From the
The molecular interaction involved in the ligand binding of the
rat angiotensin II receptor (AT
The octapeptide angiotensin II (Ang II)
The three-dimensional structure of
bacteriorhodopsin (bRh), a seven transmembrane domain protein, has been
obtained by high-resolution electron cryomicroscopy
(28) . It
provided a modeling template for the overall structure of
GPCRs
(29, 30, 31, 32, 33, 34, 35) .
Although, the sequence homology between GPCRs and bRh is low, several
relevant features of the x-ray crystallographic structure of
photosynthetic reaction center
(36) were incorporated in
modeling many GPCRs including the
Amino acid sequences of a wide variety of GPCRs have
been accumulated (37-44). By multiple amino acid sequence
alignment, highly conserved domains and residues as well as unique
sequences for specific ligand-binding sites can be identified. These
data permit modeling of the AT
In prior studies, site-directed mutagenesis in AT
We
present the homology-based model of the rat AT
Additional bent helices by prolines were introduced into TM1 and
TM7. Finally, the model of the AT
Since the side chain of a lysyl residue is long, the
Phe
An analogous triad Phe
These results showed that the
nonpeptide agonist binds near the surface area. It showed that the
binding mode of Ang II and the nonpeptide antagonist is different,
which agrees with the results of Schambye et al.(69) .
Of 11 residues mutated, ionic groups in the second extracellular loop
(Glu
In summary, the present AT
1) The
aromatic amino acid residue, Trp
2) Phe
3) Ang II penetrates almost one-third of
the way into the membrane to bind the receptor.
The result indicates
that the anionic tetrazolium of EXP985 binds selectively to the
cationic side chain of Arg
The
After the initial
Data represent results of three
identical series of binding isotherms followed by Scatchard analysis.
Results are presented as means ± S.E.
We thank Drs. K. Prendergast, H. T. Schambye, and
J.-C. Bonnafous for useful discussions and comments and Dr. Erwin J.
Landon for critical review of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
APPENDIX
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) was studied by
site-directed mutagenesis and receptor model building. The
three-dimensional structure of AT
was constructed on the
basis of a multiple amino acid sequence alignment of seven
transmembrane domain receptors and angiotensin II receptors and after
the
2 adrenergic receptor model built on the template of the
bacteriorhodopsin structure. These data indicated that there are
conserved residues that are actively involved in the receptor-ligand
interaction. Eleven conserved residues in AT
,
His
, Arg
, Glu
,
His
, Glu
, Lys
,
Trp
, His
, Phe
,
Thr
, and Asp
, were targeted individually
for site-directed mutation to Ala. Using COS-7 cells transiently
expressing these mutated receptors, we found that the binding of
angiotensin II was not affected in three of the mutations in the second
extracellular loop, whereas the ligand binding affinity was greatly
reduced in mutants Lys
Ala, Trp
Ala, Phe
Ala, Asp
Ala, and Arg
Ala. These amino acid
residues appeared to provide binding sites for Ang II. The molecular
modeling provided useful structural information for the peptide hormone
receptor AT
. Binding of EXP985, a nonpeptide angiotensin
II antagonist, was found to be involved with Arg
but not
Lys
.
(
)
is well known for its important roles in the regulation of
cardiovascular functions and electrolyte homeostasis
(1) . Its
structure-function relationship has been extensively
studied
(2) , and it has been postulated that its receptor-bound
form assumes a conformation with a twisted U-shaped bend (3). The Ang
II-binding sites for which a specific ligand is displaced by nanomolar
concentrations of losartan are now referred to as the angiotensin type
1 (AT
) receptors, and those displaced by CGP42112A or
PD123319 are designated as the type 2 (AT
)
receptors
(4) . The primary sequences of AT
receptors
of various mammalian species have been
determined
(5, 6, 7, 8, 9, 10, 11, 12, 13) in recent years, and AT
receptors have also
been cloned
(14, 15) . They were found to belong to the
G-protein coupled receptor (GPCR) family. In general, AT
receptors mediate most of the responses commonly associated with
Ang II, are dithiothreitol sensitive, linked to G-protein, and not
displaced by PD123319. AT
receptors are for the most part
losartan insensitive, not inactivated by dithiothreitol, and do not
mediate physiological responses involving the second messenger systems
commonly associated with AT
receptors. AT
receptors are further divided into subtypes which are referred to
as ``A'' and ``B'' subtypes. AT
and
AT
show an approximately 95% sequence homology
(16-23). The multiple amino acid sequence alignment of several AT
receptors is shown in Fig. 1.
