U.S. patent application number 10/473681 was filed with the patent office on 2007-01-11 for g protein-coupled receptor structural model and a method of designing ligand binding to g protein-coupled receptor by using the structural model.
Invention is credited to Masaji Ishiguro.
Application Number | 20070010948 10/473681 |
Document ID | / |
Family ID | 18954809 |
Filed Date | 2007-01-11 |
United States Patent
Application |
20070010948 |
Kind Code |
A1 |
Ishiguro; Masaji |
January 11, 2007 |
G protein-coupled receptor structural model and a method of
designing ligand binding to g protein-coupled receptor by using the
structural model
Abstract
The present invention provides a method for constructing a
structural model of a complex that a G protein-coupled protein
receptor forms with a ligand capable of binding the G
protein-coupled receptor and a three-dimensional structural model
of an activated intermediate in the structural model of the
complex. The present invention also provides a method for
identifying, screening for, searching for, evaluating, or designing
a ligand capable of binding a GPCR by using the three-dimensional
model. In one specific method by the present invention, a
three-dimensional structural model of a photoactivated intermediate
of rhodopsin is constructed by using a molecule modeling software
and by using the three-dimensional structural coordinate of the
crystal structure of rhodopsin in such a manner that amino acid
residues highly conserved among GPCRs are taken into consideration.
The three-dimensional stractural model of the photoactivated
intermediate of rhodopsin is subsequently used to construct
structural models of activated intermediates of other GPCRs. The
present invention further provides a method for identifying,
screening for, searching for, evaluating, or designing a ligand
that binds a GPCR to act as an agonist or an antagonist. This
method employs the three-dimensional structural model constructed
by the above-described method.
Inventors: |
Ishiguro; Masaji; (Hyogo,
JP) |
Correspondence
Address: |
BURNS DOANE SWECKER & MATHIS L L P
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Family ID: |
18954809 |
Appl. No.: |
10/473681 |
Filed: |
April 1, 2002 |
PCT Filed: |
April 1, 2002 |
PCT NO: |
PCT/JP02/03264 |
371 Date: |
February 17, 2004 |
Current U.S.
Class: |
702/19 |
Current CPC
Class: |
G16B 15/00 20190201;
G16B 5/00 20190201; G01N 33/566 20130101; C07K 2299/00 20130101;
C07K 14/723 20130101; G01N 33/6803 20130101 |
Class at
Publication: |
702/019 |
International
Class: |
G06F 19/00 20060101
G06F019/00; G01N 33/48 20060101 G01N033/48; G01N 33/50 20060101
G01N033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 30, 2001 |
JP |
2001-101510 |
Claims
1. A method for constructing a three-dimensional structural model
of an activated intermediate of a G protein-coupled receptor for
use in identifying, screening for, searching for, evaluating, or
designing a ligand that binds the G protein-coupled receptor to act
as an agonist or an antagonist.
2. The method according to claim 1, wherein the activated
intermediate of the G protein-coupled receptor is an intermediate
of activated rhodopsin.
3. The method according to claim 1 or 2, wherein the structural
model of the intermediate of the activated rhodopsin is a
structural model of metarhodopsin II.
4. The method according to claim 1 or 2, wherein the structural
model of the intermediate of the activated rhodopsin is a
structural model of metarhodopsin I.
5. The method according to claim 1 or 2, wherein the structural
model of the intermediate of the activated rhodopsin is a
structural model of metarhodopsin Ib.
6. The method according to claim 1 or 2, wherein the structural
model of the intermediate of the activated rhodopsin is a
structural mode of metarhodopsin I.sub.380.
7. The method according to claim 1, characterized in that the
three-dimensional structural model of the activated intermediate of
the G protein-coupled receptor is constructed based on a structural
model of metarhodopsin II.
8. The method according to claim 1, characterized in that the
three-dimensional structural model of the activated intermediate of
the G protein-coupled receptor is constructed based on a structural
model of metarhodopsin I.
9. The method according to claim 1, characterized in that the
three-dimensional structural model of the activated intermediate of
the G protein-coupled receptor is constructed based on a structural
model of metarhodopsin Ib.
10. The method according to claim 1, characteristics in that the
three-dimensional structural model of the activated intermediate of
the G protein-coupled receptor is constructed based on a structural
model of metarhodopsin I.sub.380.
11. A three-dimensional structural model of the activated
intermediate of the G protein-coupled receptor obtained by the
method according to any one of claims 1 to 10, or a
three-dimensional coordinate for determining the structural
model.
12. A three-dimensional coordinate shown in Table 1 or Table 2.
13. A computer storage medium that stores all or part of the
three-dimensional coordinate according to claim 11 or 12 for use in
identifying, screening for, searching for, evaluating, or designing
a ligand that binds the G protein-coupled receptor to act as an
agonist or an antagonist.
14. A method for identifying, screening for, searching for,
evaluating, or designing a ligand that binds a G protein-coupled
receptor to act as an agonist, the method comprises the step of
using the three-dimensional strut model according to claim 11 or
the three-dimensional coordinate for determining the structural
model according to claim 11 or 12, or the computer storage medium
according to claim 13.
15. The method according to claim 14, wherein the agonist is a full
agonist of the G protein-coupled receptor.
16. The method for identifying, screening for, searching for,
evaluating, or designing the full agonist of the G protein-coupled
receptor of claim 15, characterized in that, of the
three-dimensional structural models according to claim 11 or the
three-dimensional coordinates for determining the structural models
according to claim 11 or 12, the metarhodopsin II structural model
or the three-dimensional coordinate for determining the structural
model, or the structural model constructed based on the
metarhodopsin II structural model or the three-dimensional
coordinate for determining the structural model is used.
17. The method according to claim 14, herein the agonist is a
partial agonist of the G protein-coupled receptor.
18. The method for identifying, screening for, searching for,
evaluating, or designing the partial agonist of the G
protein-coupled receptor of claim 17, characterized in that, of the
three-dimensional structural models according to claim 11 or the
three-dimensional coordinates for determining the structural medals
according to claim 11 or 12, the metarhodopsin I.sub.380 structural
model or the three-dimensional coordinate for determining the
structural model, or the structural model constructed based on the
metarhodopsin I.sub.380 structural model or the three-dimensional
coordinate for determining the structural model is used.
19. A method for identifying, screening for, searching for,
evaluating, or designing a ligand capable of binding a G
protein-coupled protein to act as an antagonist, the method
comprises the step of using the three-dimensional structural model
according to claim 11 or the three-dimensional coordinate for
determining the structural model according to claim 11 or 12, or
the computer storage medium according to claim 13.
20. The method according to claim 19, wherein the antagonist is an
inverse agonist of the G protein-coupled receptor.
21. The method for identifying, screening for, searching for,
evaluating, or designing the inverse agonist of the G
protein-coupled protein of claim 20, characterized in that, of the
three-dimensional structural modes according to claim 11 or the
three-dimensional coordinates for determining the structural models
according to claim 11 or 12, the metarhodopsin I structural model
or the three-dimensional coordinate for determining the structural
model, or the structural model constructed based on the
metarhodopsin I structural model or the three-dimensional
coordinate for determining the structural model is used.
22. The method for identifying, screening for, searching for,
evaluating, or designing the antagonist of the G protein-coupled
protein of claim 19, characterized in that, of the
three-dimensional structural models according to claim 11 or the
three-dimensional coordinates for determining the structural models
according to claim 11 or 12, the metarhodopsin Ib structural model
or the three-dimensional coordinate for determining the structural
model, or the structural model constructed based on the
metarhodopsin Ib structural model or the three-dimensional
coordinate for determining the structural model is used.
23. A method for identifying, screening for, seeing for,
evaluating, or designing a mutant of a G protein-coupled receptor,
the method comprises the step of using the three-dimensional
structural model according to claim 11 or the three-dimensional
coordinate for determining the structural model according to claim
11 or 12, or the computer storage medium according to claim 13.
24. The method according to claim 22, wherein the mutant of the G
protein-coupled receptor is a constitutively active mutant.
25. The method according to any one of claims 14 to 24, wherein the
G protein-coupled receptor is selected from the group consisting of
rhodopsin, adrenaline receptor, muscarinergic acetylcholine
receptor, histamine H2 receptor, serotonin receptor, and amine
receptor.
26. The method according to claims 1 to 6, characterized in that
the structural model of the intermediate is generated by using
coordinates of existing amino acid sequences and existing crystal
structures of amino acids and by using an ordinary molecule
modeling software in such a manner that amino acid residues highly
conserved among the transmembrane helices of the G protein-coupled
receptor are taken into consideration, and structural optimization
is performed at 300 K according to molecular kinetics and molecular
dynamics in such a manner with C .alpha. carbons of the amino acids
fixed as firmly as possible.
27. The method according to any one of claims 7 to 10,
corresponding the steps of: introducing amino acid substitution and
insertion or deletion of amino acid residues on the loop regions by
means of a three-dimensional structural model of a rhodopsin/ligand
complex or a three-dimensional model of rhodopsin in the structural
model of the complex based on the homology between the amino acid
sequence of rhodopsin and the amino acid sequence of a G
protein-coupled receptor for which to construct a model; generating
a structure using a molecule modeling software; and performing
structural optimization with C .alpha. carbons of the amino acids
fixed as firmly as possible.
