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INTRODUCTION |
G protein-coupled receptor kinases
(GRKs)1 are a unique family
of serine-threonine kinases, which are responsible for
activator-dependent phosphorylation of G protein receptors
and provide rapid desensitization of the agonist occupied receptors
(1). The GRKs are recognized to have three functional components: an
N-terminal section believed to interact directly with the
seven-trans-membrane helical receptor protein and/or other membrane
targets, a central section, which is the catalytic domain, and a
C-terminal section containing a generally conserved autophosphorylation
region and a variable region that mediates membrane association by
various means. In GRK2 (also known as
-adrenergic receptor kinase-1)
or GRK3 (
-adrenergic receptor kinase-2), the C-terminal variable
region contains a pleckstrin homology (PH) domain (2, 3), conferring
binding specificity to G
proteins (reviewed in Ref.
1).
The PH domain family (reviewed in Refs. 4-6) appears to be a very
large family of structurally homologous protein domains of moderate to
low sequence similarity. The PH domain is believed to play a role in
intracellular signal transduction, and the functional role of the PH
domain has been characterized for several systems. In phospholipase
C
, the PH domain has a high affinity (Kd < 1 µM) site for phosphatidylinositol 4,5-bisphosphate
(PI(4,5)P2) and inositol 1,4,5-trisphosphate
(Ins(1,4,5)P3) (7), which forms a crystallographically
observed, well defined structural interaction (8). Other PH domains
have different lipid specificities, and a well defined set of binding
motifs does not readily emerge (9-15). One hypothesis is that PH
domains present a framework with a polymorphic surface used for
specific recognition, analogous to immunoglobulins (5, 9). In addition,
the overall fold of the PH domain was observed to be common with that
of the PTB (phosphotyrosine binding) domain (16, 17), a protein domain that shows little sequence homology to PH domains. In light of these
developments, it is of significance to establish whether the nominal PH
domain of GRK-2 (
-adrenergic receptor kinase (
ARK-1)), which
clearly binds (both in vivo and in vitro) to a
protein partner, G
subunits of the heterotrimeric G
protein family (18), truly has the common structural motif of the
PH/PTB domains, and what the relationship of putative lipid and protein
binding sites might be in such a structure.
In this paper, we present the solution structure of an extended PH
domain from human
ARK1, determined at 0.4 Å r.m.s.d. by high
resolution NMR using heteronuclear triple resonance methods. Although
the overall fold and topology clearly establishes that the
ARK1
extended PH domain is of the same family as other PH domains, there are
several significant alterations (most notably an extension of the
C-terminal
-helix, which in solution presents as a "molten
helix" having a clear gradient of mobility) on the subnanosecond, as
well as millisecond to microsecond, time scales, increasing toward the
free C terminus. The polarity of the surface charge observed in other
PH domains is altered by positively charged residues in the extended
-helix. This unusual clustering may be complemented by a highly
negatively charged area on G
subunits. Although a
direct study of the G
/PH domain complex is beyond the
range of current NMR technology, the structure presented here supports
a model in which the C-terminal portion of
ARK PH domain in
particular, and PH domains in general, participate in protein-protein
interactions.
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MATERIALS AND METHODS |
Sample Preparation--
Recombinant human
ARK PH domain
(h
ARK1-(556-670)) was obtained by GST fusion expression from
pGEX-2T (Pharmacia Biotech Inc.) in BL21 (DE3) Escherichia
coli cells (Novagen, Madison, WI) and subsequent bacterial
expression and protein purification as described previously (19) on a
larger scale. The full-length h
ARK1 cDNA clone was provided by
Dr. Antonio DeBlasi (Mario Negri Sud, Santa Maria Imbaro, Italy). The
sequence of the 119-residue construct used in the present study is
shown in Fig. 1. It contains both the PH domain and the
G
-binding motif (20). The first four residues are not
from the natural sequence. Uniform 15N and
15N,13C labeling was achieved by growing the
cells in M9 minimal medium using standard procedures.
Solutions used for NMR studies contained 1-2 mM protein in
10 mM acetate buffer at pH 4.5 (uncorrected for isotope
effects), 0.02% sodium azide, 1 mM
[U-2H]EDTA, 5 mM
[U-2H]dithiothreitol, and 10%
2H2O in the H2O samples. These low
salt and low pH conditions were necessary to prevent protein
aggregation. CD data indicated no changes in the protein secondary
structure, between the buffer used for NMR studies and
phosphate-buffered saline, pH 7.2. The external 1H chemical
shift reference used was sodium 2,2-dimethyl-2-silapentane-5-sulfonate, and indirect referencing was used for 15N (21) and
13C. Spectra were essentially identical among several
preparations of the PH domain.