Figure 1:
Alignment
of amino acid sequences of angiotensin II receptors (AT).
Positions of the putative transmembrane domains I-VII are indicated by
solid lines. The sequences of rAT and rAT
are aligned in parallel. Skipped sequences as indicated by
hyphens are introduced for maximizing sequence homology, and
residues identical with those of rAT
are indicated by
asterisks. rAT2, rat AT
(14, 15);
rAT1a, rat AT
(6, 16); rAT1b, rat
AT
(18); hAT1, human AT
(7);
bAT1, bovine AT
(5); tAT1, turkey
AT
(26); xAT, XenopusAT
(25).
Recently, Ang II receptors have
been cloned from an amphibian
(24, 25) and avian
species
(26) , and they were found functionally similar to
mammalian AT receptors. However, these receptors do not
recognize AT
- or AT
-specific nonpeptide
antagonists
(8, 9, 10) . The binding site for the
AT
-specific antagonist losartan was explored by replacing
nonconserved amino acid residues of the rat AT
receptor
with amino acid species at the corresponding positions in the amphibian
receptor
(27) . The losartan-binding site was defined in several
membrane spanning domains of the rat AT
receptor. A recent
study of chimeric human-amphibian Ang II receptors showed that the
binding mode for peptide and nonpeptide ligands is rather different and
that competitive and insurmountable (noncompetitive) antagonists
presumably bind to overlapping but distinct sites located in the sixth
and seventh transmembrane (TM) domain
(28) . The objective of the
present studies is to determine the binding sites for Ang II utilizing
site-directed mutagenesis and incorporating a computer-assisted
receptor modeling approach.
-adrenergic receptor
(
2-AR).
receptor on the template of
an existing model such as that of
2-AR
(45, 46) .
of a
limited number of residues in the transmembrane domains indicated that
Lys
in the fifth transmembrane domain (TM 5th) as a key
residue for ligand binding
(47) . In order to analyze ligand
binding, a model for the docking of Ang II was constructed on the basis
of positional and possible functional interactions of a limited number
of residues in the receptor model. A hypothesis for the docking mode
was then tested on the basis of mutagenesis of several highly conserved
amino acid residues considered critical for Ang II binding.
receptor
constructed after the model of
2-AR which was used in the present
study as a template. We propose a mechanism for docking of a ligand to
the AT
receptor by determining the effects of mutagenesis
of critical receptor amino acid residues on the binding affinity.
Molecular Modeling of the AT
The multiple amino acid sequence alignment of
several Ang II receptors is shown in
Fig. 1(5, 6, 7, 8, 9, 10, 11, 12, 13, 16, 17, 18, 19, 20, 21, 22, 23) .
Information on conserved and non-conserved amino acid sequences were
used for homology-based modeling of AT Receptor
on the template of
2-AR model. The modeling was performed using the molecular
modeling software SYBYL 5.5 on an UNIX workstation model IRIS/indigo
(Silicon Graphics Inc., Mt. View, CA). The extracellular and
intracellular loops were constructed, and the restraint minimization
was performed as described above for
2-AR. A restrained and full
conjugate gradient geometry optimization was then carried out using
Discover 2.9. Following this step, loop regions were removed.
Molecular Modeling of Ang-II and a Docking
Model
The full conjugate gradient geometry optimization of the
Ang II derivative, (Pen, Pen
)Ang II
(48, 49) was performed using Discover 2.9 so that this derivative
forms a twisted U-shape conformation
(3) (Pen is penicillamine).
The conformation of the
-carboxyl and
-amino groups of
Asp
of Ang II was fixed so as to form an intramolecular
salt. Docking studies were carried out using SYBYL 5.5 by placing
Phe
of Ang II on Trp
, His
on
Asp
, Arg
on Asp
, Tyr
on Arg
(see ``Results and Discussion'').