Description
TECHNICAL FIELD
[0001] The present invention relates to a structural model for
receptor/ligand complexes of G protein-coupled receptors (which may
be referred to simply as `GPCRs,` hereinafter) and ligands capable
of binding to G protein-coupled receptors. It also relates to a
method for creating a three-dimensional structural model for
activated intermediates of G protein-coupled receptors in the
structural model for the receptor/ligand complexes, as well as to
structural models of the complexes or the activated intermediates
of G protein-coupled receptors obtained by this method. The present
invention further relates to a three-dimensional coordinate for
determining these structural models.
[0002] The present invention further relates to a method for using
the three-dimensional structural model of G protein-coupled
receptors or a method for using the three-dimensional coordinates
for determining the structural model in identifying, screening for,
searching for, evaluating, and designing ligands that act as an
agonist (full agonist or partial agonist) or an antagonist
(antagonist or inverse antagonist) upon binding to G
protein-coupled receptors.
[0003] The present invention further relates to a method for using
the three-dimensional structural model of the G protein-coupled
receptors or a method for using the three-dimensional coordinate
for determining the structural model in designing mutants of G
protein-coupled receptors (e.g., constitutively active mutants) or
in screening for and searching for orphan receptors and identifying
their ligands in vivo.
BACKGROUND ART
[0004] Transmission of extracellular information into the cell in
most cases requires mediation by membrane proteins that have
transmembrane domains. G protein-coupled receptors (GPCRs) are
signal-transmitting membrane proteins that have seven transmembrane
domains and make up a receptor family that can bind various
physiological peptide ligands, including biological amines such as
dopamine and serotonin, lipid derivatives such as prostaglandin,
nucleic acids such as adenosine, amino acids such as GABA,
angiotensin II, bradykinin, and cholecystokinin. Serving also as
receptors for extracellular transmitters responsible for the senses
of vision, taste and smell, GPCRs are important membrane proteins
that play a key role in signal transduction. The recent progress in
completing the human genome sequence is expected to lead to
discovery of many orphan receptors that are suspected of being
GPCR. If successfully identified, the ligands for these GPCRs will
allow for more effective development of pharmaceutical products.
Thus, devising a structural model for G protein-coupled
receptor/ligand complexes and devising a three-dimensional
structural model for G protein-coupled receptors in the structural
model of the Complexes will provide an important approach to the
future development of pharmaceutical products, as will the
identification, screening, searching, evaluation, and designing
methods of ligands that take advantage of these structural
models.
[0005] In fact, a number of patent applications entitled "novel G
protein-coupled receptor protein and its DNA" have recently been
filed, including Japanese Laid-Open Patent Publications No.
2001-29083, No. 2001-29084, No. 2001-54388, No. 2001-54389, No.
2000-23676, No. 2000-23677, No. 2000-50875, No. 2000-152792, No.
2000-166576, No. 2000-175690, No. 2000-175691, and No. 2000-295995,
to name a few. Some applications, such as Japanese Patent Laid-Open
Publication No. 2000-354500, disclose methods for screening for
ligands that bind to G protein-coupled receptors while other
applications concern methods for cloning expression of G
protein-coupled receptors.
[0006] Ligands that bind to a particular G are generally classified
into agonists and antagonists. According to the latest
pharmacological classification standards, the former is further
divided into full agonists and partial agonists and the latter into
inverse agonists and antagonists.
[0007] These ligands are classified not by their affinity for the
receptor, but by the degree to which the ligand activates the
receptor. For example, assuming the activity elicited by binding of
a full agonist to be 100%, a partial agonist elicits a 50 to 70%
activity.
[0008] In comparison, binding of an antagonist suppresses the
activity to 5 to 10% of what is elicited by the binding of a full
agonist, and binding of an inverse agonist completely eliminates
the activity (0% activity).
[0009] Even when unbound to ligands, many GPCRs exhibit 5 to 10% of
the activity led by the binding of a full agonist. Thus, it is
believed that antagonists bind to physiologically inactive receptor
conformations. This suggests that binding of other types of ligands
brings about conformational change of GPCR. Thus, the binding of
ligands and subsequent conformational change of receptors are
believed to play an important role in information transmission
mediated by GPCR.
[0010] G protein-coupled receptors (GPCR), which share seven
transmembrane domains, are classified into different families based
on the homology of their amino acid sequences. In one such GPCR
family, each member has high homology to rhodopsin, a photoreceptor
membrane protein. The GPC of this family share highly conserved
amino acid residues in their transmembrane domains. These amino
acid residues are believed to play an important role in the
functioning of GPCRs.
[0011] Structural and functional studies of GPCR have been
conducted by analyzing three-dimensional structure of rhodopsin
through two-dimensional cryoelectron diffraction crystallography
and X-ray crystallography (Palczewski, K. et al., Science 289,
739-745. (2000)). Also, structures of the receptor proteins and the
chromophores to serve as ligands, as well as the receptors'
conformational changes, have been studied using FT-IR and Raman
spectroscopy (Sakmar, T. P., Prog. Nucleic Acid Res. 59, 1-34
(1998)).
[0012] Based on the results of two-dimensional, low-resolution,
cryoelectron diffraction crystallography, a three-dimensional
structural model of rhodopsin was first constructed. More recently,
more detailed three-dimensional structure of rhodopsin was revealed
by X-ray crystallography. This structure was consistent with the
structural characteristics previously expected from the results of
FT-IR and Raman spectroscopy and made it possible to formulate
assumptions about the roles of some parts of the highly conserved
amino acid residues of GPCRs.
[0013] For example, of the highly conserved amino acid residues of
rhodopsin, the Glu134-Arg135-Tyr136 triplet (ERY triplet, which
corresponds to Asp-Arg-Tyr, or DRY triplet, in other GPCRs) of the
third transmembrane helix (TM3) (hereinafter, each of the seven
transmembrane helixes may be denoted by abbreviation followed by
respective consecutive numbers: n th helix is denoted as TMn (e.g.,
TM3)) located on the inside of the cell plays a significant role in
the activation of G protein. It has been shown that the protonation
of ionized Glu134 in metarhodopsin II (described later), an
activated conformation of rhodopsin, triggers activation of
G-protein (Arnis, S. & Hofmann, K. P., Proc. Natl. Acad. Sci.
USA, 90, 7849-7853, 1993). Also, a significant involvement of Glu
and Arg in the activation of G is suggested.
[0014] On the other hand, it is suggested that a highly conserved
Pro residue found in TM6 and TM7 (Pro 267 in TM6) is responsible
for the kink structure characteristic of these two helices.
However, the role of the kink in the functioning of GPCRs still
remains unclear.
[0015] Hydrophilic amino acid residues Asn55, Asp83, Asn302 found
in TM1, TM2, and TM7, respectively, are linked to one another via
hydrogen bonds. Also, Tyr306 residue conserved among TM7s is
linked, through hydrophobic interaction, to a residue of C-terminal
helix located on the inside of the cell. These interactions are
believed to contribute to stabilizing the structure.
[0016] Rhodopsin is also one of the G closely studied for its
conformational change and functions. Rhodopsin consists of
11-cis-retinal, a chromophore, and rhodopsin, a protein component
with the seven transmembrane domains. 11-cis-retinal is covalently
bonded to Lys296 to form a Schiff base. This Schiff base is
protonated and is thus responsible for the shift of the maximum UV
absorbance (.lamda.max) of the chromophore to a long-wavelength
range of 498 nm.
[0017] When illuminated, rhodopsin is converted to highly unstable
bathorhodopsin (which may be referred to simply as `Batho,`
hereafter), which has the UV absorbance shifted to an even longer
wavelength range. Upon this, 11-cis-retinal is converted to
11-trans-retinal, an all-trans chromophore. The unstable,
high-energy Batho is then sequentially converted to different
intermediates in the order of lumirhodopsin (`Lumi,` hereinafter),
metarhodopsin I (`Meta I,` hereinafter), metarhodopsin Ib (`Meta
I,` hereinafter), and metarhodopsin II (`Meta II,` hereafter) as
the chromophore and opsin thermally undergo conformational changes
(Tachibanaki, S. et al., Biochemistry 36, 14173-14180 (1997)) (the
photoreaction process is shown in FIG. 1).
[0018] Under physiological conditions, Lumi is converted to Meta II
via an intermediate known as metarhodopsin I.sub.380 (`Meta
I.sub.380,` hereinafter) (T. E. et al., Biochemistry 32,
13861-13872 (1993)) (FIG. 1).
[0019] Because the activation of G protein (transducin) takes place
at Meta II stage, 11-cis-retinal attached to rhodopsin is regarded
as an inverse agonist while all-trans retinal attached to Meta II
can be regarded as a full agonist. Since the same chromophore of
rhodopsin changes from an inverse agonist to a full agonist upon
illumination of light, its conformational changes can be studied by
observing changes in absorption spectrum.
[0020] The conversion of rhodopsin to Batho is a rapid process that
takes place within 200 fs. Each conformational change leading to
Meta II takes about a few milliseconds, which is long enough to
allow a protein to undergo a significant conformational change
involving spatial displacement of the secondary structures of the
protein. It has been shown that the conformational change of opsin
causes the beta-ionon moiety of the retinal chromophore to change
its direction from the 6th helix (TM6) to the 4th helix (TM4)
(Bean, B. et al., Science, 288, 2209-2212 (2000)). This implies
that the arrangement of helices has been altered as a result of
photoisomerization.
[0021] Also, Khorana and Hubbell in their experiment illuminated
light onto a mutant rhodopsin, which has been spin-labeled in a
site-directed manner by taking advantage of SH groups in the mutant
site-specifically substituted with cysteine, and demonstrated that
the conformational changes of rhodopsin to Meta II are accompanied
by conformational changes of the intracellular loops and helices.