NMR Spectroscopy--
NMR experiments were run on Bruker DMX-500
and DMX-600 spectrometers. Quadrature detection was achieved by the
States or States-time proportional phase incrementation methods. Some
of the pulse schemes implemented pulse field gradients for coherence
selection (HCCH-TOCSY, 13C-separated NOESY-HMQC), and some
used the sensitivity enhancement method (HSQC, heteronuclear NOE) (22).
The water signal was suppressed either by the WATERGATE method (23) or
by using selective on-resonance irradiation during a relaxation delay
of ~1.3 s. Experiments were run at 35 °C with sweep widths of 8000 and 2000 Hz for 1H and 15N (at 600 MHz),
respectively, unless indicated otherwise.
The homonuclear experiments, HOHAHA and NOESY, were run in both
H2O and 2H2O using standard pulse
sequences and phase cycling. A range of t1
increments from 200 to 512, each consisting of 2048 complex points, was
typically acquired with 32-128 scans/increment.
The heteronuclear experiments consisted of two-dimensional HSQC
(1H-15N and 1H-13C),
HSQC-J, and HTQC, three-dimensional CBCA(CO)NH, CBCANH, HNCA, HN(CO)CA,
HNCO, HCCH-TOCSY, and 13C-separated NOESY-HMQC, and two-
and three-dimensional 15N-separated NOESY-HMQC (in
H2O and in 2H2O) and HOHAHA-HMQC.
The mixing time in the NOESY-HMQC experiments was 100 and 150 ms, and
the spin lock duration was 30 ms in the HOHAHA-HMQC and 19 ms and 6 ms
in the HCCH-TOCSY. The three-dimensional spectra were recorded with a
32 × 100 × 1000 hypercomplex matrix, with 32 scans/increment. The degree of amide hydrogen protection was assessed
(a) by measuring hydrogen-deuterium exchange rates by
following the intensity of cross-peaks in HMQC experiments after
exchanging a fully protonated, lyophilized sample with 99.996% 2H2O, and (b) by comparison of
cross-peak intensities in two HSQC experiments, with and without water
presaturation (24). The three-bond H
-H
coupling was assessed by the
method of (25). Heteronuclear 15N{1H} NOEs
were measured using standard methods as described elsewhere (26).
Two-dimensional H2O-selective heteronuclear
15N-edited ROESY experiments (27) were performed to map
those amide hydrogens in the
ARK PH domain that are exposed to and interacting with water molecules. Signal processing and assignment were
done as discussed previously (26, 28).
Structure Calculation--
Structure calculations used DIANA
with REDAC strategy (29) or DYANA (30) with ECEPP stereochemistry, with
structurally significant constraints of 1956 upper and 76 lower
distance bounds (from ~3000 NOEs), 38 hydrogen bonds chosen within
strands or helix with slowed exchange, 99
-angle constraints derived
from 3JHNH
coupling constants,
and 99
-angle constraints (derived from C
chemical shift data)
corresponding to conservative ranges of allowed torsion angles, in
those regions of strand or helix that were well defined (Fig. 2). All
peptide bonds were assumed to be trans. A final selection of
20 structures from 400 starting structures was done by using the lowest
target functions (the ensemble statistics are shown in Table
I). DIANA and DYANA use no assumptions
about protein energetics, other than van der Waals repulsion;
structures are unrefined and only adjusted by rotation/translation for
comparison purposes. Structures were aligned using XPLOR or in-house
software written in MATLAB (MathWorks) and displayed and analyzed with
the INSIGHTII package (Biosym) or with MOLMOL (31).
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Table I
Root-mean-square deviation from the mean structure calculated for the
ensemble of 20 structures of the ARK1 PH domain
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Alignment of the PH/PTB Domains--
Pairwise comparison of the
PH/PTB domain structures was performed by superposition of the backbone
heavy atoms (N, C
, C
, and O) of the residues from regions of
regular secondary structure, as indicated by boxes in Table II. The
-helical insertions in the loop regions of the PH domains were not
taken into account. The alignment was done by direct calculation of
r.m.s.d. values and optimized by relative shift of the protein
sequences within each secondary structure element (
1-
7 strands,
-helix), as well as by adding or removing individual residues.