Following the docking of Ang II to the AT
receptor model,
and modifying the backbone and side chain conformation, 100 cycles of
the steepest descent optimization were performed followed by about 1000
cycles of the conjugate gradient optimization with strong backbone
restraints (1000 iterations) for the AT
receptor as a
routine. Later, the backbone conformation of Ang II reported by
Nikiforovich et al.(50, 51) was adopted.
Further details of the modeling procedures involving a
-AR model as an intermediate step are provided under
the ``Appendix.''
Site-directed Mutagenesis of Rat Angiotensin II
Receptor
A 2-kb KpnI-EcoRI fragment of the rat
AT cDNA
(16) was subcloned into the polylinker site
of the plasmid vector pBluescript II KS
, and a
single-stranded DNA was prepared using the helper phage R408.
Site-directed mutagenesis was performed following the procedure of
Kunkel
(52) . Sites of mutations were confirmed by Sanger's
dideoxynucleotide sequencing method
(53) . The mutant DNA insert
was excised from the plasmid vector and introduced into the mammalian
expression vector pcDNAI. Eleven conserved residues in AT
were targeted individually for site-directed mutation to alanine.
Cell Culture
COS-7 cells (American Type Culture
Collection) were cultured in Dulbecco's modified Eagle's
medium containing 10% fetal bovine serum (FBS) penicillin and
streptomycin under 5% CO, 95% air at 37 °C. Cells were
seeded at 3
10
cells in 9-cm dishes and split every
3 days.
Expression of the Mutant Angiotensin II Receptors and
Ligand Binding Assay
COS-7 cells were transfected with mutated
AT cDNAs by electroporation as described
elsewhere
(54) . These cells were cultured for 3 days, and the
mutated receptors were allowed to express transiently. Ligand binding
studies were performed at 4 °C following the procedure reported
using
I-Ang II as an agonist ligand,
I-EXP985 as an antagonist ligand, and results were
analyzed by Scatchard analysis
(55) .
Flow Cytometric Confirmation of Receptor Expression in
Transfected COS-7 Cells
COS-7 cells transfected with the
receptor cDNAs used in the binding study were collected and washed once
with Hanks' balanced salt solution. Cells (1 10
cells) were incubated in Dulbecco's modified Eagle's
medium containing 2% fetal calf serum and
-globulin for 30 min at
4 °C. Rabbit polyclonal anti-AT
antibodies against a
15-residue epitope (Lys
-Tyr
) in the
N-terminal region of rat AT
were produced by immunization
with the peptide coupled to thyroglobulin. Cells were incubated with
the anti-rat AT
antibodies at 4 °C for 60 min, washed
three times with phosphate-buffered saline, then incubated with the
fluorescence-labeled goat anti-rabbit IgG F(ab`)2 fragment at a 1:50
dilution for 30 min at 4 °C, and subjected to an Epics Profile flow
cytometer equipped with a 488 nm laser beam (Counter Electronics Co.,
Ltd.). Cells transfected with pcDNAI containing a mutated AT
cDNA insert expressed immunoreactive rat AT
as shown
in Fig. 2. Expression levels of mutated AT
receptors
were comparable with that of the unmutated AT
Figure 2:
Flow
cytometric analysis of mutated rat AT expressed on the
surface of COS-7 cells. Distribution of fluorescence positive cells
with fluorescence intensity higher than the basal level (the dotted
line) is indicated by shaded peaks. Percentage of
positive cells is also indicated. pcDNAI, pcDNAI vector
transfected; wild AT
, nonmutated rat AT
transfected; Mut-HR166,167AA, mutant
His
Arg
Ala
Ala
, Mut-W253A, mutant
Trp
Ala; Mut-F259A, mutant Phe
Ala; Mut-D263A, mutant Asp
Ala. Other mutants used in this experiment showed similar
results.
Molecular Modeling of the AT
The multiple amino acid sequence alignment of
several Ang II receptors in
Fig. 1A(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) was used to build an AT Receptor
receptor model based
on the
-AR model as the template. A number of amino acids in the
putative TMs are conserved in a large number of GPCRs and probably are
of functional or structural importance. The existence and function of
conserved residues are best explained if they are situated on the
inside of the TM bundle or in an area which is facing other helices.
