(Received for publication, October 6, 1994; and in revised form, December 9, 1994)
From the
To identify specific interactions between either the tetrazole
or carboxylate pharmacophores of non-peptide antagonists and the rat
AT receptor, 6 basic residues were examined by
site-directed mutagenesis. Three of the mutants (H183Q, H256Q, and
H272Q) appeared to be like wild type. Lys
and Arg
mutants displayed reduced binding of the non-peptide antagonist
losartan. Examination of their properties employing group-specific
angiotensin II analogues indicated that their effects on binding were
indirect. Interestingly, the affinity of losartan was not altered by a
K199Q mutation, but the same mutation reduced the affinity of
angiotensin II, the antagonist
[Sar
,Ile
]angiotensin II, and several
carboxylate analogues of losartan. An Ala
substitution
reduced the affinity of peptide analogues to a larger extent as
compared to the affinity of losartan. Thus, the crucial acidic
pharmacophores of angiotensin and losartan appear to occupy the same
space within the receptor pocket, but the protonated amino group of
Lys
is not essential for binding the tetrazole anion. The
binding of the tetrazole moiety with the AT
receptor
involves multiple contacts with residues such as Lys
and
His
that constitute the same subsite of the ligand
binding pocket. However, this interaction does not involve a
conventional salt bridge, but rather an unusual lysine-aromatic
interaction.
The octapeptide hormone Ang II ()plays a central role
in the regulation of blood
pressure(1, 2, 3) . Two distinct angiotensin
receptor subtypes, AT
and AT
, have been
identified(2) . Because the AT
receptor is solely
responsible for mediating response of cardiovascular system to Ang II
it is a major target for drug design efforts for the treatment of
hypertension, congestive heart failure, and cardiac hypertrophy (1, 2, 3) . The AT
receptor is a
G-protein-coupled receptor characterized by a putative
seven-transmembrane helical structure motif (Fig. 1A; (4) ). Besides Ang II-peptide analogues, a family of
non-peptide antagonists binds to the AT
receptor with high
affinity (Fig. 1B). These non-peptides presumably
function like the classical G-protein-coupled receptor antagonists that
utilize residues located in the putative transmembrane helices for
their binding. Because Ang II is substantially larger, its binding is
likely to involve the extracellular loops of the AT
receptor in addition. Lys
has been suggested to be
involved in binding of the C-terminal carboxylate group of Ang
II-peptides but has not been implicated in binding the non-peptide
ligands(5) . Furthermore, residues located in transmembrane
helices 3-7 involved in binding non-peptide antagonists do not
influence peptide
binding(5, 6, 7, 8, 9) .
Because pharmacological competition between non-peptides and peptides
is clearly established, an overlapping but non-identical binding site
model is currently
favored(3, 5, 6, 7, 8, 9) .
Figure 1:
A, a proposed secondary
structure model of rat AT receptor in a lipid bilayer.
Residues relevant to this work are highlighted. B, diagram of
Ang II, losartan, and EXP7711 denoting their acidic
``pharmacophore'' in color.
The non-peptide AT receptor-antagonists available today
are rational improvements of the imidazole carboxylic acid lead
compounds, S8307 and S8308(3, 10) . They contain the
core structure of S8307 with a biphenyl-acidic extension, which is
thought to be a better mimic of the Ang II structure(3) .
Modeling of the three-dimensional structural overlay of several
non-peptides indicates that a common geometry of critical
pharmacophores is present in all high affinity angiotensin receptor
antagonists(11) . Peptide antagonists have higher affinity
toward the AT
receptor, suggesting that they bind in a
unique conformation to the receptor(2, 11) . Therefore
it is possible that the same pharmacophore geometry exists in the
receptor bound conformation of Ang II-peptide antagonists as well (Fig. 1B). Presumably the same residues on the receptor
are utilized for docking the pharmacophore presented by peptide and
non-peptide antagonists. For example, the
-carboxyl group of the
Ang II-peptides and the carboxylic acid or carboxamide and sulfonamide,
or tetrazole group on the biphenyl portion of various non-peptides, are
equivalent in structure-activity
relationship(3, 11, 12, 13) . In
this report we present evidence to suggest that the peptide and
non-peptide antagonists indeed occupy the same AT
receptor
binding pocket, but may employ dissimilar interactions for
stabilization of bound ligands. The relevance of these results to the
pharmacophore overlay of peptide and non-peptide AT
receptor ligands is discussed.