They proposed a model in which the entire TM6 helix undergoes
significant rotation. The model implies considerable conformational
changes of membrane proteins (Farms, D. L. et al., Science 274,
768-770 (1996)).
[0022] Light energy absorbed by the chromophore is harnessed to
cause initial conformational change of opsin. Transition to the
final active form, the Meta II conformation, begins with proton
transfer from the protonated Schiff base to its counterion, Glu 134
in TM3, to form neutral Schiff base. The neutralization of the
Schiff base allows movement of the helix and, ultimately, the
rotation of TM6, causing the shift to the Meta II conformation.
[0023] Of the different photoactivated intermediates of rhodopsin,
the final Meta II conformation has proven to be the only form that
has been fully activated (Khorana, H. G. J. Biol. Chem., 267, 1-4
(1992)). Ha, However, opsin without the chromophore is known to
exhibit approximately 5% activity, and mutant opsin in which
Glu134, which serves as a counterion of the protonated Schiff base,
has been substituted with Gln exhibits approximately 50% activity
even in the absence of the chromophore.
[0024] This mutant opsin is known to be deactivated when
11-cis-retinal is added and irrational with light converts it to
all-trans-retinal, which in turn is converted to fully activated
Meta II conformation. Thus, it has been shown that opsin has
several active forms (Kim, J.-M. et al., Proc. Natl. Acad. Sci.
USA, 94, 14273-14278 (1997)).
[0025] It is also known that G-protein (transducin) does not bind
opsin when rhodopsin in is in its Meta I state while it binds opsin
without activating it when rhodopsin is in its Meta Ib state
(Tachibanki, S. et al., Biochemistry 36, 14173-14180 (1997)).
[0026] As described, a series of events, including conformational
changes of opsin and its interaction with G-protein, and suit
activation of G-protein, take place over the curse of the process
from Lumi to Meta II. During this process, the rotation of TM6,
essential for the activation of rhodopsin, provides the G
protein-coupled receptor with the structural specificity required
for ligand recognition. Specifically, it has been shown that the
amino acid residues in the ligand binding site involved with TM6
before the rotation of TM6 are different than the ones involved
with TM6 after the rotation of TM6, and amino acid residues that
serve to recognize full agonists are different than those that
serve to recognize antagonists.
[0027] In fact, mutants are often found in which alteration of some
of the amino acid residues in TM6 affects the binding of full
agonists but not the binding of antagonists. Such phenomenon will
be explained by taking into account the conformational changes of
the receptors.
[0028] Studies on conformational changes of rhodopsin suggested
that the arrangement of TMs is significantly different between the
receptors that bind antagonists and the receptors that bind
agonists. For this reason, the crystal structure of rhodopsin does
not solely provide a structural model for every receptor/ligand
complex.
[0029] A comparison between the crystal structure of rhodopsin and
a structural model for Meta II in accordance with the present
invention is shown in FIG. 2. The significant displacement of
highly conserved Trp265 in TM6 suggests that different amino acid
residues are involved in recognizing agonists and antagonists.
[0030] As described above, several experiments demonstrated that
photoactivation of rhodopsin brings about conformational changes of
opsin (See, for example, Farrens, D. L. et al., Science 274,
768-770 (1996), Kim, J.-M. et al., Proc. Natl. Acad. Sci. USA, 94,
14273-14278, (1997)). Nonetheless, the nature of specific
conformational change has yet to be understood.
[0031] Accordingly, it is an objective of the present invention to
simulate three-dimensional structures of these photoactivated
intermediates of rhodopsin by means of computer graphics and
scientific calculation and to thereby construct structural models
for their complexes formed with ligands (chromophores) that can
bind rhodopsin as well as three-dimensional structural models for
the activated rhodopsin intermediates in the structural models of
the complexes.
[0032] It is another objective of the present invention to provide
a method for identifying, screening for, searching for, or
evaluating whether a given ligand is a fell agonist, a partial
agonist, an antagonist, or an inverse agonist by constructing
three-dimensional models for general G protein-coupled receptors
(GPCRs) other than rhodopsin from the three-dimensional structural
models for the activated intermediates of rhodopsin and, for each
of the three-dimensional constructing structural models for their
complexes formed with ligands and analyzing the interaction of
GPCRs with corresponding ligands. It is still another objective of
the present invention to provide a method for designing a novel
ligand molecule that acts as an agonist or an antagonist of a
GPCR.
DISCLOSURE OF THE INVENTION
[0033] In an effort to achieve the aforementioned objectives, the
present inventor has succeeded in constructing structural models
consistent with available experimental data for each of the known
photoactivated intermediates of rhodopsin: Lumi, Meta I, Meta Ib,
Meta I.sub.380 and Meta II.
[0034] Specifically, the present inventor has directed his
attention to amino acid residues highly conserved among GPCRs that
show high homology to rhodopsin and has succeeded in revealing
their role by generating and then optimizing the structural models
for rhodopsin intermediates by means of a molecule modeling
software Insight II-Discover 3 (Molecular Simulations Inc., USA)
using the three-dimensional structural coordinates for the crystal
structure of rhodopsin (Palczewski et al., Science, 289, 144-167
(2000)). In this manner, the present inventor has successfully
simulated the conformational changes of rhodopsin and analyzed its
interaction with ligands.
[0035] The conformational change of rhodopsin takes place in the
following manner: TM3 of the seven transmembrane helixes (TM1-7),
which strongly interacts with TM7, is first mobilized, and the
disulfide bond that Cys110, a highly conserved residue on the
extracellular side of TM3, forms with Cys187 causes the helix on
the cytoplasmic side to swivel about Cys110 in a pendulum-like
fashion toward the extracellular side. This in turn causes the
movement of adjacent TM4.
[0036] On the other hand, TM1, TM2, and TM7 are not subjected to
conformational changes because of hydrogen bonds between the highly
conserved amino acid residues and form a cluster of helices less
susceptible to the movement of TM3.
[0037] TM3 and TM4 move in such a manner that the ligand-binding
site is enlarged. This movement is controlled by the interaction
between Glu134-Arg135-Tyr136, a highly conserved sequence on the
cytoplasmic side of TM3, and Glu247 on the cytoplasmic side of TM6.
The movement of TM3 and TM4 lasts until the Meta I.sub.380 stage,
during which time structures corresponding to Lumi, Meta I, and
Meta Ib are formed.
[0038] The structure of Meta II is generated from Meta I.sub.380 or
similar structures: TM6, as viewed form the cytoplasmic side,
rotates clockwise by 100.degree. and then translate to come close
to TM3. Upon this, the conformational change in TM6 causes TM5 to
move to where it is free from structural interference. Finally, TM4
moves toward TM5 to form the structure of Meta II.
[0039] As described, the seven transmembrane helixes (TMs) of
rhodopsin are divided into three domains depending on the role that
they play in the conformational change: a first domain including
TM1, TM2, and TM7, a second domain including TM3 and TM4, and a
third domain including TM5 and TM6. By investigating contribution
of each of the three dins to the conformational change of
rhodopsin, it has been made possible to generate structures of all
of the intermediates between rhodopsin and Meta II.
[0040] Accordingly, the present invention provides a
three-dimensional structural model or a three-dimensional
coordinate for determining the structural model used for
identifying, searching for, screening for, evaluating, or designing
a ligand that can bind a G protein-coupled receptor to act as an
agonist or an antagonist.
[0041] Specifically, the present invention provides a
three-dimensional structural model or a three-dimensional
coordinate for determining the structural model, in which the
activated intermediate of the G protein-coupled receptor is an
intermediate of activated rhodopsin.
[0042] More specifically, the present invention provides a
three-dimensional structural model or a three-dimensional
coordinate for determining the structural model, in which the
structural model of the activated rhodopsin intermediate is a
metarhodopsin II structural model, a metarhodopsin I structural
model, a metarhodopsin Ib structural model, or a metarhodopsin
I.sub.380 structural model.
[0043] The present invention further provides a method for
constructing three-dimensional structural models of activated
intermediates of G protein-coupled receptors other than rhodopsin
by means of the structural model of the four activated rhodopsin
intermediates.
[0044] More specifically, the present invention provides a method
for constructing a structural model, the method comprising the
steps of introducing amino acid substitution and insertion or
deletion of amino acid residues on the loop regions by means of the
structural model of the four activated rhodopsin intermediates
based on the homology between the amino acid sequence of rhodopsin
and the amino acid sequence of different G protein-coupled
receptor; and subsequently optimizing the structure by using a
molecule modeling software to construct a structural model.
[0045] The present invention further provides a computer storage
medium that stores all or part of the above-described coordinate of
the three-dimensional model for use in identifying, screening for,
searching for, evaluating, or designing a ligand that binds the G
protein-coupled receptor to act as an agonist or an antagonist.
[0046] The present invention further provides a method for
identifying, screening for, searching for, evaluating, or designing
a ligand that binds a G protein-coupled receptor to act as an
agonist, the method comprises the step of using the above-described
three-dimensional structural model, the three-dimensional
coordinate for determining the structural model, or the computer
storage medium storing the coordinate.
[0047] In particular, the present invention provides a method for
identifying, screening for, searching for, evaluating, or designing
the agonist, characterized in that, of the three-dimensional
structural models or the three-dimensional coordinates for
determining the structural models, the metarhodopsin II (Meta II)
or the metarhodopsin I.sub.380 (Meta I.sub.380) structural model or
the three-dimensional coordinate for determining the structural
model, or the structural model constructed based on the
metarhodopsin II (Meta II) or the Metarhodopsin I.sub.380 (Meta
I.sub.380) structural model or the three-dimensional coordinate for
determining the structural model is used.