Residues facing the membrane environment can be mutated more easily
without affecting the function of the receptor and should be less
conserved. The AT
receptor has two relatively short loops
between TM1 and TM2 and also between TM6 and TM7. This was helpful in
determining the exact position of the TMs connected to these loops.
receptor TM bundle thus
obtained was energy minimized (Discover Force Field) with the backbone
fixed with the aim of releasing side chain repulsion. Further
minimization with no restriction of the helix bundle (1000 iterations)
was performed. These minimizations did not significantly change the
relative positions of the TMs.
Amine-Aromatic (NH-
The
amine-aromatic interaction, discovered recently in hemoglobin
(56) and repeatedly confirmed in x-ray crystallographic
structures of a number of proteins (57), were incorporated in the
modeling of AT) Interaction
. The interaction of amine, amide, or
guanido nitrogen with aromatic
electrons within 3-4 Å
of the flat face of aromatic ring was computed by molecular orbital
calculation according to the Density Functional Theory.
(
)
-amino group of Lys
(TM5), which is important for
ligand binding (), might not be located near the outer
surface. Frequently, there are aromatic amino acid residues surrounding
the ionic bridge formed between GPCRs and their ligands
(30) as
shown in Fig. 3. Tryptophyl residues are found in TM4, 6, and 7
in most
GPCRs
(37, 38, 39, 40, 41, 42, 43, 44, 59) .
It is possible that many of them stabilize an ammonium salt by an
aromatic-amine interaction. In the x-ray crystallographic structure of
the phosphotyrosine recognition domain, SH2, of v-src complexed with a
tyrosine-phosphorylated peptide, one of the guanidine nitrogens of an
arginyl residue is situated immediately above an aromatic ring, and the
-amino group of a lysyl residue is immediately below the
phosphorylated tyrosine phonemic ring
(60) . A similar
ammonium-aromatic interaction has been observed by other
investigators
(61, 62) . Our molecular orbital
calculation showed that the interaction produces a stabilization of
about 5.3 kcal/mol,
virtually indistinguishable from a
hydrogen bond which averages 6.1 kcal/mol
(63) . Thus, it is
likely that the
-amino group of Lys
may be located
on Trp
in TM6 or Trp
in TM4. From the
docking model shown below, Trp
looked more likely
(Fig. 3).
Figure 3:
Schematic representation of the
interaction between rat AT in the sixth transmembrane
region and angiotensin II.
The mutation of Lys to Ala resulted in
a greater than 50-fold increase in K
for
Ang to from 1.7 nM to >100 nM; Trp
to Ala increased K
7-fold to 12.5
nM (). These results led to the plausible
postulate that the C-terminal carboxylate anion of Phe
of
Ang-II and the
-amino group of Lys
which is located
on top of the indole ring of Trp
form an ionic link and
that Trp
stabilizes the salt bridge.
in TM6 of
2-AR is very important for ligand binding.
His
of AT
is considered to be an equivalent
of Phe
of
2-AR because of its position. We mutated
His
to Ala. The K
value for
Ang II increased to 4.5 nM from 1.7 nM of the native
receptor. The mutation of neighboring residues also affects the ligand
binding (). In mutagenesis studies of nonconserved residues
in AT
, H. Ji et al.(27) found that
Ser
Cys resulted in a considerable
reduction in the binding affinity of saralasin.
Binding Site for His
We studied the binding of an imidazole group to various
receptors and enzymes in search of a general pattern for imidazole
binding. The imidazole ring of histamine is known to bind to
Thr of Angiotensin
II
, Asn
, and Phe
in TM5 of
the histamine H1 receptor
(64) , and to Asp
,
Thr
, and Phe
in TM5 of the histamine H2
receptor
(65) . The protonation-tautomerism of an imidazole
sandwiched between two hydrogen-bonding ligands seems to be stabilized
by an adjacent aromatic ring.
