtdIns(4,5)P2 vesicle complex
(Fig. 3B) could be
ascribed to Ala-21, although an effort of direct assignment from a removal of
the peak by the replacement of Ala-21 was unsuccessful due to the intense
signal of the methyl carbon of the lipid resonated at 14.20 ppm either in the
DD-MAS or CP-MAS spectra of the A21L mutant PH
domain-PtdCho/PtdIns(4,5)P2 vesicle complex.

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FIG. 4. CP-MAS NMR spectra of the [3-13C]Ala-labeled
PLC- 1 PH domain-PtdIns(4,5)P2 complexes
measured at 20 °C. The wild-type (A) the A112G mutant
(B) and the A88G mutant (C) PH domains. The assignments of
the signals of Ala-88 and Ala-112 are shown at the top of the
spectra. The peaks arising from lipid molecules are indicated by
asterisks. The spectrum shown in A is the same as the
spectrum shown in Fig.
3B.
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The similarity in chemical shifts of Ala-116, Ala-118, and Ala-21 between
the PH domain-Ins(1,4,5)P3 complex and the PH
domain-PtdCho/PtdIns(4,5)P2 vesicle complex indicates that the
conformations of the Ala residues contained in the C- and N-terminal
helices of the PH domain are not affected by the binding of the PH domain to
PtdIns(4,5)P2 in liposomes. Conversely, the upfield displacement of
0.50 ppm from 17.49 ppm in the spectrum of the PH
domain-Ins(1,4,5)P3 complex
(Fig. 2A) observed for
the peak at 16.99 ppm in the CP-MAS spectrum
(Fig. 3B) indicates a
significant conformational change of the Ala-88 in the
5/
6 loop.
The slight upfield displacement of this signal from 16.99 ppm in the CP-MAS
NMR spectrum to 16.89 ppm in the DD-MAS NMR spectrum
(Fig. 3A) could be the
result of a minor contribution to the signal from a small amount of the
aggregated cluster of the denatured PH domain observed at 16.79 ppm in the
DD-MAS NMR spectra (Fig. 2, A and
B). The broad signal resonating between 18.5 and 19.4 ppm
in the DD-MAS and CP-MAS NMR spectra of the PH
domain-PtdCho/PtdIns(4,5)P2 vesicle complex
(Fig. 3, A and
B), corresponding to the peak of the PH
domain-Ins(1,4,5)P3 complex at 18.46 ppm
(Fig. 2A), is
attributed to the coexistence of a variety of different conformations of
Ala-112 at the C terminus of the
7 strand. Notably, the higher relative
signal intensity of the peak at 18.82 ppm in the CP-MAS NMR spectrum
(Fig. 3B) compared
with that in the DD-MAS spectrum (Fig.
3A) indicates that the Ala-112 taking the conformation
corresponding to this peak is highly immobile.
Fig. 6 (A and
B) shows the DD-MAS and CP-MAS NMR spectra of the PH
domain-PtdCho/PtdIns(4,5)P2 vesicle complex, respectively, at 4
°C (solid trace) and 20 °C (dotted trace). In the
CP-MAS NMR spectrum at 4 °C, a peak at 14.27 ppm originating from the
phospholipid methyl groups is enhanced due to the improved cross-polarization
efficiency caused by decrease in mobility of the lipid molecules at 4 °C
(Fig. 6B). In the
DD-MAS spectrum at 4 °C, the relative intensity of the Ala-112 signal
increased to form a new peak at 18.78 ppm
(Fig. 6A). In the
CP-MAS spectrum at 4 °C, the line width of the Ala-112 signal resonating
at 18.77 ppm is narrower than that in the spectrum at 20 °C
(Fig. 6B). These
changes indicate that the conformation of Ala-112 corresponding to the
chemical shift of 18.7718.78 ppm, which is most immobile at 20 °C,
becomes dominant at 4 °C.

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FIG. 6. DD-MAS (A) and CP-MAS (B) NMR spectra of the wild-type
[3-13C]Ala-labeled PLC- 1 PH
domain-PtdIns(4,5)P2 complex measured at 4 °C (solid
lines) and 20 °C (dotted lines). The peaks arising from
lipid molecules are indicated by asterisks.
