Binding
Pocket Specificity and Key Amino Acids
Zach Morrow '14 and Kotiba Malek '14
Contents:
I.
Introduction
The Androgen
receptor (AR), is a member of the nuclear receptor superfamily. The
protein activated by binding natural and artificial
androgenic hormones such as testosterone and
dihydrotestosterone (DHT), both of which are naturally occuring
steriods, and tetrahydrogestrinone
(THG), an artifical anabolic steroid (1).
After binding, the AR translocates from the cytoplasm into the nucleus
and bindings to Androgen
Response Elemets
(ARE's) as a homodimer. ARE's
typically found in the regulatory regions of genes involved in male
sexual differentiation.
When bound at ARE's, the AR has been found to interact directly with
basal transcription facotrs, most notably, TFIIF (2).
The
AR has been exploited
for years by athletes looking to gain a competitive edge. Artificial
steriods have been
synthesized to bind the AR, and thus increase the expression of genes
which lead to increased muscle mass and strength (3). THG has been
demonstrated to bind with such high affinity to the AR, that at equal
concentrations,
THG will displace natural hormones with 100% efficiency (1). THG
was found to have
the highest binding affinity, followed by DHT and then testosterone.
Much
research has been done to illucidate the structural features of these
steriods and the binding domain of the AR that lead to such increases
in binding affinity.
This tutorial investigates the structural features of testosterone and
THG that
lead to differing binding affinities to the AR.
II.
General Structure
The
AR is composed of three functional domains: the N-Terminal Domain,
the DNA binding domain, and the Ligand Binding Domain (LBD). The
N-terminal domain is responsible for the recruitment of various
transcription factors (2). The
%nbsp
is
composed of two helix turn helix motifs, each belonging to one of the
AR subunits of the homodimer. One alpha
helix is
inserted
into the
major groove of an ARE, and the second
alpha helix,
separated from the
first by a variable chain and fits across the DNA phosphate backbone,
thus
stabilizing the binding interaction
(4).
The
LBD is composed of eleven alpha
helices,
and four
short beta sheets
connected
by
short amino acid chains (in white).
Most important to the LBD
is the Ligand Binding Pocket (LBP). It is here that the interactions
between the AR and
different steroids occur (1). Of interest is the manner in which
the LBD interacts
with different ligands. When
liganded, the LBP adopts a conformation typical of most nuclear
receptor proteins. Three layers of alpha helices arrange in an
anti-parallel manner to form a pocket that embraces the ligand (DHT
shown here)
.
Specific interactions between residues and ligands are dependant on
ligand structure. Click
Here
for Ligand Structures.
III.
Hydrogen Bonding Interactions
All
androgens that bind the AR with high affinity possess a
carbonyl oxygen
at
C3,
and a hydroxy function
at C17
.
The carbony oxygen at C3
has lone pairs of electrons capable of being hydrogen bond acceptors,
and the hydroxy function at C17 can act as both a donor and
acceptor. It is the hydrogen bonding of these
functionalities to amino acid side chains that orients the
ligand molecule in the proper position for other interactions to occur.
Two amino acid side chains are capable
of making h-bond interactions with the carbonyl oxygen on C3. Arg752 and Gln711
both
interact with the carbonyl oxygen, but mutations in Arg752 have
been shown to cause Androgen Insensitivity Syndrom, while mutations
in Gln711
have minimal
effects. Arg752 and Gln711
make
water mediated hydrogen bonds to the carbonyl oxygen
.
At the other end of the steroid molecule, Asn705
and Thr877 make
hydrogen bonds with the C17 hydroxyl group.
IV.
Determinants of Binding Specificity
Within the LBP of the AR, a large,
apolar pocket is formed, being
composed of many amino acid side chains that interact
the ligand, thus creating an energetically stable
environment. Loss of these interactions energetically
costly and disfavored. The LBP is composed of mainly hydrophobic amino
acid residues
(except for those involved in h-bonding). Extensive van der Waals
interactions
between the ligand and LBP are made. Here, testosterone is shown in the
LBP of the AR
. Testosterone is composed of four
carbon rings.