Thr
Asp
is present in AT
in TM6. We established a model illustrated in Fig. 4, in
which the imidazole of His
and the C-terminal carboxylate
of Phe
of Ang II are accommodated by TM6. The imidazole
group was placed between the
-carboxyl group of Thr
and
-carboxyl group of Asp
, and the C-terminal
carboxylate anion on the
-amino group located on the indole ring
of Trp
. The model accommodated this configuration well.
The interactive centers of Trp
and Asp
are
16 Å apart. This distance is compatible with that between the
C-terminal carboxylate and imidazole groups of Ang II plus the
calculated distances of interaction between Phe
of Ang II
and Trp
(4.7 Å), and that between His
of Ang II and Asp
(3.9 Å). Significant
increases in K
accompanying mutations of
individual amino acid residues in this triad, except for
Thr
, to an alanyl residue are compatible with probable
roles of these residues in ligand binding. His
and
Phe
are 2 of the functionally important residues for the
agonistic activity of Ang II
(66) . Although a small effect on
K
of mutation Thr
Ala is somewhat different from a larger change due to a similar
mutation in the histamine H2-receptor, it is significant compared with
the mutation of the next residue Ile
, which had no effect
on saralasin binding as reported by H. Ji et al.(27) .
The small effects of Asp
Ala and Thr
Ala are also compatible with a relatively small
contribution of His
to the binding of Ang II
(67) .
It looks as if AT
binds its ligand with a somewhat
different manner from H2-R. The hydrogen bonding of the imidazole of
His
to Thr
is weak or not essential in case
of AT
().
Figure 4:
View of rat AT receptor model
from the side (A) and the top of receptor well (B).
The transmembrane helices are represented by the solid ribbon.
The oxygen atom is shown in red, and the nitrogen atom in
purple. The extracellular space is in the top of the
figure.
Contribution of Arg
In the Ang II molecule, the phenolic side chain
of Tyr to the Binding of
Angiotensin II
has a very strong interaction with the
receptor
(67, 68) . It is likely that phenolic group
interacts with guanido group as reported in various x-ray
crystallographic structures. In our model with Phe
and
His
fixed, the side chain of Arg
extends
toward the phenolic group of Tyr
of Ang II. Thus we propose
that Arg
in TM4 is important for Ang II binding. This
notion is supported since Arg
is conserved throughout Ang
II receptor isoforms.
The Binding Mode of Nonpeptide Antagonist
EXP985
Finally, the present approach brought out an interesting
contrast between the binding of the peptide agonist Ang II and the
non-peptide antagonist EXP985, an analog of losartan. As shown in
, mutations of many residues had comparable effects on the
binding of the agonist Ang II and the nonpeptidic antagonist. However,
mutations, Lys
Ala and Trp
Ala had hardly any effect on the binding of EXP985, whereas these
individual mutations drastically weakened Ang II binding. As discussed
above, Lys
and Trp
are most likely hydrogen
bonded in the ligand-free state. Because of mutual proximity and close
interaction, the mutation of either one of these residues will disrupt
the ligand binding function of the two associated side chain groups in
parallel. Thus, the specific and parallel change of Lys
Ala and Trp
Ala on the binding of Ang
II, and a lack of effect of either of these mutations on the binding of
the nonspecific antagonist EXP985 gave further support to the present
hypothesis that Lys
and Trp
function as a
unit in association with each other. These observations indicate that
the anionic tetrazolium widely used in many nonpeptidic antagonists
binds selectively to the cationic side chain of Arg
rather than Lys
. Further, contribution of
Asp
, presumably in the binding of the imidazole ring of
His
of Ang II, seems to play a relatively minor role for
the binding of nonpeptidic antagonist.
, His
, Gln
) seem to play
only minor, if any, roles in the binding for both the agonist and
antagonist.
receptor
modeling and mutagenesis presented the following hypotheses.
stabilizes the ionic
bridge formed between Lys
of AT
and the
carboxylate anion of Phe
of Ang II.
and Asp
in TM6 provide the binding site for
His
of Ang II.
of AT
. In
AT
, mutants, Val
Ile, Ala
Ser, and Ser
Cys had a marked effect
on losartan binding
(27) . It shows that the mutation of
neighboring amino acids also reduces ligand binding ability, and our
present results are in agreement with these observations.
