Homodimeric
DNA-Binding
and Ligand-Binding Interactions of Human Thyroid Hormone
Receptor
Justin Taft '13, Andrew Gipson '13, and Patrick Mershon '14
Contents:
I.
Introduction
The organization
of a
complex multicellular organism
requires a sophisticated system of chemical communication to coordinate
the
cooperation
of various tissues. Depending on stage
of development,
dietary intake, or even mood, the profile of signaling molecules
in an
animal will change as a response to
these conditions. These responses
are regulated by a group of chemical-synthesizing organs known
collectively as the endocrine
system.
The thyroid is an endocrine
gland
responsible for regulating metabolism and growth. Its primary mode of
effect is the production of the thyroid hormone thyroxine (
),
which is converted
to triiodothyronine (
)
in a tissue-specific manner. These
hormones both act as transcriptional activators, binding the nuclear
receptor known as thyroid hormone receptor (TR), a transcription factor
constitutively bound to hormone response elements (HREs). Like most
nuclear receptors, TR does not bind to DNA alone; rather, it most
frequently forms a heterodimer with the retinoid X receptor (RXR),
another nuclear receptor. The heterodimer acts as a transcriptional
activator.
Occasionally it forms a homodimer with another TR molecule and acts as
a repressor until a hormone ligand arrives and causes it to dissociate.
TR
proteins are encoded by two genes, TR alpha and TR beta,
with multiple isoforms for each gene. Although their central role as a
transcription factor is the same in all forms, they exhibit
differential expression at different times in development and in
different tissues. Despite these differences, the TRs all share a
domain scheme common to the nuclear receptors. The A/B domain at the
amino terminus is involved in regulation of tertiary structure. Moving
towards the carboxy terminus, the DNA binding domain (DBD) binds to the
HREs with sequence specificity. The hinge domain is a flexible linker
that connects the DBD to the ligand binding domain (LBD). The LBD
mediates dimerization interactions and binds a wide variety of ligands,
including endogenous hormones and drugs that affect its function as a
transcriptional activator or repressor.
II.
Comparison of TR-β
LBD with T3 Vs. T4
TR
binds the active form of thyroid hormone triiodothyronine (T3) with
high affinity, but also binds its precursor thyroxine (T4) with a
roughly 30-fold lower affinity. TR-T4 complexes are less stable than
TR-T3 complexes, and dissociate more readily. While the protein adopts
a different conformation with each ligand, T4 can also act as an
agonist in the cell. T4’s agonist activity is only about 10%
that
of T3, but 4- to 6-fold higher T4 levels mean that T4 likely plays an
active role in TR activation.
When
bound to T3 or T4, the ligand binding domain (LBD) undergoes a
conformational
change that packs the
C-terminal helix 12
against
the LBD, burying the hormone
in the ligand-binding pocket.
The T3-bound LBD has a smaller binding pocket than the T4-bound
structure; the pocket expands to accommodate the bulky 5’
iodine group
not
present in T3. In order to accommodate this group, backbone shifts
occur in Helix
12,
the H11-H12
loop and the wall of the
binding pocket near the
5’ iodine
.
Other backbone shifts
involve residues
199-212, Helix
2 residues 234-243, regions
of beta
sheet (residues 318-321 and
325-339), the H2-H3
loop (232-236), and regions
of Helix
3
(248-267)
.
The
conformational change in helix 12 that occurs in either T3 or T4
binding is sufficient to cause folding into the active conformation.
This causes the region of the LBD involved in dimerization to assume
the proper positioning.
Both ligands therefore stimulate the association of coactivators, the
release of
corepressors, and the dissociation of TR homodimers (although not
TR-RXR
heterodimers).
III.
TR-β
LBD + HPPE (Inhibitor)
There are several diseases associated with nuclear receptor activity,
but some of the more dangerous involve excess production of thyroid
hormone, the most severe cases of which are called thyroid storms. Many
drugs used in the treatment of these conditions resemble agonists
of TR
and so bind to the LBD to prevent activation of TR by those agonists,
but they lack specificity and will bind the LBD of other nuclear
receptors, causing unwanted side effects. This problem can be
circumvented by targeting other regions more specific to TR to inhibit
functions such as DNA or coactivator binding. This latter approach is
the method by which the surface-interacting drug DHPPA and its active
form HPPE act on
TRβ.
TRβ
is normally activated by dimerizing with the steroid receptor
coactivator 2 (SRC2) after hormone binding induces a conformational
change in the collection
of proximate residues
called the
ligand-dependent transactivation function
AF-2
,
which then recruits
SRC2. The addition of
DHPPA prevents this dimerization and thus inhibits the activating
function of TRβ.
