Summarised: http://creationismvevolution.blogspot.com/2007/08/molecular-evolution-proven.html
From:
http://www.bay-of-fundie.com/archives/208/molecular-evolution-proven#comments
my CommentI've just started blogging (today) on Creationism v Evolution
I bought Michael Behe 'Darwins Black Box' a few years ago - but its still in my 'to be read' book section.
From Science Daily: http://www.sciencedaily.com/releases/2007/08/070816143825.htm
"The work involving the protein is detailed in a paper appearing online Aug. 16 in Science Express (abstract here): http://www.sciencemag.org/cgi/content/abstract/1142819
where the journal Science promotes selected research in advance of regular publication."
The University of Oregon have the press release: http://www.uoregon.edu/newsstory.php?a=8.16.07-Ancient-Proteins.html
Supporting Online Material for
Crystal Structure of an Ancient Protein: Evolution by Conformational
Epistasis: http://www.sciencemag.org/cgi/data/1142819/DC1/1
contents: Materials and Methods, Figures, Tables and References
It would be interesting to read the full Science article - cost $10
http://www.sciencemag.org/cgi/rapidpdf/1142819.pdf
www.sciencexpress.org / 16 August 2007/ Page 1 / 10.1126/science.1142819
downloaded here: C:\Users\cstreet\Documents\Home\Interests\Creationism v Evolution\1142819_Science.pdf
I've reposted some of this article here:-Supporting Online Material for
Crystal Structure of an Ancient Protein: Evolution by Conformational
Epistasis:
http://www.sciencemag.org/cgi/data/1142819/DC1/1(download FOC for Figures, Tables and References)
The structural mechanisms by which proteins have
evolved new functions are known only indirectly. We
report x-ray crystal structures of a resurrected ancestral
protein—the ~450 million-year-old precursor of
vertebrate glucocorticoid (GR) and mineralocorticoid
(MR) receptors. Using structural, phylogenetic, and
functional analysis,
we identify the specific set of
historical mutations that recapitulate the evolution of
GR’s hormone specificity from an MR-like ancestor.
These substitutions repositioned crucial residues to create
new receptor-ligand and intraprotein contacts. Strong
epistatic interactions occur because one substitution
changes the conformational position of another site.
“Permissive” mutations—substitutions of no immediate
consequence, which stabilize specific elements of the
protein and allow it to tolerate subsequent function switching
changes—played a major role in determining
GR’s evolutionary trajectory.
A central goal in molecular evolution is to understand the
mechanisms and dynamics by which changes in gene
sequence generate shifts in function and therefore phenotype
(1, 2). A complete understanding of this process requires
analysis of how changes in protein structure mediate the
effects of mutations on function. Comparative analyses of
extant proteins have provided indirect insights into the
diversification of protein structure (3–6), and protein
engineering studies have elucidated structure-function
relations that shape the evolutionary process (7–11). To
directly identify the mechanisms by which historical
mutations generated new functions, however, it is necessary
to compare proteins through evolutionary time.
Here we report the empirical structures of an ancient
protein, which we “resurrected” (12) by phylogenetically
determining its maximum likelihood sequence from a large
database of extant sequences, biochemically synthesizing a
gene coding for the inferred ancestral protein, expressing it in
cultured cells, and determining the protein’s structure by xray
crystallography.
Specifically, we investigated the
mechanistic basis for the functional evolution of the
glucocorticoid receptor (GR), a hormone-regulated
transcription factor present in all jawed vertebrates (13). GR
and its sister gene, the mineralocorticoid receptor (MR),
descend from the duplication of a single ancient gene, the
ancestral corticoid receptor (AncCR), deep in the vertebrate
lineage ~450 million years ago (Ma) (Fig. 1A) (13).
GR is
activated by the adrenal steroid cortisol and regulates stress
response, glucose homeostasis, and other functions (14). MR
is activated by aldosterone in tetrapods and by
deoxycorticosterone (DOC) in teleosts to control electrolyte
homeostasis, kidney and colon function, and other processes
(14). MR is also sensitive to cortisol, though considerably
less so than to aldosterone and DOC (13, 15). Previously,
AncCR was resurrected and found to have MR-like
sensitivity to aldosterone, DOC, and cortisol, indicating that
GR’s cortisol specificity is evolutionarily derived (13).
