The
Ancestral Protein of the Glucocorticoid
Receptor (GR) and
Mineralcorticoid Receptor (MR)
Haley
Adcox '11 and
Cami Odio '11
Contents:
I.
Introduction
The
glucocorticoid
receptor (GR)
and its sister protein,
mineralcorticoid receptor (MR),
are transcription factors present in all jawed vertebrates
(1).
GR is activated by adrenal steroid cortisol and regulates stress
response, glucose homeostasis, and other functions (2). MR,
on
the other hand, is activated by aldosterone in tetrapods and by
deoxycorticosterone (DOC) in teleosts. It then acts as a
hormone signal regulating
sodium and water homeostasis as well as
cardiovascular function, neuronal fate, and adipocyte
differentiation
(3).
Similarities between the two receptors
and their
ligands
suggests that they descended from a single ancestor.
II.
AncCR (Ancestrol Corticoid Receptor): The ancestor of the sister
proteins MR and GR
A
phylogenetic analysis of existing GR and MR sequences revealed that the
receptors descend from the duplication of a single ancient gene, the
ancestrol
corticoid receptor (AncCR),
in
the vertebrate lineage ~450
million years
ago . This phylogenetic analysis was also used to
infer a
genetic
sequence for the AncCR that was biochemically synthesized,
expressed in
cultured cells, and the resulting protein was crystallized. Overall,
the structure of AncCR
consists of
nine
helices and an activation-function
helix(AF-H).
The
study of
its structure was then used to understand the evolution of MR and GR.
Importantly, the “resurrected” AncCR binds
aldosterone
and
DOC with highest
affinity, while it’s affinity to cortisol is much lower.
Further,
AncCR’s
network of hydrogen bonds
for
aldosterone binding
is
conserved in the
modern MR and GR.
Thus, GR's hormone specificity evolved from the AncCR
through a
specific set of historical mutations without altering these three
residues.
III.
Conformational Epistasis: The evolutionary switch in binding preference
from aldosterone to cortisol (mutations S106P
and
L111Q)
Conformational
epistasis is a mode of structural evolution
where one substitution changes the position and thus, the functional
effect
of a
second substitution. Two mutations of the ancestor protein, S106P
and L111Q,
are characterized as epistasic changes and are conserved in modern GR.
In
combination, the two substitutions destabilize binding with aldosterone
or DOC and re-establish stability in a cortisol-specific fashion. Thus
they switch the binding preference from aldosterone to
cortisol.
Specifically,
these mutations
occur on helices 6
and 7
of
the ancestor protein. The position of helix 7
of
the ancestor protein is
stabilized
by a
hydrogen bond between Ser106
and the
carbonyl backbone
of Met103.
The
S106P
mutation breaks the H-bond and creates a sharp kink in the
backbone.
This kink repositions the loop downward, and partially
unwinds helix
7
(see
'mutations' link above).
Thus, through this destabilization of a crucial region of the
receptor, S106P
impairs activation by all ligands. However, the
movement of helix
7
brings site 111 into
close proximity with
the
binding ligand.
Further, the
mutation of
this site, L111Q,
creates a hydrogen
bond with
cortisol’s C17-hydroxyl (see
'mutations'
link
above). Because
aldosterone and
DOC lack this
hydroxyl, the new bond is cortisol specific. Alone, the L111Q
mutant
has no
effect on the binding
of any of the ligands because it is too far from
the
ligand binding
site. However, the repositioning provided by S106P,
brings
position 111
close to the ligand and increases the effect of the L111Q
mutation.
These
mutations can be
characterized as conformational
epistasis
because both mutations are required for the switch in binding
preference from aldosterone to cortisol.
IV.
Disruption
of the activation-function
helix (AF-H): Three mutations (L29M, F98I,
and
deletion S212∆)
that complete the functional switch
Although
S106P
and
L111Q
mutations
result in
stabilization
of cortisol binding, receptors with these mutations still retain some
affinity
to aldosterone and DOC. Thus, further mutations are required to
decrease
affinity for MR’s ligands, and complete the
switch to the singular cortisol binding that
characterizes
GR. Specifically, the transcriptional activity of all steroid receptors
depends
on the stability of the activation-function
helix (AF-H),
whose
repositioning
due to ligand binding creates an interface for transcriptional
co-activators. The stability of the
AF-H
depends
on interactions between S212,
the loop preceding the AF-H,
the
ligand, and helix
3.
Three
mutations (L29M,
F98I,
and
deletion S212∆
)
decrease
the receptor’s affinity for
aldosterone and
DOC by disrupting
these interactions. In particular, by removing a serine (that contains
an
H-bonding hydroxyl), S212∆
eliminates
a hydrogen bond that stabilizes
the AF-H
loop.