The resulting alignment and r.m.s.d. values are presented in Tables II
and III, respectively.
Protein Backbone Dynamics--
The backbone dynamics were
assessed via 15N spin relaxation studies comprising
T1, T2, and heteronuclear
steady state NOE measurements using previously described protocols
(26). Fifteen two-dimensional spectra with the relaxation delays of 4 (×2), 200, 400 (×2), 600, 900 (×2), and 1200 ms (positive initial
15N magnetization), and 4, 200 (×2), 400, 600 (×2), and
900 ms (negative initial 15N magnetization) were acquired
in the alternate-sign T1 experiment (duplicate
experiments are indicated by ×2) (26). Eleven two-dimensional spectra
were collected for the T2 measurements, with the
relaxation delays of 8 (×2), 16, 32 (×2), 48, 64 (×2), 80, 96 (×2),
112, 128 (×2), and 160 ms. The heteronuclear
{1H}15N steady state NOEs were assessed as
a ratio of cross-peak intensities in the experiments with and without
proton presaturation. The relaxation data analysis was performed using
programs RELAXFIT and DYNAMICS (26), extended to include anisotropic
character of the overall motion of the protein (32).
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RESULTS |
h
ARK-PH domain corresponding to residues 556-670 of human
ARK1 (Fig. 1) was produced in E. coli and isolated as a GST fusion protein, cleaved, and purified.
Solubility and stability limited observation to a narrow range of
conditions, and the majority of studies were conducted in 20 mM acetate buffer at pH 4.5, 35 °C. Under these
conditions, binding of the construct to G
is
maintained (data not shown). The 546-670 construct was also produced,
and NMR spectra indicated that the additional N-terminal residues did
not belong to the domain fold, and were apparently unstructured. It was
concluded that the first construct contained the essential domain.
Assignment used standard triple resonance methods, complemented by
study of the [U 13C, 15N; 12C,
14N-Met]PH domain to help identify methionine residues
that underwent partial oxidation during sample preparation. Assignment
and NOE data are summarized in Fig.
2.

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Fig. 1.
The sequence of the protein construct used in
the present work and its relation to the nominal PH domain and to the
G -binding region of ARK1. At the
top is the nominal length PH domain section; below is the
domain demonstrated previously (50) to be sufficient and optimal for
G binding, below which is the construct used here,
which has the same G binding. The lowercase
"gshm" residues are from the GST construct, and are not
further referred to. At the bottom, the complete sequence of
h ARK1 PH domain, and the similar h ARK2 are compared, with the
secondary structural elements of h ARK1 superimposed in color. The
more flexible region of the C-terminal -helix is shown in light blue.
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Fig. 2.
Summary of NMR data used for identification
of the secondary structure. From top to bottom, deviations from
standard chemical shift (in random coil peptides) (53, 54), of
-hydrogens ( H ), - and -carbons ( C , C ,
C - C ), and carbonyl carbons ( C ) (54);
3JHNH , the magnitude (in Hz) of
the three-bond scalar (intraresidue) spin-spin coupling between the
- and the amide hydrogens; intensities of the NOE cross-peaks (on an
arbitrary log scale) between the - and amide hydrogens,
d ,N(i,i+1), and between
amide hydrogens, dN,N(i,i+1), of the
adjacent residues; horizontal bars indicate NOEs observed
between the - and amide hydrogens three
(d ,N(i,i+3)) or four
(d ,N(i,i+4)) residues
apart, and between the - and -hydrogens three residues apart
(d , (i,i+3))
characteristic for the -helix; heteronuclear
15N{1H} steady state NOE;
circles indicate amides protected from exchange with
(solid circles) or exposed to (open circles)
solvent. -Helices are typically characterized by H < 0, C > 0, C < 0, and C > 0, whereas chemical shift
deviations of the opposite sign are expected for the -strands (54).
3JHNH is directly related to the
intervening torsion angle , so that
3JHNH < 6 Hz is characteristic
of the -helix. Based on all the data, seven -strands ( 1,
561-568, 2, 578-584; 3, 587-591; 4, 600-602; 5,
606-613; 6, 618-624; 7, 628-634) and a C-terminal -helix
(residues 638-655) were identified. A representation of the secondary
structure of the ARK PH construct is shown on the bottom.