To identify the structural mechanisms by which GR
evolved this new function, we used x-ray crystallography to
determine the structures of the resurrected AncCR ligandbinding
domain (LBD) in complex with aldosterone, DOC,
and cortisol (16) at 1.9, 2.0, and 2.4 Å resolution, respectively
(table S1). All structures adopt the classic active
conformation for nuclear receptors (17), with unambiguous
electron density for each hormone (Fig. 1B and figs. S1 and
S2). AncCR’s structure is extremely similar to the human MR
[root mean square deviation (RMSD) = 0.9 Å for all
backbone atoms] and, to a lesser extent, to the human GR
(RMSD = 1.2 Å). The network of hydrogen-bonds supporting
activation in the human MR (18) is present in AncCR,
indicating that MR’s structural mode of action has been
conserved for >400 million years (fig. S3).
Because aldosterone evolved only in the tetrapods, tens of
millions of years after AncCR, that receptor’s sensitivity to
aldosterone was surprising (13). The AncCR-ligand structures
indicate that the receptor’s ancient response to aldosterone
was a structural by-product of its sensitivity to DOC, the
likely ancestral ligand, which it binds almost identically (Fig.
1C). Key contacts for binding DOC involve conserved
surfaces among the hormones, and no obligate contacts are
made with moieties at C11, C17, and C18, the only variable
positions among the three hormones. These inferences are
robust to uncertainty in the sequence reconstruction: We
modeled each plausible alternate reconstruction [posterior
probability (PP) > 0.20] into the AncCR crystal structures and
found that none significantly affected the backbone
conformation or ligand interactions. The receptor, therefore,
had the structural potential to be fortuitously activated by
aldosterone when that hormone evolved tens of millions of
years later, providing the mechanism for evolution of the
MR-aldosterone partnership by molecular exploitation, as
described (13).
To determine how GR’s preference for cortisol evolved,
we identified substitutions that occurred during the same
period as the shift in GR function. We used maximum
likelihood phylogenetics to determine the sequences of
ancestral receptors along the GR lineage (16). The
reconstructions had strong support, with mean PP >0.93 and
the vast majority of sites with PP >0.90 (tables S2 and S3).
We synthesized a cDNA for each reconstructed LBD,
expressed it in cultured cells, and experimentally
characterized its hormone sensitivity in a reporter gene
transcription assay (16). GR from the common ancestor of all
jawed vertebrates (AncGR1 in Fig. 1A) retained AncCR’s
sensitivity to aldosterone, DOC, and cortisol. At the next
node, however, GR from the common ancestor of bony
vertebrates (AncGR2) had a phenotype like that of modern
GRs, responding only to cortisol. This inference is robust to
reconstruction uncertainty: We introduced plausible
alternative states by mutagenesis, but none changed function
(fig. S4). GR’s specificity therefore evolved during the
interval between these two speciation events, ~420 to 440 Ma
(19, 20).
During this interval, there were 36 substitutions and one
single-codon deletion (figs. S5 and S6). Four substitutions
and the deletion are conserved in one state in all GRs that
descend from AncGR2 and in another state in all receptors
with the ancestral function. Two of these—S106P and L111Q
(21)—were previously identified as increasing cortisol
specificity when introduced into AncCR (13). We introduced
these substitutions into AncGR1 and found that they
recapitulate a large portion of the functional shift from
AncGR1 to AncGR2, radically reducing aldosterone and
DOC response while maintaining moderate sensitivity to
cortisol (Fig. 2A); the concentrations required for halfmaximal
activation (EC50) by aldosterone and DOC increased
by 169- and 57-fold, respectively, whereas that for cortisol
increased only twofold. A strong epistatic interaction between
substitutions was apparent: L111Q alone had little effect on
sensitivity to any hormone, but S106P dramatically reduced
activation by all ligands. Only the combination switched
receptor preference from aldosterone and DOC to cortisol.
Introducing these historical substitutions into the human MR
yielded a completely nonfunctional receptor, as did reversing
them in the human GR (fig. S7). These results emphasize the
importance of having the ancestral sequence to reveal the
functional impacts of historical substitutions.