Second, L29M
on
helix
3 creates
a
steric
clash and unfavorable interactions between the inserted methionine and
the D-ring
of aldosterone.
This mutation also enhances
cortisol
specificity by forming an H-bond
between the Met29
and cortisol’s
unique C-17
hydroxyl.
The last mutation, F98I
opens
up
space between the ligand, helix
3 and helix
7 through the elimination
of base
stacking by Phe98
and the insertion of the longer side chain Ile29.
This indirectly de-stabilizes the AF-H thus
impairing activation by all
ligands. However, the space created by F98I
does
stabilize the crucial H-bond
between Q111
and
cortisol's C -17
hydroxyl
by relieving a
steric clash
between the repositioned loop
and Met108
.
V.
Permissive
mutations
(N26T, Q105L, and Y27R): Stabilizing receptors to tolerate the
functional
switch
The
three
mutations (L29M,
F98I,
and
deletion S212∆
)
that
decrease binding affinity for MR’s
ligands and thus complete the functional switch to singular cortisol
binding,
actually result in nonfunctional receptors that cannot activate
transcription.
Thus, more mutations are required to stabilize specific elements of the
protein
allowing it to tolerate the function switching changes. These
mutations, which
have no consequence in the ancestor receptor but stabilize the
functional-switches of the new form, are called permissive mutations.
The first
of these, N26T,
creates a new hydrogen bond on the loop between
helix
3 and
the AF-H
.
The
next
permissive mutation, Q105L,
allows helix 7
to
pack more
tightly against helix
3 by
increasing Van-der-waals interactions.
As
a
result,
this mutation stabilizes helix
3 and
indirectly stabilizes AF-H.
These
two
permissive substitutions restore transcriptional activity to the
receptor in a
fully GR-like fashion. A final permissive mutation that may have
evolved even
earlier than those previously noted is Y27R.
The
permissive character
of this
mutation is supported by the finding that it has a negligible effect on
AncCR,
but it’s reversal in receptors containing cortisol binding
preference
abolishes ligand
activation.
Specifically, the inserted arginine stabilizes
helix
3 and
the ligand
pocket by
forming a
cation-π;
interaction with Tyr17
.
VI.
The
Overall
Evolution by Epistasis: from AncCR to MR and GR
The
overall evolution from the AncCR
to
MR and GR
resulted
from a gene duplication of the ancestor ~450 million years ago. One of
these
duplicates, which resulted in MR, has been conserved for ~400 million
years.
Consequently, it binds preferentially to aldosterone and DOC in a
similar
fashion as AncCR. The other gene duplicate evolved to GR through
multiple
mutations that resulted in high affinity binding for cortisol
and very
low
activation by aldosterone and DOC.
Because
some of these
mutations (namely those that complete the functional
switch: L29M,
F98I,
and
deletion S212∆)
abolish ligand binding all
together, evolution by epistasis was crucial to maintaining an active
receptor.
Specifically, the permissive mutations (N26T,
Q105L,
and Y27R)
had to occur on the same
receptors as
the evolutionary switch mutations (S106P
and
L111Q) and
before the AF-H
disrupting
mutations
could occur. If the
AF-H
disrupting
mutations occurred
before the
other two groups of mutations could be
coupled
on a receptor a non-functional intermediate would have resulted. In
general,
non-functional intermediates are less likely to support evolutionary
trajectories. Thus, a
specified order
of the three mutations in which
permissive substitutions occurred before the destabilizing mutations
that
confer new function was crucial to GR development. This
order of
general mutations along with a high incidence of conformational
epistasis
indicates two factors that may be crucial to all genetic evolution and
should
be considered in studies that involve evolutionary trajectories.
References:
(1) Eric
A. Ortlund, Jamie T.
Bridgham, Matthew R. Redinbo, and Joseph W. Thornton (2007). "Crystal
structure of an ancient protein: evolution by conformational
epistasis." Science
317: 1544-1548
(2)
Lombes, M., L.
Pascual-Le
Tallec. (2005). "The mineralocorticoid receptor: a journey
exploring its diversity and specificity of action." Molecular
Endrocrinology. 9:2211-21
(3) Lu NZ, Wardell SE, Burnstein KL, Defranco D, Fuller PJ, Giguere V,
Hochberg RB, McKay L, Renoir JM, Weigel NL, Wilson EM, McDonnell DP,
Cidlowski JA (2006). "International Union of Pharmacology. LXV. The
pharmacology and classification of the nuclear receptor superfamily:
glucocorticoid, mineralocorticoid, progesterone, and androgen
receptors". Pharmacol Revl
58 (4):782–97.
Back to
Top