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 |
DISCUSSION
|
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In this study, we aimed to gain insights into the structural features of
the PH domain at the lipid bilayer surface when the domain forms a complex
with PtdIns(4,5)P2. The PH domain of PLC-
1 forms a high
affinity complex with either PtdIns(4,5)P2 or
Ins(1,4,5)P3 with comparable binding constants
(10) through a specific
interaction between the phosphoinositol group and the side chains located at
the basic surface of the PH domain (e.g. the
1/
2 and
3/
4 loops) (11).
There is a possibility, however, that additional interactions between the PH
domain and the lipid bilayer contribute to the characteristics of the PH
domain at the membrane surface, such as binding affinity, conformation,
mobility, and orientation. Such nonspecific interactions might be responsible
for the dependence of binding affinity of the PH domain to inositol compounds
on assay conditions, such as pH, ionic composition of buffer, or lipid
composition of membrane (4).
Possible candidates for such interactions are electrostatic interactions
between the positively charged surface of the PH domain, which is not involved
in the specific interaction with PtdIns(4,5)P2 and the lipid head
groups, and hydrophobic interactions between the hydrophobic surfaces of the
PH domain and the hydrophobic inner layer of the lipid bilayer. These
interactions, if any, would modify the conformation, dynamics, and orientation
of the PH domain at the lipid bilayer surface, which in turn, would influence
lipid-protein and protein-protein interactions at the membrane surface
involved in signal transduction pathways. In the case of multidomain proteins
such as PLC-
1, which contains the PH domain, EF-hands, an active site
domain and a C2 domain, intramolecular interaction between the domains would
also be affected by the conformational characteristics of the domains at the
membrane surfaces.
As shown in Fig. 3, changes
in the 13C NMR spectra of the [3-13C]Ala-labeled
PLC-
1 PH domain clearly revealed conformational changes induced by
localization of the domain at the surface of the
PtdCho/PtdIns(4,5)P2 vesicle. The changes in the PH domain
structure were found to occur in the vicinity of the
5/
6 loop
containing Ala-88 and the C terminus of the
7 strand containing Ala-112.
In contrast, the N- and C-terminal
-helices located at the surface
opposite to the ligand binding site of the PH domain showed virtually no
conformational change.
The side chains of Leu-84 and Ala-88 in the
5/
6 loop and
Ile-111 in the
7 strand form a hydrophobic cluster between the
-helix in the
5/
6 loop (
2-helix;
Fig. 7A) and the
7 strand according to the three-dimensional model structure of the PH
domain-Ins(1,4,5)P3 complex
(11). The proximity of Ala-88
and Ala-112 in the model structure suggests a relationship between these
residues in the conformational changes. The origin of the upfield displacement
of the Ala-88 signal is ascribed to a direct interaction between the
2-helix and the membrane, considering the close location of the
5/
6 loop to the positively charged lipid-binding surface of the PH
domain (Fig. 7A).
Taking into account the highly amphipathic nature of the
2-helix (as
shown in Fig. 7B by a
helical wheel), it is plausible to predict a hydrophobic interaction
between the hydrophobic face of the
2-helix and the hydrophobic inner
layer of the lipid bilayer. In fact, x-ray diffraction studies of membrane
binding states of synthetic model peptides
(26,
27) have suggested that such a
hydrophobic interaction facilitates the orientation of an amphipathic
-helix parallel to the membrane plane at the interface between the
layer of the polar head groups and the inner layer of the membrane. The
reorientation of the
2-helix is thought to cause an opening of the
hydrophobic cluster between the
2-helix and the
7 strand
(Fig. 7C). As a
result, hydrophobic interaction between the newly exposed hydrophobic surface
and the inner layer of the lipid bilayer may induce tilted orientation of the
PH domain (Fig. 7C,
right). The downfield displacement of the Ala-112 signal to the
chemical shift of the typical
sheet, together with the reduction of
mobility at the membrane surface, suggests the formation of an additional
hydrogen bond at the C terminus of the
7 strand between the carbonyl
group of Ala-112 and the amide group of Arg-95. The formation of a new
hydrogen bond elongates the anti-parallel
sheet structure of the
6 and
7 strands and stabilizes it
(Fig. 8). This conformational
change is consistent with the above-mentioned "open and tilt"
model. The opening of the
5/
6 loop would result in the breakage of
a hydrogen bond between the carbonyl group of Pro-92 and the amide group of
Arg-95 contained in the
turn connecting the
5/
6 loop and
the
7 strand. As shown in Fig.