A rat AT receptor gene with 41 unique restriction
sites was designed by strategies used previously(14) , and
synthesized and cloned into the shuttle expression vector
pMT2(15) . The synthetic gene encodes 359 amino acids of the
rat vascular AT
receptor with an 8-residue epitope tag
(ETSQVAPA) at the C terminus. The epitope is the binding site for the
monoclonal antibody 1D4, which can be used to detect polypeptide
expression in transfected cells as described previously(16) .
Total membrane preparations from transfected COS cells were frozen at
-85 °C in 50 mM HEPES, 12.5 mM MgCl
, 1.5 mM EGTA, and 10% glycerol until
assayed(16) . The K
and B
of the receptor were estimated by
I-[Sar
,Ile
]Ang II
equilibrium binding and Scatchard plot analysis. Extensive analysis of
affinity, expression levels and agonist-induced inositol phosphate
formation suggests that the AT
receptors expressed from
cDNA and the epitope-tagged synthetic DNA exhibit identical properties.
Mutations were constructed in the synthetic gene by the technique of
restriction fragment replacement, and all of the mutants were confirmed
by DNA sequence analysis(16, 17) .
AT receptor binding was determined using total membrane prepared
from transfected COS cells as described earlier(18) .
[Sar
,Ile
]Ang II and
[Sar
,Ile
]Ang II-amide were
radioiodinated by the lactoperoxidase method, and the radiolabeled
peptides were purified by the reverse-phase high performance liquid
chromatography method(19) . The specific activity of both these
peptides was 2200 Ci/mmol. [
H]Losartan (specific
activity 42.3 Ci/mmol) was obtained from Amersham. All binding data
were analyzed and IC
values determined by nonlinear
regression analysis. K
values for
radiolabeled [Sar
,Ile
]Ang II were
estimated from competition binding data with 8-10 different
concentrations of the corresponding unlabeled
[Sar
,Ile
]Ang II using the equation: K
= IC
/(1 - L/K
), where L is
concentration of radioligand, and IC
is the concentration
of competing ligand required to reduce specific radioligand binding by
50%, and K
is the dissociation constant
for
I-[Sar
,Ile
]Ang
II(20) . K
values (nanomolar)
represent mean ± S.E., n = 3-10.
Multiple interactions of Ang II with the receptor contribute
to its binding enthalpy. Among these, the ion pair interaction of the
C-terminal carboxyl group of Ang II with the receptor is well defined.
The contractile response to
[acetyl-Asn,Val
]Ang II, an
agonist analogue of Ang II that contains a single carboxylate at the
peptide C terminus, is pH-sensitive, and the modification of its
carboxylate to hydrogen bonding or hydrophobic groups reduces
biological activity(1, 21) . Structure-activity
analysis and modeling studies of non-peptide antagonists predict that a
similar ion pair interaction is essential for them to bind with high
affinity to the AT
receptor(3, 11, 13) . We decided to
define this interaction by systematic mutagenesis of receptor combined
with group-specific modification of the ligand, since an earlier study (5) did not investigate all potential residues in the receptor
that could be involved in this interaction. We restricted our search to
basic residues (Arg, Lys, and His in its protonated state) located in
the putative transmembrane helices and the extracellular loops of the
AT
receptor because the majority of small molecule ligands
bind to G-protein-coupled receptors within this region(22) .
Basic residues conserved in all losartan-selective AT
receptors were selected for mutagenesis (Fig. 1A). Several different single residue
substitutions at each position were tested.
Figure 2:
The effect of pH on the binding of I-[Sar
,Ile
]Ang II,
I-[Sar
,Ile
]Ang II-amide
and
H-losartan to the wild-type AT
receptor and
its mutants. The upperpanel shows binding to
wild-type AT
receptor. The middlepanel shows binding of
I-[Sar
,Ile
]Ang II to
wild-type and mutant receptors. The lowerpanel shows
binding to wild-type receptor only.