[0048] The present invention also provides a method for
identifying, screening for, searching for, evaluating, or designing
a ligand capable of binding a G protein-coupled protein to act as
an antagonist, the method comprising the step of using the
above-described three-dimensional structural model or the
three-dimensional coordinate for determining the structural models
or the computer storage medium storing the coordinate.
[0049] In particular, the present invention provides a method for
identifying, screening for, searching for, evaluating, or designing
the antagonist, characterized in that, of the above-described
three-dimensional structural models or the three-dimensional
coordinates for determining the structural models, the
metarhodopsin Ib (Meta Ib) or the metarhodopsin I (Meta I)
structural model or the three-dimensional coordinate for
determining the structural model or the structural model
constructed based on the metarhodopsin Ib (Meta Ib) or the
metarhodopsin I (Meta I) structural model or the three-dimensional
coordinate for determining the structural model is used.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIG. 1 is a diagram showing the photoreaction process of
rhodopsin.
[0051] FIG. 2 is a diagram showing a comparison between crystal
structural model of rhodopsin and a structural of Meta II in a
accordance with the present invention.
[0052] FIG. 3 is a structural model for a Meta II-ligand
(chromophore) complex in accordance with the present invention.
[0053] FIG. 4 is a structural model for a Meta I-ligand
(chromophore) complex in accordance with the present invention.
[0054] FIG. 5 is a structural model for a Meta Ib-ligand
(chromophore) complex in accordance with the present invention.
[0055] FIG. 6 is a structural model for a Meta I.sub.380-ligand
(chromophore) complex in accordance with the present invention.
[0056] FIG. 7 is a structural model in one embodiment of the
present invention showing a complex that an adrenergic beta-2
receptor to serve as a Meta I-like structure of the present
invention forms with an inverse agonist propranolol.
[0057] FIG. 8 is a structural model in another embodiment of the
present invention showing a complex that an adrenergic beta-2
receptor to serve as a Meta II-like structure of the present
invention forms with a full agonist (S)-isoproterenol.
[0058] FIG. 9 is a structural model in another embodiment of the
present invention showing a complex that a muscarinic acetylcholine
receptor to serve as a Meta II-like structure of the present
invention forms with a full agonist acetylcholine.
[0059] FIG. 10 is a structural model in another embodiment of the
present invention showing a complex that a muscarinic acetylcholine
receptor to serve as a Meta Ib-like structure of the present
invention forms with an antagonist N-methylscopolamine.
[0060] FIG. 11 is a structural model in another embodiment of the
present invention showing a complex that a histamine H2 receptor to
serve as a Meta II-like structure of the present invention forms
with a full agonist histamine.
[0061] FIG. 12 is a structural model in another embodiment of the
present invention showing a complex that a histamine H2 receptor to
serve as a Meta Ib-like structure of the present invention forms
with an antagonist tiotidine.
[0062] FIG. 13 is a structural model in another embodiment of the
present invention showing a complex that a serotonin receptor to
serve as a Meta II-like structure of the present invention forms
with a full agonist serotonin.
[0063] FIG. 14 is a structural model in another embodiment of the
present invention showing a complex that a serotonin receptor to
serve as a Meta I.sub.380-like structure of the present invention
forms with a partial agonist lysergic acid diethylamide (LSD).
[0064] FIG. 15 is a structural model in another embodiment of the
present invention showing a complex that a serotonin receptor to
serve as a Meta Ib-like structure of the present invention forms
with an antagonist ketanserine.
[0065] FIG. 16 is a structural model in another embodiment of the
present invention showing a complex that a dopamine receptor to
serve as a Meta II-like structure of the present invention forms
with a full agonist dopamine.
[0066] FIG. 17 is a structural model in another embodiment of the
present invention showing a complex that a dopamine receptor to
serve as a Meta Ib-like structure of the present invention forms
with an antagonist sulpiride.
[0067] FIG. 18 is a diagram showing a homology in amino acid
sequences of the seven transmembrane domains among rhodopsin and
other GPCRs.
[0068] FIG. 19 is a structural model of a human adrenergic alpha-1A
receptor bound to an antagonist.
[0069] FIG. 20 is a structural model of a human adrenergic alpha-1B
receptor bound to an antagonist.
[0070] FIG. 21 is a structural model of a human adrenergic alpha-1D
receptor bound to an antagonist.
[0071] FIG. 22 is a structural model of a human adrenergic alpha-2A
receptor bound to an antagonist.
[0072] FIG. 23 is a structural model of a human adrenergic alpha-2B
receptor bound to an antagonist.
[0073] FIG. 24 is a structural model of a human adrenergic
alpha-2C-1 receptor bound to an antagonist.
[0074] FIG. 25 is a structural model of a human adrenergic
alpha-2C-2 receptor bound to an antagonist.
[0075] FIG. 26 is a structural model of a human adrenergic beta-1
receptor bound to an antagonist.
[0076] FIG. 27 is a structural model of a human adrenergic beta-2
receptor bound to an antagonist.
[0077] FIG. 28 is a structural model for a human adrenergic
alpha-1A receptor isoform 4 bound to an antagonist.
[0078] FIG. 29 is a structural model of a human adrenergic alpha-1C
receptor isoform 2 bound to an antagonist.
[0079] FIG. 30 is a structural model of a human adrenergic alpha-1C
receptor isoform 3 bound to an antagonist.
[0080] FIG. 31 is a structural model of a human adrenergic
alpha-1C-AR receptor bound to an antagonist.
BEST MODE FOR CARRYING OUT THE INVENTION
[0081] In this specification, amino acids are represented by
three-letter codes or single-letter codes as defined by IUPAC and
IUB.
[0082] By "identifying a ligand," it is meant to determine whether
a certain compound is an agonist (a full agonist or a partial
agonist), an antagonist (an antagonist or an inverse antagonist),
or neither of these.
[0083] By "screening or searching for a ligand," it is meant to
find compounds having activity as an agonist or an antagonist in a
set of naturally occurring or synthetic compounds.
[0084] Although same may agree that the term "screening" refers to
selecting desired compounds from an available set or a library of
compounds whereas the term "searching" refers to finding new
compounds existing in nature, these terms are used interchangeably
in this specification.
[0085] As used herein, the term "evaluation" has substantially the
same meaning as "identification." Nonetheless, the term is
preferentially used when a certain compound is discussed in terms
of the magnitude of its activity as an agonist or an
antagonist.
[0086] As used herein, the phrase "a structural model based on the
structural models of activated rhodopsin intermediates" is meant to
encompass not only the structural models for the activated
intermediates of G protein-coupled receptors (GPCRs) other than
rhodopsin that are constructed based on the above-describe
structural model of rhodopsin, but also the structural models for
the mutants of the G protein-coupled receptors and the activated
intermediates of the mutants.
[0087] Using three-dimensional coordinates with a molecule modeling
software Insight II-Discover 3 (Molecular Simulations Inc., USA)
that can determine the crystal structure of rhodopsin by means of
X-ray diffraction crystallography (Palczewski et al., Science, 289,
144-167 (2000)), a structural model was generated for each of the
intermediates and each structure was optimized.
[0088] Specifically, this is done as follows: TM3 is swung about
the C .alpha. carbon of Cys110 to serve as the pivot point while
the distance to TM2 is kept at 5 .ANG. or more. The magnitude of
the swing is determined by taking into consideration the
interaction of TM6 with Glu247 for each of Lumi, Meta I, Meta II,
Meta Ib, and Meta I.sub.380 structures. Specifically, in each of
Lumi, Meta I, Meta Ib, and Meta I.sub.380, Cys140 on TM3 is swung
in such a manner that Cys140 is spaced from TM6 by a distance of
1.6 .ANG., 4.3 .ANG., 6.8 .ANG., and 9.0 .ANG., respectively.
Furthermore, N-terminal (Glu150) of the portion of TM4 that would
interfere with TM3 is swung toward TM5 about Gly174 on the
C-terminal of the helix to serve as the pivot point by a distance
of 3.5 .ANG., 7.4 .ANG., 12.1 .ANG., and 17.1 .ANG., respectively,
to avoid interference. The structures so generated are optimized at
300 K by means of molecular kinetics and molecular dynamics so that
C .alpha. carbons of the amino acids can be fixed as firmly as
possible.
[0089] As for the structure of Meta II, TM6 is rotated clockwise by
an angle of 100 degrees as viewed from the intracellular side, and
the distance between the residues on TM6 and the residues on TM3 is
monitored and is decreased to a minimal distance that does not
cause steric interference. Upon this, TM5 is twisted about Asn200
in a direction that can avoid steric interference resulting from
the rotation of TM6. TM4 is then translated by a distance of 4.1
.ANG. to place it between TM3 and TM5.
[0090] As a result, the distance between the C .alpha.-carbon of
Cys140 on TM3 and the C .alpha.-carbon of Ala246 on TM6 becomes
12.7 .ANG. and the C .alpha.-carbon of Cys140 on TM3 is positioned
at 4.8 .ANG. from Glu150 on TM4. Leu226 on TM5 is positioned at a
distance of 10.5 .ANG. from Ala246 on TM6. TM5 and TM4 are moved so
that they do not sterically interfere with TM6. The structures so
generated are optimized at 300 K by means of molecular kinetics and
molecular dynamics so that C .alpha. carbons of the amino acids can
be fixed as firmly as possible.