.This
general structure is consistant with most steriods that interact with
the AR. The
four carbon rings provide a surface for van der Waals interactions to
occur. Differing numbers of
van der Waals contacts are made between the
ligand and LBP for different steriods. The number of hydrophobic
interactions is hypothesized to be a
factor leading to differing binding affinities.
The side chains of
amino acid residues
within the LBP are highly mobile. It is
hypothesized that the mobility of these residues is a major factor that
enables the AR to bind
many different ligands. For instance, here we compare the structure of
the LBP amino acid residues when the AR is complexed with testosterone,
which shows low binding affinity (about 93% less), to
another ligand, THG. When bound to testosterone, the distance of Met745
side chain to C4 where it is thought to make van der Waals
contacts was not resolved due to its high mobility and distance from
the steroid
.
The methyl group on C19 is thought to cause steric clash with the side
chain, and thus an energetically disfavorable interaction there,
influencing the spacial arrangement of this side chain. Another factor
leading to high mobility of this residue when bound to testosterone is
the lack of conjugated
pi systems within the ligand molecule. Without conjugated pi systems,
the ligand adopts many differing conformations within the LBP,
thus creating an environment where Met745 must
similarly adopt many
conformations. This differs from Met745 in
the AR
complexed with
THG. This ligand has considerably more conjugated pi systems, confering
a
higher level of rigidity, thus disfavoring the mobility of Met745 when
the
AR is complexed
with this ligand
.
The distance of this side chain to THG was determined to be 3.77
angstroms, close enough to have extensive non-polar interactions. Click
both buttons in the above section in sequence to observe the differing
conformations of Met745 in complex
with testosterone and then THG.
Trp741 is
another amino acid that
plays a role in determining binding affinity
for differing ligands. When testosterone is bound to the AR, it
has been shown that Met745 sterically
clashes
with Trp741 due
to it's high
mobility and distance from the ligand
,
while when THG is bound, Met745
takes a much
more stable conformation near the ligand, allowing Trp741 to
make apolar
interactions with the face of the ligand.
The observed
difference in Trp741
location
is
a nearly 60 degree rotation about it's beta carbon.
Further adding to the equation of
binding specificity is Met895, which
in the AR bound to testosterone, adopts a position
that is 1.2 angstroms closer to the ligand than when THG is bound. It
is not well understood the effect this different conformation has on
binding specificity, but the change is still noteworthy. Also of
interest is Leu701 which also adopts differing conformations depending
on the bound ligand.
Based upon electron density map analysis, it has been determined that
testosterone makes 20 van der Waals contacts with the
LBP
,
and THG makes 26
.
An increased
number of favorable contacts confers increased stability and thus a
higher binding affinity as is observed. Comparison of the AR bound to
both ligands shows that fewer side chains contact testosterone than
THG. Possible reasons for this are steric clash of the less rigid
testosterone molecule with these side chains as well as THG being
slighly larger, with two ethyl groups protruding from C13 and C18,
providing more surface for van der Waals interactions. Further research
into the
effects of the differing
ligand structures and side chain conformations in the LBP of
the AR will enable the determination of
more factors that affect binding specificity based upon the structure
of the
LBD.
V.
References
(1) Jesus-Tran, K. P, P. C. Cote, L. Cantin,
J. Blanchet, F. Labrie, R.
Breton. “Comparison of crystal
structures of human androgen
receptor ligand-binding domain complexed
with various
agonists reveals molecular determinants
responsible for
binding affinity.” Oncology
and Molecular Endocrinology
Research Center. (FEB 2006)
(2) McEwan, I.J., J. A. Gustafsson.
“Interaction of the human
androgen
receptor transactivation function with the general
transcription factor TFIIF.”
Department of Biosciences. Vol. 94,
(AUG 1997):8485-8490.
(3) Kadi, F., P. Bonnerud, A. Eriksson, L.E.
Thornell. “The
expression of androgen receptors in
human neck and limb
muscles:
effects of training and self-administration of
androgenic-anabolic steroids.”
Histochem Cell Biol (2000). 113:
25-29.
(4) Shaffer,
P.L., A. Jivan, D. E. Dollins, F. Claessens, D. T. Gewirth.
“Structural basis of androgen receptor binding to selective
androgen response elements.”
Proc. Natl. Acad. Sci USA. Vol.
101, No. 14. (APR 2004):
4758-4763.
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