Substitution of 3 residues in the putative transmembrane domain
(Lys, Arg
, and Lys
) leads to
a decrease of [Sar
,Ile
]Ang II binding
affinity. All of the Arg
mutants expressed receptor
proteins at the same level, which were glycosylated as for the
wild-type receptor. The expressed mutant proteins did not specifically
bind any of the peptide and non-peptide antagonists. Therefore, the
role of Arg
in ligand binding remains unclear. The K102Q
mutant showed loss of binding affinity toward all ligands (Table 1). Interestingly, K102Q mutation did not alter pH profile
of [Sar
,Ile
]Ang II binding (Fig. 2) and the ability to discriminate differences between Ang
II and [Sar
,Ile
]Ang II, indicating
that Lys
does not interact with the carboxylate group of
the Asp
side chain of Ang II (Table 1). Thus, these
results are consistent with the requirement of a positively charged,
long side chain at this position for stabilization of the AT
receptor conformation.
The substitution of Lys with Gln did not significantly alter the binding of losartan.
Surprisingly, however, the affinity of this mutant (K199Q) for
[Sar
,Ile
]Ang II and Ang II was about
10- and 48-fold lower, respectively, than that of the wild-type
receptor (Table 1, Fig. 3). The effect of pH on
[Sar
,Ile
]Ang II binding of the K199Q
mutant is shown in Fig. 2. The binding profile shows a shift of
maximal binding to pH 7.0. When the pH is raised to 8.0, specific
binding dropped to about 40% of that at pH 7.0 for the mutant, while it
did not change for the wild type. This indicates that the positive
charge of Lys
plays a predominant role in the pH
dependence of [Sar
,Ile
]Ang II
binding. Similar profile is observed when
[Sar
,Ile
]Ang II-amide binds to
wild-type AT
receptor (Fig. 2). This clearly
establishes the complementarity of interaction between Lys
and C-terminal carboxylate of
[Sar
,Ile
]Ang II. However, the
pH-binding profiles of losartan with the mutant K199Q and the wild-type
receptor were identical. [Sar
,Ile
]Ang
II binding to wild-type receptor at pH 6-9 is consistent with a
salt bridge involving a Lys, but the pH optimum of 6.5-7.0 for
losartan binding suggests that its negatively charged tetrazole group
(pK
= 6, see (13) ) interacts with
a basic residue that deprotonates closer to neutral pH. These
observations lead us to question if Lys
is indeed the
common counterion for both carboxylate- and tetrazole-containing
ligands. We investigated this question by substitution of Ala (K199A),
Glu (K199E), or Arg (K199R) for Lys
(Fig. 3, Table 1). Electrostatic interaction and hydrogen bonding could
account for interaction of
[Sar
,Ile
]Ang II and
[Sar
,Ile
]Ang II-amide with these
mutants (21) . We conclude that the effects of modifying the
Lys
positive charge are consistent with its being the
exclusive counterion for peptidyl ligands. This conclusion supports the
observation of Yamano et al.(5) .
Figure 3:
Effect of substitution of Gln, Glu, Arg
and Ala for Lys on the binding affinity of different
antagonists. The K
of
[Sar
,Ile
]Ang II-amide (also see Table 1) was used as a common denominator for comparison (SIA
II-amide, sarcosine-R-V-Y-I-H-P-I-CONH
).
, sarcosine-R-V-Y-I-H-P-I-COOH;
, sarcosine-R-V-Y-I-H-P-I-CONH
;
, DUP753;
, EXP7711.
The role of the
positive charge of Lys in losartan binding is unclear
because a Gln substitution does not affect its binding and an Arg
substitution unexpectedly led to reduction of binding affinity. It is
possible that the binding interaction of tetrazole might be different
from a conventional salt bridge interaction. This viewpoint is
supported by the observation that the affinity of a variety of
carboxyl-containing non-peptide antagonists is reduced (5-fold for
Exp7711 and 14-fold for L-159,810; see (11) ) in the K199Q
mutant (Table 1, Fig. 3). Therefore, the
carboxyl-containing non-peptides display all properties of a salt
bridge interaction with Lys
. Moreover, this indicates
that peptide to non-peptide differences do not drive non-peptide
antagonists to bind at a different basic residue on the receptor.