Shaded in gray is the C-terminal extension of the
-helix (656-658) where some of the helical features are still
preserved (see text), characteristic of a "molten" helix.
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In Figs. 3 and
4, the overall fold and the electrostatic
potential of the h
ARK PH domain are shown, along with the PH domain of PLC
(8) for comparison. The topology of the fold is typical for
PH domains, and consists of seven
-strands forming a
-sandwich flanked on one end by a C-terminal
-helix. The termini of the construct are disordered and highly flexible, with large amplitudes of
backbone motion on a nanosecond time scale (Fig. 3). The
1/
2 and
3/
4 loops are disordered and display increased amplitudes of
backbone dynamics on a subnanosecond to nanosecond time scale, as well
as motions on a millisecond to microsecond time range (data not
shown).

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Fig. 3.
Three-dimensional structure of the ARK1 PH
domain. A, view of the backbone (N, C , C ) of 20 superimposed NMR-derived structures of the ARK1 PH domain. Parts of
the backbone belonging to the elements of secondary structure are
colored ( -strands, yellow; -helix, blue)
and labeled. The termini of the construct and some residues in the
loops 1/ 2 and 3/ 4 are disordered (see also D and
E). B, a ribbon representation of the tertiary solution structure of the ARK1 PH domain, the same orientation as in
A. C, the structure of PLC H domain from x-ray
diffraction (8). D and E, ribbon representation
of the backbone of the ARK1 PH domain. The ribbon width in
D represents local backbone r.m.s.d. values in the ensemble
of 20 calculated structures and, in E, the amplitude of
nanosecond-subnanosecond backbone motion (as inferred from
1-S2, S is the order parameter). The
15N relaxation data indicate a dynamic character of
structural disorder in the N and C termini (S2 < 0.5 for the backbone NH groups in residues 552-557 and 659-670) and in the loops 1/ 2 and 3/ 4. The backbone mobility in the elements of secondary structure is restricted
(S2 > 0.85, local correlation time in a
subnanosecond time range). The ratio of the principal components of the
rotational diffusion tensor of the molecule is
Dz/Dx = 1.30; the z
axis of the diffusion tensor is tilted by ~20° angle from the C-terminal helix axis. Residues 556-670 ( ARK1 PH domain) are represented in A, 556-664 in B and D.
Unanalyzed residues (unassigned or with insufficiently resolved
1H-15N correlations) are colored
gray in E. The drawings were performed with
INSIGHTII (Biosym) (A, D, and E) and
with SETOR (55) (B and C).
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Fig. 4.
The effect of the C terminus on the
electrostatic potential of the h ARK1 PH domain, and comparison with
the PLC PH domain. Surfaces are contoured at 2
kT/e (red) and 2 kT/e (blue) (GRASP; Ref. 56) for
various lengths of the C-terminal extension: a, full-length
construct, 556-670; b, residues 556-666; c,
residues 556-661; d, residues 556-656; e,
nominal PH domain, residues 556-651. The ARK1 PH domain constructs
in b-d correspond to C-terminal deletion studies of
G binding (b and c (50) and
d (19)). The most C-terminal residues, upon truncation, are
indicated. For comparison, the electrostatic potential of the PH domain
from PLC (Protein Data Bank entry 1MAI) is shown in f;
the arrow indicates a positively charged area at the opening
of the -barrel, which is involved in the phospholipid binding (8).
The molecular orientations are similar, as indicated by the backbone
tube diagrams.
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Of specific note, the C-terminal
-helix is clearly extended by more
than one turn compared with C-terminal
-helices of most previously
determined PH domain structures. The position/orientation of the helix
appears to be fixed by interactions with the protein core, namely by a
hydrophobic strip formed by Leu-640, Trp-643, Leu-647, Ala-650,
Tyr-651, and Ala-654, which are located on the side of the helix facing
the
-sandwich and are involved in contacts with several residues in
the first two
-strands. The aromatic ring of Trp-643, the only
conserved residue among PH domains, is buried in the protein core and
exhibits numerous NOE contacts to residues in
1 and
2. Another
aromatic residue in the helix, Tyr-651, is also oriented toward the
interior of the
-sandwich. The NOESY data indicate several close
contacts between the aromatic ring of Tyr-651 and the residues in the
4/
5 loop and in the strand
5. Both the structure of this loop
and the orientation of the Tyr-651 ring are well defined, as indicated
by low r.m.s.d. values in these parts of the structure, and by chemical
shift non-equivalence of all four ring hydrogens of Tyr-651.