8, the rupture of this hydrogen bond is expected to facilitate
formation of a hydrogen bond between the amide group of Arg-95 and the
carbonyl group of Ala-112. Because only changes of three torsion angles,
of Asp-94 and
and
of Arg-95, are required for the
reorientation of the amide group of Arg-95, this reconstitution of the
hydrogen bond is expected to occur readily. The chemical shift of the Ala-112
signal at 4 °C (18.7718.78 ppm;
Fig. 6) reflects the
conformation with the lowest energy at the membrane surface that might include
the hydrogen bond between Arg-95 and Ala-112. Another reason for the origin of
the conformational change at the C terminus of the
7 strand is
interaction between the
6/
7 loop located at the positively charged
surface of the PH domain and the polar head groups of the lipids. Because the
6/
7 loop does not contribute to the specific interaction between
the PH domain and Ins(1,4,5)P3 in the model structure, except by an
indirect hydrogen bond between the carbonyl group of Thr-107 and the
4-phosphate of Ins(1,4,5)P3, nonspecific electrostatic interactions
between the charged side chains of this loop (Lys-102, Asp-103, and Arg-105)
and the polar head groups of phospholipids are likely to occur at the membrane
surface. A variety of these nonspecific interactions might induce a variety of
conformations of Ala-112 at the C terminus of the
7 strand through
conformational change of the main chains of the
6 and
7
strands.
The nonspecific hydrophobic or electrostatic interactions described above
would contribute to the affinity of the PLC-
1PH domain to
PtdIns(4,5)P2 in the membrane. These auxiliary mechanisms for
membrane binding are probably susceptible to assay conditions, such as the
structures of the hydrophobic acyl-chains of the lipids, surface charges of
the membrane, pH value, and ionic strength of the buffer
(4). For example, although it
has been reported that the Kd value of the
PLC-
1PH domain-PtdIns(4,5)P2 interaction measured using
dimyristoyl phosphatidylcholine (DMPC)-PtdIns(4,5)P2 vesicles is
1.66 µM, eight times larger than that of the PLC-
1 PH
domain-Ins(1,4,5)P3 interaction (210 nM)
(10), the affinity of the
PLC-
1 PH domain to PtdIns(4,5)P2 in the natural membrane
could be different and might be higher than that observed for the DMPC vesicle
system due to the presence of unsaturated acyl chains.
Although more detailed structural information about the membrane binding
state of the PH domain is required to judge the validity of these models, the
results of the solid state NMR experiments clearly indicate that the
PLC-
1 PH domain has an unique conformation and dynamics at the lipid
bilayer surface, which are different from those in solution. The structural
information at the membrane surface is indispensable for gaining insights into
the molecular mechanisms of the functions of peripheral membrane proteins. Our
results also proved that solid state NMR spectroscopy is a powerful tool for
obtaining structural information about peripheral membrane proteins at the
membrane surface under conditions mimicking physiological conditions.
 |
FOOTNOTES
|
---|
* This work was supported in part by a Grant-in-aid for Scientific Research
13033035 from the Ministry of Education, Culture, Sports, Science and
Technology of Japan (to H. Y.). The costs of publication of this article were
defrayed in part by the payment of page charges. This article must therefore
be hereby marked "advertisement" in accordance with 18
U.S.C. Section 1734 solely to indicate this fact. 
To whom correspondence should be addressed. Tel.: 81-791-58-0180; Fax:
81-791-58-0182; E-mail:
tuzi{at}sci.himeji-tech.ac.jp.
1 The abbreviations used are: PH, pleckstrin homology; PLC, phospholipase C;
PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate;
Ins(1,4,5)P3, D-myo-inositol
1,4,5-trisphosphate; PtdCho, phosphatidylcholine; DD-MAS, dipolar
decoupled-magic angle spinning; CP-MAS, cross polarization-magic angle
spinning; TMS, tetramethylsilane; DMPC, dimyristoyl phosphatidylcholine; GST,
glutathione S-transferase; MES, 4-morpholineethanesulfonic acid. 
 |
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