However, DHPPA is not
the molecule contributing directly to this effect; rather, a derived
form called
HPPE (Inhibitor
Structures) is the
compound responsible for forming a covalent bond at the AF-2 site and
occluding SRC2. DHPPA rapidly converts to HPPE at the AF-2 surface to
form the intermediate structure shown here
,
and it may be that the
protein itself catalyzes this reaction by an as yet undetermined
mechanism.
HPPE is positioned in the AF-2 pocket through electrostatic
interactions
between the
carbonyl oxygen of HPPE and residues K306 and
E457, whereas the alkyl chain and aromatic ring of HPPE are held in
place by hydrophobic interactions with residues L454,
V284,
and I302
.
The beta carbon of the enone group of HPPE is positioned 6.5 angstroms
from
.
Surprisingly, though, it is C298 that is covalently bound
to
HPPE
.
The proximity of the
residue to the carbonyl group of HPPE would
suggest that the sulfhydryl group of the cysteine attacks the carbonyl
directly to form the bond; however, the inactivity of the HPPE analog
HPPA, which lacks the alpha-beta unsaturated moiety
(see Inhibitor Structures), gives
evidence that the sulfhydryl undergoes 1,4-nucleophilic addition with
the beta carbon of HPPE. How the molecule is oriented to fit this
predicted bonding configuration is unclear, but the distance to C298
is only 10.0 angstroms; this implies that it would not be necessary for
the
entire molecule to shift in order to accommodate the
bond.
Regardless of the exact position of covalently bound HPPE, its presence
in the AF-2 pocket occludes the binding of SRC2 through steric
interference. Short domains of the coactivator that have the consensus
sequence L-X-X-L-L normally interact with the six hydrophobic
residues
of AF-2
.
Similar
to drugs that act on the ligand-binding pocket, the bulky hydrophobic
features of HPPE act as an insurmountable
barrier to coactivator
binding
.
IV.
Homodimeric DNA Binding
TR,
like all other nuclear receptors, interacts with DNA in a sequence
specific
manner, binding to thyroid responsive elements (TREs) located near the
promoter
region of
the target gene. Specifically, a monomer of the TR DNA
binding domain
(DBD) recognizes a highly conserved core hexameric sequence of the TRE
(5’-AGGTCA-3’),
often referred to as a half-site,
that is the generic sequence
of all steroidal and non-steroidal nuclear receptors.
This consensus
sequence
often exists as two repeats in the genome that can be arranged in
multiple
ways, and this arrangement appears to dictate the inter-subunit
interactions of
the DBD. Thus, these differing TRE conformations provide an additional
means of
gene regulation beyond ligand-binding. For example, the TRβ
homodimer
selectively binds
a less common palindromic repeat separated by 6 bp,
while the
more common TR/RXR heterodimer preferentially binds a direct repeat
separated by
4 bp. Mutational experiments have shown that insertions and deletions
in the
DNA separating the consensus
sequences disrupt the optimal spatial
arrangement
that allows inter-subunit contacts by the C-terminal extension (CTE)
that can
be vital for dimerization.
In
this instance, each TRβ
homodimer subunit binds a palindromic
half-site
of DNA separated by a 6
bp insert
.
The TR-DBD is composed of
two nonequivalent
zinc finger
motifs, characteristic
of nuclear receptors
,
and contains three main helices. The N-terminal helix (Cys 18 - Lys31)
or recognition
helix,
inserts
into the major groove and is responsible for the vast
majority of DNA
contacts. Base-Protein
Interactions
in the recognition
helix occur
between
Glu19, Arg26, Arg27 and G4, A5, C6 on the
5’ to 3’ strand, and
between Lys22, Arg26 and G16, G17 on the 3’ to 5’
strand
.
Helix
3 (Cys55 - Val66) lies
perpendicular to helix one and
participates in stabilizing
hydrophobic interactions with aromatic residues (Phe24, Phe25, Phe50,
Ile29 and Leu64) from both the recognition helix
and helix
2(Leu33 - Ser36)
.
The T-box
region
(69 - 76) is located at the 5’ end of the
CTE and acts as a
hinge between the CTE and the DBD via the helical
turn (Thr
70 - Leu72)
.
The hinge action of the helical turn allows the CTE to
interact with
the phosphate backbone as well as insert into the minor grove and make
direct
base contacts outside of the consensus repeat sequence.
V.
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