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DISCUSSION |
Relation to Other PH/PTB Domain Structures--
The h
ARK1 PH
domain has very low sequence similarity to other PH domains of known
three-dimensional structure, and therefore cannot be satisfactorily
homology-modeled from known structures. The structure-based alignment
of the h
ARK1 PH with these PH domains (Tables
II and III)
demonstrates the same overall topology of the protein fold. The
expected range of r.m.s.d. values between sequences of the same
structural class, but with varying degrees of homology, has been
derived previously (33). The r.m.s.d. values between different members
of the PH/PTB domain family (Fig. 7) are
within the range expected for such homologous sequences of low
identity. The charge distribution is, however, different among the PH
domains, and the large positive charge associated with the C-terminal
helix of
ARK is unusual.
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Table II
Structure-based alignment of the PH/PTB domains
Domains are as follows (top to bottom): GRK-2 (this work); human
dynamin PH domain (35); PH domain of human SOS (41); PH domain of mouse
-spectrin (36); PH domain of Drosophila -spectrin
(37); N-terminal PH domain from pleckstrin (38); PLC- PH domain (8);
PTB domain of Shc (16); PTB domain of IRS (17). The alignment was done
by pair-wise superposition of the structures and direct calculation of
aligned RMSD (Table III), based on the elements of regular secondary
structure, as described in the text. Residues belonging to the elements
of secondary structure used for the alignment are enclosed in boxes,
labeled on the top. Numbers indicate residue positions within the
corresponding secondary structure element. Conserved hydrophobic
residues are colored green. The only conserved residue in PH domains,
Trp 1-7, is colored blue and underlined. In the Shc PTB domain,
residues 58-114, belonging to an insertion in the 1/ 2 loop are
omitted.
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Table III
Pairwise root-mean-square deviations (in Å) between the PH/PTB domain
structures
The elements of regular secondary structure of the proteins used for
the comparison and their alignment are indicated in Table II. Numbers
shown above the diagonal were obtained with only C atoms selected
for the alignment and r.m.s.d. evaluation, whereas numbers below the
diagonal correspond to all heavy backbone atoms (N, C , C , and O) in
the selected core elements taken into account. The percent of sequence
identity between core elements of the compared proteins is indicated in
the parentheses. PH/PTB domain notation is the same as in Table 2. Protein atom coordinates were obtained from the Protein Data Bank, PDB
entries 1DYN (dynamin), 1AWE (hSOS), 1PLS (pleckstrin), 1BTN
( -spectrin, mouse), 1DRO ( -spectrin, Drosophila), 1MAI (PLC ),
1SHC (Shc PTB), and 1IRS (IRS
PTB).
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Validity of the Derived Structure of the
ARK PH Domain--
The
low pH (4.5) required for this study is close to the
pKa values for both glutamate and aspartate, so
variations in the side chain charges of these residues compared with
physiological conditions are expected. Since this might result in a
perturbed structure in those regions containing negatively charged
residues, a question arises of whether the NMR structure derived under
these conditions represents the protein structure under physiological conditions. As mentioned above, the circular dichroism data indicate no
changes in the protein structure as compared with the more physiological conditions in a phosphate-buffered saline pH 7.2. To
address this issue in greater detail, the
1H-15N correlation maps (HSQC) were also
recorded for the PH domain dissolved in the phosphate-buffered saline
(pH 6.0, temperature 25 °C) or in 0.1 M Tris buffer (pH
7.9, 35 °C). The minor chemical shift changes (up to 0.06 ppm in
1H and 0.6 ppm in 15N) are consistent with
expectations of variations in pH, temperature, and buffer content. The
absence of significant chemical shift perturbations in this fingerprint
region suggests no significant changes in the protein structure. The
ARK PH domain tertiary structure here is also generally similar to
other PH domain structures measured in the range pH 6.0-9.0 (8, 11,
34-39). The high flexibility of the C terminus in the extended
ARK
PH domain construct reported here is also preserved at the more
physiological conditions, as indicated by negative steady-state
heteronuclear NOEs observed for the C-terminal residues (663-670) in
phosphate-buffered saline (pH 6.0, 25 °C). The binding to
G
subunits is also retained at pH 4.5, with c. 100 nM affinity of the GST fusion protein at pH 4.5 and 7.5, from an immunoblotted Western assay (19).