These results
indicate that the positive charge of the 199 residue is the most
important electrostatic interaction required for
[Sar,Ile
]Ang II binding affinity. By
contrast, losartan binding requires a side chain bearing an amino
group, but it is not necessary that this group be protonated. The
straightforward explanation may be that both Lys
and
His
participate in tetrazole binding. The role of
His
may be secondary in the wild-type receptor.
Therefore, in the K199Q and K199A mutants, removal of the charge is
presumably compensated by His
through a change of its
pK
(23) , which then allows it to function
as an alternate counterion in the same microenvironment as Lys
(see (24) for similar examples). In the double mutant
K199A/H256A, this environment for tetrazole binding may be altered
substantially, since the Ala side chain is smaller than that of either
Lys
or His
(Table 2). This could
result in the formation of a cavity in the receptor
structure(25) , such that the tetrazole group probably cannot
make van der Waals contacts with either of the substituted Ala
residues. The cavity in the pocket is expected to affect
[Sar
,Ile
]Ang II binding affinity as
well (Table 2). The Arg
substitution in the mutant
K199A/H256R probably allowed the restoration of
[Sar
,Ile
]Ang II binding by filling
the cavity in addition to restoring the positive charge. The positive
charge of Arg could satisfy losartan but the bulkier Arg side chain may
cause steric problems, explaining partial improvement of affinity
compared to that of K199A/H256A mutant.
A conventional hydrogen bond
interaction between tetrazole and receptor must also be considered
because such an interaction is probably involved in the binding of
[Sar,Ile
]Ang II-amide and Ang
II-amide(21) . However, since conventional hydrogen bonds are
weak interactions, loss of such an interaction in the mutant K199A, for
example, could not account for the observed 10-fold decrease in binding
affinity. The observation that a Gln can perfectly substitute for
Lys
must indicate that either the
-amide group of
Gln
or the
-amino group of Lys
are
involved in a direct interaction with the tetrazole of losartan.
Receptor/ligand interactions in which sp
nitrogen atoms
form a stacked interaction with aromatic rings or form an
amino/aromatic hydrogen bond are known(26, 27) . Lys
with its sp
nitrogen cannot form stacked interaction but
could interact with planar aromatic rings. (
)This might
explain why Lys
and Gln
side chains have
the same effect on losartan binding affinity and on the apparent pH
dependence of binding. Such interactions may be further stabilized by
additional interactions, with His
, for instance. A
Lys-aromatic interaction between receptor and ligand has not been
reported before. In support of this suggestion, Duncia et al.(12) have reported that an acidic aromatic group
substituted in the meta position of the terminal phenyl ring in
biphenyl series has a binding affinity comparable to that of tetrazole
analogues. Furthermore, disruption of the conjugation in the terminal
ring of the biphenyl reduces the binding
affinity(3, 13) . Therefore, aromaticity is a crucial
determinant. Thus, exploitation of the novel interaction in the
tetrazole binding subsite of the AT
receptor might be
worthy of consideration in future drug design.
The conclusion that
the same space is occupied by tetrazole and the carboxylate groups
seems justified. This provides an experimental basis for docking
peptides and non-peptide ligands to a common site on the AT receptor in modeling the receptor-ligand complex. In structurally
related peptide hormone receptors for the neurokinins, research so far
has shown that critical binding determinants for peptide ligands do not
participate in the binding of the non-peptide ligands(22) .
Similar conclusions on the interaction of losartan with AT
receptor have
appeared(5, 6, 7, 8, 9) .
However, unlike the non-peptide antagonists of the neurokinin receptor,
losartan was developed through model-based drug development and
pharmacophore overlay(3) , a concept that is further supported
by the experiments in this report.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z28391[GenBank].