To determine the mechanism by which these two
substitutions shift function, we compared the structures of
AncGR1 and AncGR2, which were generated by homology
modeling and energy minimization based on the AncCR and
human GR crystal structures, respectively (16). These
structures are robust to uncertainty in the reconstruction:
Modeling plausible alternate states did not significantly alter
backbone conformation, interactions with ligand, or
intraprotein interactions. The major structural difference
between AncGR1 and AncGR2 involves helix 7 and the loop
preceding it, which contain S106P and L111Q and form part
of the ligand pocket (Fig. 2B and fig. S8). In AncGR1 and
AncCR, the loop’s position is stabilized by a hydrogen bond
between Ser106 and the backbone carbonyl of Met103.
Replacing Ser106 with proline in the derived GRs breaks this
bond and introduces a sharp kink into the backbone, which
pulls the loop downward, repositioning and partially
unwinding helix 7. By destabilizing this crucial region of the
receptor, S106P impairs activation by all ligands. The
movement of helix 7, however, also dramatically repositions
site 111, bringing it close to the ligand. In this conformational
background, L111Q generates a hydrogen bond with
cortisol’s C17-hydroxyl, stabilizing the receptor-hormone
complex. Aldosterone and DOC lack this hydroxyl, so the
new bond is cortisol-specific. The net effect of these two
substitutions is to destabilize the receptor complex with
aldosterone- or DOC and restore stability in a cortisolspecific
fashion, switching AncGR2’s preference to that
hormone.
We call this mode of structural evolution
conformational epistasis, because one substitution remodels
the protein backbone and repositions a second site, changing
the functional effect of substitution at the latter.
Although S106P and L111Q (“group X” for convenience)
recapitulate the evolutionary switch in preference from
aldosterone to cortisol, the receptor retains some sensitivity to
MR’s ligands, unlike AncGR2 and extant GRs. We
hypothesized that the other three strictly conserved changes
that occurred between AncGR1 and AncGR2 (L29M, F98I,
and deletion S212Δ) would complete the functional switch.
Surprisingly, introducing these “group Y” changes into the
AncGR1 and AncGR1 + X backgrounds produced completely
nonfunctional receptors that cannot activate transcription,
even in the presence of high ligand concentrations (Fig. 3A).
Additional epistatic substitutions must have modulated the
effect of group Y, which provided a permissive background
for their evolution that was not yet present in AncGR1.
The AncCR crystal structure allowed us to identify these
permissive mutations by analyzing the effects of group Y
substitutions (Fig. 3B). In all steroid receptors, transcriptional
activity depends on the stability of an activation-function
helix (AF-H), which is repositioned when the ligand binds ,
generating the interface for transcriptional coactivators. The
stability of this orientation is determined by a network of
interactions among three structural elements: the loop
preceding AF-H, the ligand, and helix 3 (17). Group Y
substitutions compromise activation because they disrupt this
network. S212Δ eliminates a hydrogen bond that directly
stabilizes the AF-H loop, and L29M on helix 3 creates a steric
clash and unfavorable interactions with the D-ring of the
hormone. F98I opens up space between helix 3, helix 7, and
the ligand; the resulting instability is transmitted indirectly to
AF-H, impairing activation by all ligands (Fig. 3B). If the
protein could tolerate group Y, however, the structures
predict that these mutations would enhance cortisol
specificity: L29M forms a hydrogen bond with cortisol’s
unique C17-hydroxyl, and the additional space created by
F98I relieves a steric clash between the repositioned loop and
Met108, stabilizing the key interaction between Q111 and the
C17-hydroxyl (Fig. 3B).