[0091] As described, the seven transmembrane helices of rhodopsin
are divided into three drains depending on the role that they play
in the conformational change of rhodopsin: a first domain including
TM1, TM2 and TM7, a second domain including TM3 and TM4, and a
third domain including TM5 and TM6. By investigating contribution
of each of the three domains to the conformational change of
rhodopsin, it has been made possible to generate structures of all
of the intermediates between 3 rhodopsin and Meta II. In this
manner, three-dimensional structural model coordinates were
obtained for Meta II, Meta I, Meta Ib, and Meta I.sub.380. Of
these, the coordinates for Meta II, the structure that binds a full
agonist, and for Meta I, the structure that binds an inverse
agonist, are shown in Tables 1 and 2, respectively.
[0092] Based on the coordinates so obtained, three-dimensional
structural models were constructed for complexes bound to ligands.
The structural model for the complexes of Meta II, Meta I, Meta Ib,
and Meta I.sub.380 are shown in FIGS. 3 to 6, respectively.
TABLE-US-00001 LENGTHY TABLE REFERENCED HERE
US20070010948A1-20070111-T00001 Please refer to the end of the
specification for access instructions.
TABLE-US-00002 LENGTHY TABLE REFERENCED HERE
US20070010948A1-20070111-T00002 Please refer to the end of the
specification for access instructions.
[0093] The Meta II structure constructed here has the arrangement
of the extracellular helices very similar to that of
bacteriorhodopsin, the crystal structure of which has already been
known. The arrangement of the helices of Meta II, however, is
significantly different from that of helices of bacteriorhodopsin
on the cytoplasmic side.
[0094] A method for constructing structural models for complexes of
other G protein-coupled proteins formed with respective ligands, as
well as a method for constructing three-dimensional structural
models for these GPCRs in the structural models for the complexes,
will now be described with reference to specific examples using the
structural models for the four activated intermediates of
rhodopsin: Meta II, Meta I, Meta Ib, and Meta I.sub.380.
[0095] For other G inverse agonists, antagonists, partial agonists,
and full agonists exist as different compounds. For this reason,
the degree of activation can be defined for each receptor
conformation that binds each of the ligands. Accordingly, of the
structural models of other GPCRs that have been constructed based
on the structural modes for the photoactivated intermediates of
rhodopsin, namely, the four activated intermediates of rhodopsin of
the present invention, the structural models for GPCRs of
adrenaline, muscarinic acetylcholine, histamine H2, serotonin, and
dopamine, for which inverse agonists, antagonists, partial
agonists, and full agonists are known to exist as different
compounds, are used as specific examples in studying interactions
between the structural models of GPCRs of the present invention and
their respective ligands.
[0096] This study demonstrates the usefulness and viability of the
structural models for GPC provided in accordance with the present
invention.
[0097] (1) Propranolol, an inverse agonist of adrenergic beta-2
receptors, inactivates the receptor completely. The fact that most
of the light energy absorbed by rhodopsin is used to generate Meta
I suggests that the structure to which an inverse agonist of beta-2
receptor binds has a structure similar to Meta I.
[0098] A structural model for a complex that propranolol forms with
an adrenergic beta-2 receptor is shown in FIG. 7.
[0099] The amino group of the ligand interacts with the Asp residue
conserved on TM3, whereas the naphthyl group of propranolol forms a
cluster of aromatic rings with aromatic amino acid residues of TM5
and TM6. This interaction stabilizes inactive structure of the
receptor.
[0100] (2) In a complex that an adrenergic beta-2 receptor forms
with its full agonist (S)-isoproterenol, the amino group interacts
with the Asp residue similarly conserved on TM3. Meanwhile, the
catechol group interacts with the two Ser groups on TM5. This
serves as a model for stabilizing the structure of fully activated
Meta II-like structures.
[0101] A structural model for the complex formed with
(S)-isoproterenol is shown in FIG. 8.
[0102] (3) A complex that a muscarinic acetylcholine receptor forms
with acetylcholine serves as a typical example of stabilization of
Meta II-like structure by a full agonist (FIG. 9).
[0103] Cationic moiety of acetylcholine interacts with the Asp103
residue similarly conserved on TM3. The model for complex on the
other hand implies the interaction between Tyr403 residue on TM6
and the acetyl group of the ligand. The site-specific mutation of
this Tyr residue has been shown to result in a reduced binding
activity of acetylcholine.
[0104] On the other hand, this mutation does not affect the binding
of antagonists, which is consistent with the fact that the Tyr
residue cannot interact with the acetyl group in the Meta I-like
inactive structural but is positioned so that it can interact only
in the fully active structural model. Thus, it is believed that
this interaction contributes to the stabilization of the fully
active structure that results from the conformational change of
TM6.
[0105] (4) On the other hand, an antagonist N-methylscopolamine
readily binds the Meta Ib-like inactive structural model and, in
particular, binds the Asn404 on TM6 to stabilize the structure
bound to the antagonist (FIG. 10).
[0106] (5) Likewise, the nitrogen atom of the imidazole group of
histamine interacts with the Tyr 250 on TM6 in the Meta II-like
structural model (which is present at the same position as the
Tyr403 in the muscarinic acetylcholine receptor) of histamine H2
receptor to stabilize the structure bound to the agonist (FIG.
11).
[0107] (6) an the other hand, tiotidine, an antagonist of the
histamine H2 receptor, interacts with the Asp186 on TM5 in the Meta
Ib-like structural model- to stabilize the structure bound to the
antagonist (FIG. 12).
[0108] (7) In a model for a serotonin receptor/serotonin complex,
NH in the indole backbone, a characteristic functional group of
serotonin, interacts with the carbonyl oxygen in the peptide bond
of the Met335 on TM6 in the Meta II-Like structural model to
stabilize the structure bound to the agonist (FIG. 13).
[0109] (8) Lysergic acid diethylamide (LSD), known as a partial
agonist of serotonin receptors, includes a characteristic
diethylamide group, which effectively interacts with the Asn343 of
TM6 in the Meta I.sub.380-like structural model to stabilize the
structure bound to the partial agonist. The indole ring of the
lysergic acid diethylamide, which is stacked with the highly
conserved Trp336 on TM6, also contributes to stabilization of the
partial agonist-bound structure. The stacking with tryptophan is
unique to the partial agonist-bound structure (FIG. 14).
[0110] (9) Ketanserine, a serotonin receptor antagonist, interacts
both with the Asp155 on TM3 and with Ser242 on TM5 in the Meta
Ib-like structural model. This interaction brings about interaction
between the amine moiety of the piperidine ring, which is often
found in serotonin receptor antagonists such as ketanserine, and
the Asn343 on TM6 to stabilize the structure bound to the
antagonist (FIG. 15).
[0111] (10) Dopamine receptors bind dopamine at Ser193 and Ser194
on TM5 in the Meta II-like structural model to stabilize the
structure bound to the agonist (FIG. 16).
[0112] (11) Ligands including a sulfone group, such as sulpiride,
which act as antagonists of dopamine receptors, interact with the
His 393 on TM6 in the Meta Ib like structural model. This
interaction is possible only in the antagonist-bound structure and
thus proves to be a major specific interaction with the ligands
having sulfone groups (FIG. 17).
[0113] As shown in the above-described examples, the structural
models for complexes provided in accordance with the present
invention allows identification of a set of inverse agonists or
full agonists that can stabilize the inactive structure or the
fully active structure of the receptor. As for partial agonists, a
typical model postulates that they bind both of the inactive and
active structures, and the resulting two different complexes exist
in equilibrium. However, the fact that mutations on the amino acid
residues that specifically bind an antagonist or a full agonist do
not affect the activity of partial agonists in either direction
implies the presence of a specific receptor structure for this type
of ligand. Indeed, it is one of several points that the present
invention has demonstrated to be true. Likewise, it appears that
inverse agonists also as the receptors to undergo conformational
change from a conformation to bind an antagonist. This also implies
the presence of a specific receptor structure, as evidenced by the
present invention.
[0114] A description will now be given of a method for identifying,
screening for, searching for, evaluating or designing a ligand
(either an agonist or an antagonist) for a G protein-coupled
receptor by the use of either a structural model for the activated
intermediates of rhodopsin obtained above or a three-dimensional
coordinate for determining such a structural model, or a structural
model for a G protein-coupled receptor other than rhodopsin or a
three-dimensional coordinate for determining such a structural
model.
[0115] It should be appreciated that the method described herein is
also applicable when it is desired to construct a structural model
for a G protein-coupled receptor other than rhodopsin by the use of
the structural model for the activated intermediates of rhodopsin
or the three-dimensional coordinate for determining such a
structural model.
[0116] A three-dimensional structural model coordinate is
determined for each of the structural models for the intermediates
between rhodopsin and Meta II, namely, Meta II, Meta I, Meta Ib,
and Meta I.sub.380. The coordinates are then entered into a
computer operated by a computer program capable of displaying
three-dimensional structural coordinates of molecules or suitable
storage median for use with such a computer. This allows visual
observation or calculation of energy, which are required steps for
identifying, screening for, searching for, evaluating, or designing
a ligand that binds the above-described receptors to act as an
antagonist or an agonist.
[0117] Specifically, an agonist or an antagonist can be identified,
screened for, searched for, evaluated, or designed for example by
examining interactions between ligands and amino acid residues that
have specificity to the above-described receptors and are highly
conserved among TMs 1 through 7. In particular, compounds that
exhibit a higher biological activity and stability than the
original ligands that bind GPCRs can be identified, screened for,
searched for, evaluated, or designed.