Structure of the C-terminal Extension: Molten Helix--
The
C-terminal segment shows an unusual structural feature that, to our
knowledge, has not been reported previously in proteins. NOEs
characteristic for an
-helix are preserved for residues toward the C
terminus despite the gradual loss of other NMR characteristics of
helical structure (deviations from standard chemical shifts, heteronuclear 15N{1H} NOEs,
3JHNH
coupling) (Fig. 2).
Increased mobility is indicated by both relaxation and solvent
exchange/accessibility data, suggesting that the C-terminal part of the
-helix is present as a "molten helix" in solution. Molecular
dynamics calculations (40) of
-helical melting appear to be
qualitatively consistent with our NMR observations.
The hydrophobic residues C-terminal to Gln-656 (Leu-657, Val-658, and
Val-661) are located at proper sequence positions to extend the already
existing hydrophobic strip on the helix surface. However, being
extended beyond possible interaction with the protein core's
-sandwich and therefore exposed to solvent, these residues lack
proper hydrophobic contacts to other residues that might stabilize the
helix formation. It is possible, however, that the C-terminal extension
of the helix becomes structured under certain conditions,
e.g. in the presence of a binding partner such as G
subunits. It is possible also, that the apparent
"molten helix" is a consequence of the expression of the C-terminal
region of
ARK in the absence of its native protein context, and
therefore might be stabilized through tertiary contacts with residues
N-terminal to the PH domain. It is unlikely, however, that the
thermodynamic stability of the protein is a result of special
conditions of this study (low salt, pH4.5), since both the CD and NMR
data (see above) indicate no significant perturbations in the
ARK PH
domain structure upon exchange into phosphate buffer.
In the human SOS PH domain, residues N-terminal to the normal PH domain
(in the Dbl homology domain) make well defined structural contacts with
the PH domain, (41) and in the Btk PH domain, the "Btk motif"
C-terminal to its nominal PH domain packs against the C-terminal
-sheet (39).
Binding of Phosphatidylinositides and Inositol Phosphates--
A
general hypothesis that has been advanced in the literature is that the
PH domain recognizes specifically and with high affinity highly anionic
phospholipids, especially phosphatidylinositol (4, 5) bisphosphate
(42). This has been illustrated for the PLC
PH domain, in which the
binding of PI(4,5)P2 and Ins(1,4,5)P3 is
submicromolar, and a well defined structural interaction with the PH
domain has been characterized (8). However, other PH domains appear to
have significantly weaker affinity (11, 12, 42). There is also
sensitivity to the isomer identity of the inositide, since the PH
domain of Akt (13, 14) binds to phosphatidylinositol 3,4-bisphosphate,
and a newly identified GRP1 binds to phosphatidylinositol 3,4,5-triphosphate (43). It is evident that the basic residues in the
N-terminal section involved in the PLC
PH domain/ligand complex (8)
are not generally present in the structure-based sequential alignment
of Table II. The physiological relevance of phosphatidylinositol
phosphates and phosphoinositides binding to PH domains remains
unresolved for the PH domains of
-spectrin, N-pleckstrin, dynamin,
and
ARK.
Possible PI(4,5)P2 Binding Site on
ARK-1 PH Domain
and Its Significance--
Using Ins(1,4,5)P3 as a model
compound for PI(4,5)P2, the 15N and
1H spectral perturbations upon titration were mapped for
h
ARK-1 PH domain using a previously published procedure (12). Under the experimental conditions, the
ARK PH domain binds
Ins(1,4,5)P3 with Kd of 207 µM, according to our protein fluorescence titration
measurements using the protocol described in Ref. 12. The data (not
shown) indicated maximal shift perturbations at residues Gly-569,
Trp-576, Arg-578
, Tyr-580, and Ala-596, located in the N-terminal
segment of the domain, a pattern seen similar to, but different in
detail from, other PH domains. The amide 1H chemical shift
of Asp-635 is also perturbed (0.02 ppm), that is probably caused by
variation in the distance between this site and the closely located (in
the unbound state) positively charged side chain of Arg-578.
The association of
ARK with membranes is complex. It has been
suggested (44) that the high affinity binding of
ARK to microsomal
membranes depends on a segment of the N terminus of
ARK, distinct
from the putative PH domain. However, other investigators have shown
that the
ARK PH domain binds with moderate specificity to
PI(4,5)P2 and suggest that synergistic interactions of
binding to both PI(4,5)P2 and to G
proteins via the PH domain are required for activation of
ARK. (45).