We hypothesized that historical substitutions that added
stability to the regions destabilized by group Y might have
permitted the evolving protein to tolerate group Y mutations
and to complete the GR phenotype. Structural analysis
suggested two candidates (group Z): N26T generates a new
hydrogen bond between helix 3 and the AF-H loop, and
Q105L allows helix 7 to pack more tightly against helix 3,
stabilizing the latter and, indirectly, AF-H (Fig. 3B). As
predicted, introducing group Z into the nonfunctional
AncGR1 + X + Y receptor restored transcriptional activity,
indicating that Z is permissive for Y (Fig. 3A). Further,
AncGR1 + X + Y + Z displays a fully GR-like phenotype that
is unresponsive to aldosterone and DOC and maintains
moderate cortisol sensitivity. Both N26T and Q105L are
required for this effect (table S4). Strong epistasis is again
apparent: Adding group Z substitutions in the absence of Y
has little or no effect on ligand-activated transcription,
presumably because the receptor has not yet been destabilized
(Fig. 3A). Evolutionary trajectories that pass through
functional intermediates are more likely than those involving
nonfunctional steps (22), so the only historically likely
pathways to AncGR2 are those in which the permissive
substitutions of group Z and the large-effect mutations of
group X occurred before group Y was complete (Fig. 3C).
Our discovery of permissive substitutions in the AncGR1-
AncGR2 interval suggested that other permissive mutations
might have evolved even earlier. We used the structures to
predict whether any of the 25 substitutions between AncCR
and AncGR1 (fig. S5) might be required for the receptor to
tolerate the substitutions that later yielded GR function. Only
one was predicted to be important: Y27R, which is conserved
in all GRs, stabilizes helix 3 and the ligand pocket by forming
a cation-π interaction with Tyr17 (Fig. 4A). When we reversed
Y27R in the GR-like AncGR1 + X + Y + Z, activation by all
ligands was indeed abolished (Fig. 4B). In contrast,
introducing Y27R into AncCR (Fig. 4B) or AncGR1 (fig. S9)
had negligible effect on the receptor’s response to any
hormone. By conferring increased stability on a crucial part
of the receptor, Y27R created a permissive sequence
environment for substitutions that, millions of years later,
remodeled the protein and yielded a new function.
These results shed light on long-standing issues in
evolutionary genetics. One classic question is whether
adaptation proceeds by mutations of large or small effect
(23). Our findings are consistent with a model of adaptation
in which large-effect mutations move a protein from one
sequence optimum to the region of a different function, which
smaller-effect substitutions then fine-tune (24, 25);
permissive substitutions of small immediate effect, however,
precede this process. The intrinsic difficulty of identifying
mutations of small effect creates an ascertainment bias in
favor of large-effect mutations; the ancestral structures
allowed us isolate key combinations of small-effect
substitutions from a large set of historical possibilities.
A second contentious issue is whether epistasis makes
evolutionary histories contingent on chance events (26, 27).
We found several examples of strong epistasis, where
substitutions that have very weak effects in isolation are
required for the protein to tolerate subsequent mutations that
yield a new function. Such permissive mutations create
“ridges” connecting functional sequence combinations and
narrow the range of selectively accessible pathways, making
evolution more predictable (28). Whether a ridge is followed,
however, may not be a deterministic outcome. If there are few
potentially permissive substitutions and these are nearly
neutral, then whether they will occur is largely a matter of
chance. If the historical “tape of life” could be played again
(29), the required permissive changes might not happen, and
a ridge leading to a new function could become an
evolutionary road not taken.
Our results provide insights into the structural mechanisms
of epistasis and the historical evolution of new functions.
GR’s functional specificity evolved by substitutions that
destabilized the receptor structure with all hormones but
compensated with novel interactions specific to the new
ligand. Compensatory mutations have been thought to occur
when a second substitution restores a lost molecular
interaction (30). Our findings support this notion, but in a
reversed order: Permissive substitutions stabilized specific
structural elements, allowing them to tolerate later
destabilizing mutations that conferred a new function (9, 10,
31). We also observed a more striking mechanism:
conformational epistasis, by which one substitution
repositions another residue in three-dimensional space and
changes the effects of mutations at that site. It is well known
that mutations may have nonadditive effects on protein
stability (32), and fitness (9, 33), but we are aware of few
cases (11, 34) specifically documenting new functions or
epistasis via conformational remodeling. This may be due to
the lack of ancestral structures, which allow evolutionary
shifts in the position of specific residues to be determined.
Conformational epistasis may be an important theme in
structural evolution, playing a role in many cases where new
gene functions evolve via novel molecular interactions.