[0118] Many of such computer programs for constructing
three-dimensional structural coordinates of G protein-coupled
receptors are commercially acceptable. These programs typically
include means for entering a three-dimensional structural
coordinate for a molecule, means for visually displaying the
coordinate on a computer screen, means for determining for example
distances and bond angles between atoms within the displayed
molecule, and means for correcting the coordinate. A program can be
also used that includes means for calculating structural energy of
a molecule based on the original coordinate of the molecule, and
means for calculating free energy by taking into account water
molecules and other solvent molecules. In the present invention, a
molecule modeling software Insight II-Discover 3 (Molecular
Simulations Inc., USA) was used.
[0119] One method for identifying, screening for, searching for,
evaluating, or designing an agonist or an antagonist provided in
accordance with the present invention is executed by entering a
three-dimensional structural coordinate of a structural model for
Meta II, Meta I, Meta Ib, or Meta I.sub.380, each of Which is a G
protein-coupled receptor of the present invention, into a computer
or its storage medium, and displaying, by mans of a suitable
computer program, a three-dimensional structure of the receptor on
a computer screen for visual observation.
[0120] Specifically, a complex of Meta II structural model and a
ligand is displayed on a computer screen. Interactions with amino
acid residues specific to the binding of the ligand to the receptor
is then observed an the computer screen. The ligand is then
chemically or spatially modified and the changes in the local
structural coordinate caused by the modification are corrected by
determining relative spatial positions of atoms in such a manner
that the requirements for chemical bonds are met. In doing so,
agonists or antagonists may be selected from a panel of candidates
or structures of suitable chemical modification groups displayed on
the computer. Alternatively, agonists or antagonists may be
designed by calculating chemical modification groups or structures
with a low energy state.
[0121] According to the present invention, it is also possible to
design a receptor mutant and identify, screen for, search for,
evaluate or design a ligand capable of binding such a mutant. Since
the structural models for the photoactivated intermediates of
rhodopsin are considered to correspond to different structures of
GPCRs, constructing a three-dimensional structural model for a
receptor based on the structure of each intermediate can provide a
clue to understand the specificity of binding of further ligands.
Furthermore, constructing a three-dimensional structural model for
a receptor mutant can provide a clue to understand the specificity
of binding of still further ligands.
[0122] In designing such a receptor mutant, a complex of, for
example, the Meta II structural model and a ligand is displayed on
a computer screen in the same manner as described above.
Subsequently, amino acid residues involved in the interaction with
the ligand, along with amino acid residues in an adjacent region,
are displayed on the computer screen. Mutations such as
substitutions, deletions and insertions or chemical modifications
of one or more amino acid residues are introduced on the computer
screen, and the resulting changes in the interactions with the
ligand are Monitored on the computer screen. The changes in the
local structural coordinate caused by the modification are
corrected by determining relative spatial positions of atoms in
such a manner that the requirements for chemical bonds are met. In
doing so, agonists or antagonists may be selected from a panel of
candidates or structures of suitable chemical modification groups
displayed on the computer. Alternatively, agonists or antagonists
may be designed by calculating chemical modification groups or
structures with a low energy state.
[0123] The receptor mutants so designed can interact more strongly
with ligands that act as antagonists or agonists and thus,
identifying, scanning for, searching for, evaluating, or designing
novel ligands capable of binding the receptor mutant can lead to
discovery of compounds that exhibit higher biological activity and
stability.
[0124] The three-dimensional structural model for GPCRs provided in
accordance with the present invention is based on the crystal
structure of rhodopsin, or in particular, activated intermediates
generated during the photoisomerization reaction of rhodopsin. Each
of the activated intermediates exhibits a specificity with which
the receptor recognizes a ligand either as an antagonist or as an
agonist based on the difference in the position of highly conserved
amino acids in helices that play an important role in the
interaction with the ligand.
[0125] The present invention will now be described in detail with
reference to examples, which are not intended to limit the scope of
the invention in any way. The scope of the invention is deemed to
be defined only by the foregoing description.
EXAMPLE 1
Construction of Models for Photoactivated Intermediates of
Rhodopsin
[0126] Using a molecule modeling software Insight II-Discover 3
(Molecular Simulations Inc., USA), a structural model for each of
the rhodopsin intermediates was generated and was optimized based
an the crystal structure of rhodopsin (Palczewski et al., Science,
289, 144-167, 2000). TM3 was swung about the C .alpha. carbon of
Cys110 to serve as the pivot point while the distance to TM2 was
kept at 5 .ANG. or more. The magnitude of the swing was determined
by taking into consideration the interaction of TM6 with Glu247 for
each of Lumi, Meta I, Meta Ib, and Meta I.sub.380 structures.
Specifically, in each of Lumi, Meta I, Meta Ib, and Meta I.sub.380,
Cys140 on TM3 was swung in such a manner that Cys140 is spaced form
TM6 by a distance of 1.6 .ANG., 4.3 .ANG., 6.8 .ANG., and 9.0
.ANG., respectively. Furthermore, N-terminal (Glu150) of the
portion of TM4 that would interfere with TM3 was swung toward TM5
about Gly174 on the C-terminal of the helix to serve as the pivot
point by a distance of 3.5 .ANG., 7.4 .ANG., 12.1 .ANG., and 17.1
.ANG., respectively, to avoid interference. The structures so
generated were optimized at 300 K by means of molecular kinetics
and molecular dynamics so that C .alpha. carbons of the amino acids
can be fixed as firmly as possible.
[0127] As for the structure of Meta 11, TM6 was rotated clockwise
by an angle of 100 degrees as viewed from the intracellular side,
and the distance between the resides on TM6 and the resides on TM3
was monitored and was decreased to a minima distance that does not
cause steric interference. Upon this, TM5 was twisted about Asn200
in a direction that can avoid steric interference resulting from
the rotation of TM6. TM4 was then translated by a distance of 4.1
.ANG. to place it between TM3 and TM5.
[0128] As a result, the distance between the C .alpha.-carbon of
Cys140 on TM3 and the C .alpha.-carbon of Ala246 on TM6 becomes
12.7 .ANG. and the C .alpha.-carbon of Cys140 on TM3 was positioned
at 4.8 .ANG. from Glu150 on TM4. Leu226 on TM5 was positioned at a
distance of 10.5 .ANG. from Ala246 on TM6. TM5 and TM4 were
monitored so that they would not sterically interfere with TM6. The
structures so generated were optimized at 300 K by means of
molecular kinetics and molecular dynamics so that C .alpha. carbons
of the amino acids can be fixed as firmly as possible.
EXAMPLE 2
Construction of models for GPCR and GPCR/Ligand Complex
[0129] Using the structure of Meta I, Meta Ib, Meta I.sub.380, and
Meta II and based on the homology among the amino acid sequences of
rhodopsin and other GPCRs (FIG. 18), three-dimensional
conformations for binding a full agonist, a partial agonist, an
antagonist, and an inverse agonist were constructed for each of the
GPCRs.
[0130] For each of the GPCRs, a receptor conformation for binding
an inverse agonist was generated by using the structure of Meta I
as a template. Using a homology module of Insight II, amino acid
substitution was carried out, as were insertion or deletion of
amino acid residues in the loop region. Using Discover 3, the
conformation was optimized so that the C .alpha. carbon of the
amino acids was fixed as firmly as possible.
[0131] Likewise, three-dimensional conformations for binding an
antagonist, a partial agonist, and a full agonist that correspond
to Meta Ib, Meta I.sub.380, and Meta II, respectively, were
constructed for each of the receptors and were optimized.
[0132] A ligand corresponding to each conformation of each of the
receptors was manually bound to the ligand-binding site of each
receptor by using the docking method, such as AUTODOCK, or by
mainly forming hydrogen bonds. Using Discover 3, the structure of
the resulting complex was optimized on the basis of molecular
kinetics and molecular dynamics.
EXAMPLE 3
Construction of Structural Model for Adrenaline Receptors Bound to
Antagonist
[0133] Using the structure of rhodopsin Meta Ib as a template, Meta
Ib-Like structural models of antagonist-bound receptor was
constructed for a panel of twelve adrenaline receptors, which form
a class of G protein-coupled receptors (GPCRs).
[0134] To construct the structural model for the panel of
adrenaline receptors, the amino acid sequence of rhodopsin to serve
as a template was first aligned with the amino acid sequences of
the panel of adrenaline receptors for which to construct the
structural model Clustal W was used as the alignment program
(Thompson et al., Nucleic Acids Research, 22:4673-4680(1994)). The
analysis revealed that while the amino acid sequences showed a
relatively low homology to one another, the transmembrane regions,
which include conserved hydrophobic residues and sequence motifs,
are aligned at a relatively high homology, and the less conserved
loop regions tend to include abnormal insertions and deletions.
[0135] Thus, the alignment of the regions with low homology was
carefully manually corrected by comparing with the
three-dimensional structure of rhodopsin to serve as a template and
the amino acid sequences of the other GPCRs. As for the
intracellular loops, no sequence alignment was made, nor was any
model constructed. This is because these regions are diverse among
proteins and numerous insertions and deletions make the
construction of structural models difficult. Also, these regions
are distant from what is considered to be the ligand-binding site
and thus are deemed to have no significant influence on the design
of, for example, antagonists.