This synergism was not observed in a model system of higher turnover,
where PI(4,5)P2 was inhibitory (46).
Residues perturbed by the Ins(1,4,5)P3 binding are located
mostly in the
1/
2 loop and in the N-terminal part of the
2-strand. This rather flexible loop is relatively distant from both
the putative G
binding site and the area of
hydrophobic contacts between the
-helix and the protein core. The
present data provide no direct structural evidence for a possible
relationship between the phospholipid and G
binding
regions.
Protein Interaction--
The clustering of positive residues in
the extended C-terminal helix creates a positively charged site on the
ARK PH domain surface (Fig. 4, a-e), in addition to a
cluster of positive charges at the opening of the
-barrel, a site
implicated for phospholipid binding (11) in other PH domains (Fig.
4f). This causes a different polarity of the surface charge
in the case of
ARK PH domain, so that in the fully extended
construct, the dipole moment of the molecule is aligned at ~30°
angle relative to the helical axis (Fig.
5). Truncation of a few C-terminal
residues causes a 2.5-fold reduction in the protein dipole moment and
alters the orientation of the dipole vector to close to perpendicular
to the helix axis. Further truncation causes only minor variations of
the dipole vector. This reduction could explain the observed differences of G
binding to truncated
ARK1 PH
domains (19).

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Fig. 5.
The dipole moment as a function of C-terminal
truncation. The magnitude of the dipole moment
(A) and its orientation (B) relative to the
-helix (the polar angle between the dipole vector and the -helix
axis), for various lengths of the C-terminal extension in ARK1 PH
domain. Upon truncation, the C terminus was capped with the COO-group.
The following partial charges were assigned to the side chain atoms: +1
(NZ in Lys), +0.5 (NH2, NH3 in Arg), 0.5 (OG1, OG2 in Asp and OD1,
OD2 in Glu) (56), and the protein mass centroid was used as the origin
(57). Each peptide bond was assigned a dipole moment of 3.5 Debye
aligned along the CO bond (57, 58). To account for the observed
flexibility in the loops and in the termini, the results are the
average of the values from the ensemble of 20 ARK PH domain
structures (Fig. 3A). Similar results were obtained using a
much larger ensemble of 100 NMR-derived lowest-target-function
structures from the same distance-geometry calculation. Positions
corresponding to the charged residues are labeled in A.
Insets show the orientation and relative strength (in
arbitrary units) of the effective electric dipole vector, colored
green, in the full-length (B) and in the residue
556-656 construct (A).
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A depression on the
ARK PH domain surface between the
-helix and
the
5-strand, flanked by positively charged side chains of Lys-644,
Lys-645, Arg-648, and Arg-652 (helix); Lys-623 (
6); and Arg-625
(
6/
7) (Fig. 4) resembles the site in the PTB domains involved in
the phosphopeptide binding of that protein. This topological similarity
suggests that these residues might be involved in electrostatic interaction with a negatively charged cluster on the G
surface formed by Glu-226, Asp-228, Asp-246, and Asp-247 of the WD5 and Asp-267, Asp-290, and Asp-291 of the WD6 subunits (Fig.
6). With the exception of the Asp-247 and
Asp-291, which are highly conserved among the WD40 subunits and are
involved in the formation of an inter- and intra-blade hydrogen bonds
(47, 48), other negatively charged residues in this cluster are unique
for WD5 and WD6, and highly conserved in eukaryotes. Asp-228 and
Asp-246 are directly involved in ionic interactions with the switch-II
region (
-Lys-210, Fig. 6) of G
, which plays a
critical role in G protein heterotrimer formation (47). It is possible
that in the absence of G
the PH domain of
ARK
interacts (via its positively charged C terminus) with the same
residues on the top of G
surface and therefore either
directly or indirectly interferes with G
binding to
G
.

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Fig. 6.
Electrostatic polarization of the
G heterodimer. A, molecular surface of the
G complex colored by the electrostatic potential,
from red ( 10 kT/e) to blue (+10 kT/e). A fragment of G (green)
contains the switch II region ( 2 helix), which is intimately
involved in a direct contact with the top of the G
propeller in the heterotrimer (47, 48). Those positively charged
residues of G (Lys-209, Lys-210) in the
G  complex less than ~3 Å between side chain heavy atoms from Asp-228 and Asp-246 of G are labeled.