Molecular Modeling of
For the
molecular modeling of 2-ARM
2-AR, we used the modeling program SYBYL 5.4
(Tripos Associates, St. Louis) on a micro VAX-II (Degital Equipment
Corp., Boston) and PS 390 (Evans & Sutherland, Salt Lake City), and
Discover (Biosym. Technologies Inc., San Diego) version 2.1 was used
for optimization. The result of the multiple sequence alignment of
several GPCRs was used to build the model of
2-AR on the basis of
the three-dimensional structure of bRh
(28) . The angle of a kink
in a helical column due to a proline residue in a transmembrane helical
structure was cited from the x-ray crystallographic structure of
photosynthetic reaction center (36). Results of mutation studies of
2-AR
(45, 46) were also incorporated. The
extracellular and intracellular loops were generated using the
``Loop Search'' in SYBYL, then 100 cycles of a steepest
descent optimization were performed with strong backbone restraints
(1000 iterations) as usual. These ``Loop Search'' and
restrained minimization processes were repeated until this protein
satisfied acceptable
-
angle values for the peptide
backbone. The docking model of
2-AR was constructed by fitting
each helix of
2-AR to the corresponding bRh in order to minimize
the root-mean-square (rms) value
(32) . A full conjugate gradient
optimization of the model was performed without the backbone restraints
until the rms change or gradient reached their minimum levels.
Molecular Modeling of the
Various GPCRs were cloned and
sequenced, and their molecular models were constructed based on the
overall structure of bRh alignment to
2-Adrenergic Receptor as a
Template for AT
2-AR
(29, 30, 31, 32, 33, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44) .
A mumber of residues are highly conserved throughout the GPCR family,
and other residues are conserved among Ang II receptors shown in
Fig. 1
. The effect of the mutation of the highly conserved
aspartyl residue (Asp
of
2-AR and Asp
of
AT
) in TM2 on the receptor structure and function has been
experimentally demonstrated (58, 71, 72). Many investigators speculated
that the residues conserved throughout the GPCRs are probably involved
in their general structural and functional roles for transmission of
signals from a bound ligand, whereas those conserved only within Ang II
receptors will play important roles in the specific ligand binding.
2-AR model, constructed after the three-dimensional
structure of bRh
(28) , was used as a template for the AT
receptor model in the present study according to a similar method
used for other
receptors
(29, 30, 31, 32, 33, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44) .
This two-step strategy was used because (i) the bRh is the only seven
membrane spanning domain protein for which atomic coordinates are
available; (ii) homology between bRh and AT
is very low;
(iii) a reasonable homology is seen between
2-AR and AT
in their transmembrane domains; (iv) the interaction between
2-AR and its ligands has been well
studied
(45, 46) ; (v)
2-AR and AT
have
large numbers of proline residues compared with bRh; and (vi) bRh binds
retinal, whereas GPCRs do not. Thus, we proceeded to use only the
topology of the seven transmembrane spanning helices of bRh and refined
the
2-AR model in reference to the results of site-directed
mutagenesis studies (45, 46). Because
2-AR has more proline
residues than bRh and a proline residue introduces a kink to the
transmembrane conformation of each helix, we had to modify the backbone
conformation of each helix according to the published
method
(29, 30, 31, 32, 33, 34, 35) .
Since the bRh structure is of a low resolution, we adopted the x-ray
crystallographic structure of the membrane photosynthetic reaction
center
(37, 38, 39, 40, 41, 42, 43, 44) ,
for introducing proline kinks to the helical columns of
2-AR.
2-AR model was constructed, we docked a
2 agonist and antagonists and rotated or translationally shifted
the position of each helix.
Table:
Binding
affinity of peptide and nonpeptide ligands for rat AT wild
type receptor and mutant receptors
,
angiotensin type 1 receptor; AT
, angiotensin type 2
receptor; GPCR, G-protein coupled receptor; TM, transmembrane domain;
bRh, bacteriorhodopsin;
2-AR,
2-adrenergic receptor; Pen,
penicillamine; SH2, src homology-2; kb, kilobase(s).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.