[0136] Once constructed, the initial protein structure was refined:
Calculations mere performed in terms of molecular dynamics and
energy minimization with the entire protein except for the regions
including insertions and deletions initially fixed and subsequently
only each backbone fixed. In this manner, distortions in the
initial structural was removed and, as a result, accurate model
structure was constructed.
[0137] While abnormal loop structure was observed in some of the
receptors containing relatively long insertions or deletions, the
correction of the alignment improved the accuracy of the structural
model to some extent.
[0138] For the three-dimensional structure modeling, widely used
Modeler program (Accelrys) was employed. Although making alignment
is a time-consuming process, the alignment, once completed, can be
used repeatedly and thus posed no problem to the modeling process
in terms of time required. The time that it took for the modeling
itself was appropriately one minute for constructing the initial
structure for each receptor and approximately 10 minutes for the
subsequent refinement process.
[0139] FIGS. 19 through 31 show structural models for 12 adrenaline
receptors in their antagonist-bound state.
[0140] As shown, the peptide backbones of the seven-transmembrane
domains, each existing as an .alpha.-helix, are shown by solid
liners while the side chains of amino acid residues that are highly
conserved among the GPCRs and are involved in the interaction with
ligands are shown by ball-and-stick models.
[0141] The spatial arrangement of the seven .alpha.-helices
(transmembrane domains) was identical for each of the structural
models of the antagonist-bond 12 adrenaline receptors constructed
in this embodiment. The spatial arrangement of the .alpha.-helices
was also matched in the Meta Ib structure of rhodopsin shown in
FIG. 5 and in the different antagonist-bound GPCRs shown in FIG. 10
(N-methylscopolamine), FIG. 12 (tiotidine), FIG. 15 (ketangerine),
FIG. 16 (dopamine), and FIG. 17 (sulpiride).
INDUSTRIAL APPLICABILITY
[0142] It is expected that G protein-coupled receptors (GPCRs) will
account for as much as 5% of the human genome, and, given that
those already discovered are included, 2000 or more genes encoding
GPCRs will be discovered. It is therefore known that GPCRs are most
important and diverse receptors responsible for signal transduction
of extracellular information into cells. GPCRs play a special role
in circulatory systems, central nervous systems, and immune systems
and functional implements of these receptors can lead to various
serious diseases. Many drugs are a available and are known to act
on these receptors. There is no doubt that the need for the drugs
that can control functions of these receptors will be significantly
increased in future.
[0143] According to the present invention, once the amino acid
sequence of a known or a newly discovered GPCR is known,
conformations of the receptor to bind full agonists, partial
agonists, antagonists, or inverse agonists can be readily
generated, and the structure of the ligand-binding site of the
receptor provides a clue to create a desired de novo design of
ligand and allows screening of a panel of existing compounds for
compounds that bind each conformation.
[0144] For GPCRs that are orphan receptors, screening for agonists
or antagonists relying for example on their functionalities has
been particularly difficult due to the absence of the molecules
that actually bind the receptors. The receptor structure provided
in accordance with the present invention, however, has
well-understood functionalities and thus serves as a means to
readily find agonists or antagonists. The agonists or the
antagonists can then be used to understand the functions of the
orphan receptor.
[0145] This structure also allows designing constitutively active
receptors and thus, screening for ligands using such mutants. Also,
mutations may be introduced at amino acid residues that
specifically bind an agonist or an antagonist. This allows binding
experiments for screening exclusively for the agonist or the
antagonist.
[0146] Accordingly, the present invention makes a significant
contribution to the development of future pharmaceutical products
and serves as a means to develop pharmaceutical products with less
side effects. TABLE-US-00003 LENGTHY TABLE The patent application
contains a lengthy table section. A copy of the table is available
in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070010948A1)
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Sequence CWU 1
1
42 1 31 PRT Homo sapiens TRANSMEM (1)..(31) human muscarinic
acetylcholine m2 receptor TM 1 1 Phe Glu Val Val Phe Ile Val Leu
Val Ala Ala Ser Leu Ser Leu Val 1 5 10 15 Thr Ile Ile Gly Asn Ile
Leu Val Met Val Ser Ile Lys Val Asn 20 25 30 2 31 PRT Homo sapiens
TRANSMEM (1)..(31) human histamine H2 receptor TM 1 2 Ala Cys Lys
Ile Thr Ile Thr Val Val Leu Ala Val Leu Ile Leu Ile 1 5 10 15 Thr
Val Ala Gly Asn Val Val Val Cys Leu Ala Val Gly Leu Asn 20 25 30 3
31 PRT Homo sapiens TRANSMEM (1)..(31) human serotonin 5HT2A
receptor TM 1 3 Gln Glu Lys Asn Trp Ser Ala Leu Leu Thr Ala Val Val
Ile Ile Leu 1 5 10 15 Thr Ile Ala Ala Asn Ile Leu Val Ile Met Ala
Val Ser Leu Glu 20 25 30 4 31 PRT Homo sapiens TRANSMEM (1)..(31)
human dopamine D2 receptor TM 1 4 Pro His Tyr Asn Tyr Tyr Ala Thr
Leu Leu Thr Leu Leu Ile Ala Val 1 5 10 15 Ile Val Phe Gly Asn Val
Leu Val Cys Met Ala Val Ser Arg Glu 20 25 30 5 31 PRT Homo sapiens
TRANSMEM (1)..(31) human adrenergic b2 receptor TM 1 5 Val Trp Val
Val Gly Met Gly Ile Val Met Ser Leu Ile Val Leu Ala 1 5 10 15 Ile
Val Phe Gly Asn Val Leu Val Ile Thr Ala Ile Ala Lys Phe 20 25 30 6
31 PRT Homo sapiens TRANSMEM (1)..(31) human rhodopsin TM 1 6 Trp
Gln Phe Ser Met Leu Ala Ala Tyr Met Phe Leu Leu Ile Met Leu 1 5 10
15 Gly Phe Pro Ile Asn Phe Leu Thr Leu Tyr Val Thr Val Gln His 20
25 30 7 30 PRT Homo sapiens TRANSMEM (1)..(30) human muscarinic
acetylcholine m2 receptor TM 2 7 Val Asn Asn Tyr Phe Leu Phe Ser
Leu Ala Cys Ala Asp Leu Ile Ile 1 5 10 15 Gly Val Phe Ser Met Asn
Leu Tyr Thr Leu Tyr Thr Val Ile 20 25 30 8 30 PRT Homo sapiens
TRANSMEM (1)..(30) human histamine H2 receptor TM 2 8 Leu Thr Asn
Cys Phe Ile Val Ser Leu Ala Ile Thr Asp Leu Leu Leu 1 5 10 15 Gly
Leu Leu Val Leu Pro Phe Ser Ala Ile Tyr Gln Leu Ser 20 25 30 9 30
PRT Homo sapiens TRANSMEM (1)..(30) human serotonin 5HT2A receptor
TM 2 9 Ala Thr Asn Tyr Phe Leu Met Ser Leu Ala Ile Ala Asp Met Leu
Leu 1 5 10 15 Gly Phe Leu Val Met Pro Val Ser Met Leu Thr Ile Leu
Tyr 20 25 30 10 21 PRT Homo sapiens TRANSMEM (1)..(21) human
dopamine D2 receptor TM 2 10 Thr Thr Asn Tyr Val Ser Ala Val Ala
Asp Val Ala Thr Val Met Trp 1 5 10 15 Val Val Tyr Val Val 20 11 30