The highly negatively charged area around this position may serve as a
complementary charged surface to the ARK1 PH domain. B, a
diagrammatic representation of the location of negative charges on the
G surface. The surface corresponding to the highly negatively charged area in A is indicated by a broken
line. The general sequence consensus of a WD motif and location of
negatively charged residues in the sequences of WD5 and WD6 subunits
are also shown (B, top). The residues in the WD
repeat sequence are marked: x, a non-conserved position;
h, a conserved hydrophobic position; r, a
conserved aromatic; p, a conserved polar position; t, a tight turn containing Gly, Pro, Asp, or Asn. The
superscripts indicate the range of residues observed in the various
known G subunits. Those acidic residues that are unique
for the WD5 and WD6 subunits are shaded in gray.
A was generated using GRASP (56); atom coordinates were
extracted from PDB entry 1GG2; the schematic representation of
G and the general sequence consensus of WD motifs were
adopted from Ref. 59.
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The
ARK2 (GRK-3) PH domain has a different receptor and
G
selectivity, but possesses a similar pattern of
basic residues (Lys-644, Lys-645, Lys instead of Arg at 625 and 652, and Arg instead of Lys at 623) (Fig. 1). The difference of sequence in
the C-terminal segment of the
ARK2 PH domain as compared with
ARK1 (Arg/Asn-648, Gln/Arg-656, and Gln/Arg-659) results in an increased total positive charge and thus a larger polarization of the
PH domain. Consistent with the above electrostatic model of the
ARK - G
interaction, a recent G
binding assay (49) involving the C-terminal-peptide sequences (corresponding to
the segments 643-670 and 648-665) indicates a higher potency of the
PH domain of
ARK2 compared with that of
ARK1.

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Fig. 7.
Backbone r.m.s.d. values between
the PH/PTB domains versus percent of sequence identity in
the superimposed secondary structure elements. Data are taken from
Table III. The solid line represents the relation: r.m.s.d. = 0.4 e1.87 (1 h),
where H is a fraction of identical residues (33), obtained for homologous proteins. Data corresponding to ARK1 PH domain are
indicated by solid symbols. The leftmost data point
(open square) corresponds to r.m.s.d. between DynA and DynB
(see Table III).
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The structure of the
ARK PH domain allows rationalization of the
results of
ARK truncation studies (50). The modifications in
ARK
that affect the G
binding in that assay can be
explained either by deletion of one or more core elements, hence
causing a disruption of the overall fold of the PH domain, or by
deletion of positively charged residues on the C terminus (see
below).
Several fusion proteins, containing sequences encompassing a PH domain
(from Ras-GRF, Ras-GAP, OSBP,
-spectrin, IRS-1, and others), were
shown to bind G
in vitro with varying affinities, and some of these PH domains were able to compete with the
ARK PH domain and G
for binding to
G
(18). The C-terminal extended PH domains of Btk
(51), IRS-1, and Dbl (19) were also demonstrated to interact with the
G
heterodimer in vitro. The results of
indirect assays (51, 52) suggest that some of these PH domain
constructs may also interact with G
in
vivo. The C-terminal parts of all these proteins, in particular
region corresponding to residues 644-670 in
ARK (Figs. 1 and 4),
are rich in basic residues, and this supports the proposed
ARK-G
interaction model. Other literature data are
also consistent with this model. Mutation of the last four of the five
basic residues in the highly charged C terminus (Arg-660, Lys-663,
Lys-665, Lys-667, and Arg-669) to acidic ones leads to almost complete
loss of the G
binding (20), as does a deletion of the
last nine residues (662-670) (50). A truncated fragment, ending with
Asn-666, does retain an intermediate level of G
activation, whereas further truncation up to Val-661 leads to a
significantly lower amounts of G
binding compared
with the full-length construct (50). On the other hand, the results of
deletion analysis (19) indicate that the C-terminal extension beyond
Gln-656 is not absolutely required for binding to G
;
the full-length C-terminal extension (553-689 construct) dramatically
increases the maximal extent of G
binding although
does not significantly alter the binding affinity. A 18-residue peptide
comprising the C-terminal amino acids 648-665 was recently shown to
bind G
, also suggesting that the critical C-terminal
extension in
ARK1 PH domain required for G
binding
might be shorter than initially suggested by (50). However, other
functions may be associated with the extended domain.