PRT Homo sapiens TRANSMEM (1)..(30) human adrenergic b2 receptor TM
2 11 Val Thr Asn Tyr Phe Ile Thr Ser Leu Ala Cys Ala Asp Leu Val
Met 1 5 10 15 Gly Leu Ala Val Val Pro Phe Gly Ala Ala His Ile Leu
Met 20 25 30 12 30 PRT Homo sapiens TRANSMEM (1)..(30) human
rhodopsin TM 2 12 Pro Leu Asn Tyr Ile Leu Leu Asn Leu Ala Val Ala
Asp Leu Phe Met 1 5 10 15 Val Phe Gly Gly Phe Thr Thr Thr Leu Tyr
Thr Ser Leu His 20 25 30 13 35 PRT Homo sapiens TRANSMEM (1)..(35)
human muscarinic acetylcholine m2 receptor TM 3 13 Gly Pro Val Val
Cys Asp Leu Trp Leu Ala Leu Asp Tyr Val Val Ser 1 5 10 15 Asn Ala
Ser Val Met Asn Leu Leu Ile Ile Ser Phe Asp Arg Tyr Phe 20 25 30
Cys Val Thr 35 14 35 PRT Homo sapiens TRANSMEM (1)..(35) human
histamine H2 receptor TM 3 14 Gly Lys Val Phe Cys Asn Ile Tyr Thr
Ser Leu Asp Val Met Leu Cys 1 5 10 15 Thr Ala Ser Ile Leu Asn Leu
Phe Met Ile Ser Leu Asp Arg Tyr Cys 20 25 30 Ala Val Met 35 15 35
PRT Homo sapiens TRANSMEM (1)..(35) human serotonin 5HT2A receptor
TM 3 15 Pro Ser Lys Leu Cys Ala Val Trp Ile Tyr Leu Asp Val Leu Phe
Ser 1 5 10 15 Thr Ala Ser Ile Met His Leu Cys Ala Ile Ser Leu Asp
Arg Tyr Val 20 25 30 Ala Ile Gln 35 16 35 PRT Homo sapiens TRANSMEM
(1)..(35) human dopamine D2 receptor TM 3 16 Ser Arg Ile His Cys
Asp Ile Phe Val Thr Leu Asp Val Met Met Cys 1 5 10 15 Thr Ala Ser
Ile Leu Asn Leu Cys Ala Ile Ser Ile Asp Arg Tyr Thr 20 25 30 Ala
Val Ala 35 17 35 PRT Homo sapiens TRANSMEM (1)..(35) human
adrenergic b2 receptor TM 3 17 Gly Asn Phe Trp Cys Glu Phe Trp Thr
Ser Ile Asp Val Leu Cys Val 1 5 10 15 Thr Ala Ser Ile Glu Thr Leu
Cys Val Ile Ala Val Asp Arg Tyr Phe 20 25 30 Ala Ile Thr 35 18 35
PRT Homo sapiens TRANSMEM (1)..(35) human rhodopsin TM 3 18 Gly Pro
Thr Gly Cys Asn Leu Glu Gly Phe Phe Ala Thr Leu Gly Gly 1 5 10 15
Glu Ile Ala Leu Trp Ser Leu Val Val Leu Ala Ile Glu Arg Tyr Val 20
25 30 Val Val Cys 35 19 26 PRT Homo sapiens TRANSMEM (1)..(26)
human muscarinic acetylcholine m2 receptor TM 4 19 Arg Thr Thr Lys
Met Ala Gly Met Met Ile Ala Ala Ala Trp Val Leu 1 5 10 15 Ser Phe
Ile Leu Trp Ala Pro Ala Ile Leu 20 25 20 26 PRT Homo sapiens
TRANSMEM (1)..(26) human histamine H2 receptor TM 4 20 Val Thr Pro
Val Arg Val Ala Ile Ser Leu Val Leu Ile Trp Val Ile 1 5 10 15 Ser
Ile Thr Leu Ser Phe Leu Ser Ile His 20 25 21 26 PRT Homo sapiens
TRANSMEM (1)..(26) human serotonin 5HT2A receptor TM 4 21 Asn Ser
Arg Thr Lys Ala Phe Leu Lys Ile Ile Ala Val Trp Thr Ile 1 5 10 15
Ser Val Gly Ile Ser Met Pro Ile Pro Val 20 25 22 26 PRT Homo
sapiens TRANSMEM (1)..(26) human dopamine D2 receptor TM 4 22 Ser
Ser Lys Arg Arg Val Thr Val Met Ile Ser Ile Val Trp Val Leu 1 5 10
15 Ser Phe Thr Ile Ser Cys Pro Leu Leu Phe 20 25 23 26 PRT Homo
sapiens TRANSMEM (1)..(26) human adrenergic b2 receptor TM 4 23 Leu
Thr Lys Asn Lys Ala Arg Val Ile Ile Leu Met Val Trp Ile Val 1 5 10
15 Ser Gly Leu Thr Ser Phe Leu Pro Ile Gln 20 25 24 26 PRT Homo
sapiens TRANSMEM (1)..(26) human rhodopsin TM 4 24 Phe Gly Glu Asn
His Ala Ile Met Gly Val Ala Phe Thr Trp Val Met 1 5 10 15 Ala Leu
Ala Cys Ala Ala Pro Pro Leu Val 20 25 25 26 PRT Homo sapiens
TRANSMEM (1)..(26) human muscarinic acetylcholine m2 receptor TM 5
25 Ala Ala Val Thr Phe Gly Thr Ala Ile Ala Ala Phe Tyr Leu Pro Val
1 5 10 15 Ile Ile Met Thr Val Leu Tyr Trp His Ile 20 25 26 26 PRT
Homo sapiens TRANSMEM (1)..(26) human histamine H2 receptor TM 5 26
Glu Val Tyr Gly Leu Val Asp Gly Leu Val Thr Phe Tyr Leu Pro Leu 1 5
10 15 Leu Ile Met Cys Ile Thr Tyr Tyr Arg Ile 20 25 27 26 PRT Homo
sapiens TRANSMEM (1)..(26) human serotonin 5HT2A receptor TM 5 27
Asp Asn Phe Val Leu Ile Gly Ser Phe Val Ser Phe Phe Ile Pro Leu 1 5
10 15 Thr Ile Met Val Ile Thr Tyr Phe Leu Thr 20 25 28 26 PRT Homo
sapiens TRANSMEM (1)..(26) human dopamine D2 receptor TM 5 28 Pro
Ala Phe Val Val Tyr Ser Ser Ile Val Ser Phe Tyr Val Pro Phe 1 5 10
15 Ile Val Thr Leu Leu Val Tyr Ile Lys Ile 20 25 29 26 PRT Homo
sapiens TRANSMEM (1)..(26) human adrenergic b2 receptor TM 5 29 Gln
Ala Tyr Ala Ile Ala Ser Ser Ile Val Ser Phe Tyr Val Pro Leu 1 5 10
15 Val Ile Met Val Phe Val Tyr Ser Arg Val 20 25 30 26 PRT Homo
sapiens TRANSMEM (1)..(26) human rhodopsin TM 5 30 Glu Ser Phe Val
Ile Tyr Met Phe Val Val His Phe Ile Ile Pro Leu 1 5 10 15 Ile Val
Ile Phe Phe Cys Tyr Gly Gln Leu 20 25 31 36 PRT Homo sapiens
TRANSMEM (1)..(36) human muscarinic acetylcholine m2 receptor TM 6
31 Pro Pro Ser Arg Glu Lys Lys Val Thr Arg Thr Ile Leu Ala Ile Leu
1 5 10 15 Leu Ala Phe Ile Ile Thr Trp Ala Pro Tyr Asn Val Met Val
Leu Ile 20 25 30 Asn Thr Phe Cys 35 32 36 PRT Homo sapiens TRANSMEM
(1)..(36) human histamine H2 receptor TM 6 32 Ala Thr Ile Arg Glu
His Lys Ala Thr Val Thr Leu Ala Ala Val Met 1 5 10 15 Gly Ala Phe
Ile Ile Cys Trp Phe Pro Tyr Phe Thr Ala Phe Val Tyr 20 25 30 Arg
Gly Leu Arg 35 33 36 PRT Homo sapiens TRANSMEM (1)..(36) human
serotonin 5HT2A receptor TM 6 33 Ser Ile Ser Asn Glu Gln Lys Ala
Cys Lys Val Leu Gly Ile Val Phe 1 5 10 15 Phe Leu Phe Val Val Met
Trp Cys Pro Phe Phe Ile Thr Asn Ile Met 20 25 30 Ala Val Ile Cys 35
34 36 PRT Homo sapiens TRANSMEM (1)..(36) human dopamine D2
receptor TM 6 34 Ser Gln Gln Lys Glu Lys Lys Ala Thr Gln Met Leu
Ala Ile Val Leu 1 5 10 15 Gly Val Phe Ile Ile Cys Trp Leu Pro Phe
Phe Ile Thr His Ile Leu 20 25 30 Asn Ile His Cys 35 35 36 PRT Homo
sapiens TRANSMEM (1)..(36) human adrenergic b2 receptor TM 6 35 Phe
Cys Leu Lys Glu His Lys Ala Leu Lys Thr Leu Gly Ile Ile Met 1 5 10
15 Gly Thr Phe Thr Leu Cys Trp Leu Pro Phe Phe Ile Val Asn Ile Val
20 25 30 His Val Ile Gln 35 36 36 PRT Homo sapiens TRANSMEM
(1)..(36) human rhodopsin TM 6 36 Thr Gln Lys Ala Glu Lys Glu Val
Thr Arg Met Val Ile Ile Met Val 1 5 10 15 Ile Ala Phe Leu Ile Cys
Trp Leu Pro Tyr Ala Gly Val Ala Phe Tyr 20 25 30 Ile Phe Thr His 35
37 20 PRT Homo sapiens TRANSMEM (1)..(20) human muscarinic
acetylcholine m2 receptor TM 7 37 Gly Tyr Trp Leu Cys Tyr Ile Asn
Ser Thr Ile Asn Pro Ala Cys Tyr 1 5 10 15 Ala Leu Cys Asn 20 38 20
PRT Homo sapiens TRANSMEM (1)..(20) human histamine H2 receptor TM
7 38 Val Leu Trp Leu Gly Tyr Ala Asn Ser Ala Leu Asn Pro Ile Leu
Tyr 1 5 10 15 Ala Ala Leu Asn 20 39 20 PRT Homo sapiens TRANSMEM
(1)..(20) human serotonin 5HT2A receptor TM 7 39 Phe Val Trp Ile
Gly Tyr Leu Ser Ser Ala Val Asn Pro Leu Val Tyr 1 5 10 15 Thr Leu
Phe Asn 20 40 20 PRT Homo sapiens TRANSMEM (1)..(20) human dopamine
D2 receptor TM 7 40 Phe Thr Trp Leu Gly Tyr Val Asn Ser Ala Val Asn
Pro Ile Ile Tyr 1 5 10 15 Thr Thr Phe Asn 20 41 20 PRT Homo sapiens
TRANSMEM (1)..(20) human adrenergic b2 receptor TM 7 41 Ile Asn Trp
Ile Gly Tyr Val Asn Ser Gly Phe Asn Pro Leu Ile Tyr 1 5 10 15 Cys
Arg Ser Pro 20 42 20 PRT Homo sapiens TRANSMEM (1)..(20) human
rhodopsin TM 6 42 Pro Ala Phe Phe Ala Lys Thr Ser Ala Val Tyr Asn
Pro Val Ile Tyr 1 5 10 15 Ile Met Met Asn 20
* * * * *
References