Protein Evolution by Molecular Tinkering: Diversification of the Nuclear Receptor Superfamily from a Ligand-Dependent Ancestor

Article (PDF Available)inPLoS Biology 8(10) · October 2010with38 Reads
DOI: 10.1371/journal.pbio.1000497 · Source: PubMed
Abstract
Author Summary Many protein families are so diverse that it is hard to determine their ancestral functions and to understand how their derived functions evolved. The existence of so many different functions within protein families often creates the impression that complex, novel functions must have evolved repeatedly and independently. Nuclear receptors (NRs) are a large family of related proteins that regulate key biological processes in animals by binding to specific DNA sequences and triggering expression of nearby target genes. Many NRs are activated by a specific hormone or other small molecule, but some do not require a ligand, and still others are incapable of activating gene expression and so act primarily as repressors of transcription. To understand how the functional diversity of NRs evolved, we reconstructed the structural and functional characteristics of the ancient protein from which the entire family evolved, using genomic, biochemical, functional, and structural analyses in a phylogenetic framework. We show, contrary to current belief, that the ancestral NR was a ligand-activated transcriptional activator that existed in the earliest period of animal evolution. Our analysis reveals how the extraordinary functional diversity of modern receptors was generated by subtle tinkering with this ancestral template—slightly reshaping the ligand cavity, stabilizing the protein's active conformation so it no longer required a ligand, or disabling the protein's capacity to activate transcription without affecting its other properties. We predict that, when sufficient data are gathered to allow detailed evolutionary reconstructions in other protein families, it will become apparent that most protein functional diversity evolved by tinkering with ancient functions; invoking the evolution of wholesale “novelty” will seldom be necessary.
Protein Evolution by Molecular Tinkering: Diversification
of the Nuclear Receptor Superfamily from a Ligand-
Dependent Ances tor
Jamie T. Bridgham
1,2
, Geeta N. Eick
2
, Claire Larroux
, Kirti Deshpande
4
, Michael J. Harms
2
, Marie E. A.
Gauthier
3
, Eric A. Ortlund
4
, Bernard M. Degnan
3
, Joseph W. Thornton
1,2
*
1 Howard Hughes Medical Institute, Eugene, Oregon, United States of America, 2 Center for Ecology and Evolutionary Biology, University of Oregon, Eugene, Oregon,
United States of America, 3 School of Biological Sciences, University of Queensland, Brisbane, Australia, 4 Biochemistry Department, Emory University School of Medicine,
Atlanta, Georgia, United States of America
Abstract
Understanding how protein structures and functions have diversified is a central goal in molecular evolution. Surveys of very
divergent proteins from model organisms, however, are often insufficient to determine the features of ancestral proteins
and to reveal the evolutionary events that yielded extant diversity. Here we combine genomic, biochemical, functional,
structural, and phylogenetic analyses to reconstruct the early evolution of nuclear receptors (NRs), a diverse superfamily of
transcriptional regulators that play key roles in animal development, physiology, and reproduction. By inferring the
structure and functions of the ancestral NR, we show—contrary to current belief—that NRs evolved from a ligand-activated
ancestral receptor that existed near the base of the Metazoa, with fatty acids as possible ancestral ligands. Evolutionary
tinkering with this ancestral structure generated the extraordinary diversity of modern receptors: sensitivity to different
ligands evolved because of subtle modifications of the internal cavity, and ligand-independent activation evolved
repeatedly because of various mutations that stabilized the active conformation in the absence of ligand. Our findings
illustrate how a mechanistic dissection of protein evolution in a phylogenetic context can reveal the deep homology that
links apparently ‘‘novel’’ molecular functions to a common ancestral form.
Citation: Bridgham JT, Eick GN, Larroux C, Deshpande K, Harms MJ, et al. (2010) Protein Evolution by Molecular Tinkering: Diversification of the Nuclear Receptor
Superfamily from a Ligand-Dependent Ancestor. PLoS Biol 8(10): e1000497. doi:10.1371/journal.pbio.1000497
Academic Editor: Laurence D. Hurst, University of Bath, United Kingdom
Received February 11, 2010; Accepted August 17, 2010; Published October 5, 2010
Copyright: ß 2010 Bridgham et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Science Foundation grant IOB-0546906 (JWT) and National Institutes of Health grants R01-GM081592 (JWT), F32-
GM074398 (JTB), and F32-GM090650 (MJH). JWT is an Early Career Scientist of the Howard Hughes Medical Institute. BMD is supported by the Australian Research
Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: AncNR, ancestral NR; CAR, constitutive androstane receptor; DBD, DNA-binding domain; FA, fatty acid; LBD, ligand-binding domain
* E-mail: joet@uoregon.edu
¤ Current address: Department of Earth & Environmental Sciences, Ludwig-Maximilians-University, Munich, Germany
Introduction
By sequencing genomes of taxa occupying key positions in the
metazoan tree of life, it has become possible to infer when important
animal gene families originated and proliferated [1–3]. Sequence
data alone, however, cannot yield insight into the functions and
structures of ancient proteins or the processes by which their
descendants evolved. Further, many gene families have diversified so
extensively that comparisons of extant proteins from model organisms
are insufficient to reveal which functions are ancestral and which are
derived. In principle, it should be possible to reconstruct the history of
a protein family by phylogenetically analyzing the underlying
structural mechanisms that produce functional diversity among
densely sampled members of the family. Such a strategy would be
analogous to detailed studies of the evolution of animal development,
which have revealed the deep homology of diverse morphologies in
distant lineages and the mechanisms by which they evolved from
common ancestral forms [4].
The members of the superfamily of nuclear receptor (NR)
transcription factors, for example, are regulated in diverse ways—
by ligands, postranslational modifications, and association with
other proteins or DNA—depending on the cellular context [5].
NRs have a modular domain structure, including a highly
conserved DNA-binding domain (DBD) and a moderately
conserved ligand-binding domain (LBD)—which in most receptors
contains a ligand-regulated transcriptional activation function—
along with extremely variable hinge and N-terminal domains.
There is considerable diversity in the functions of NR LBDs,
which can be roughly classified into three major groups. In one
class, the LBD’s transcriptional function can be activated by a
specific hydrophobic ligand, such as a steroid, retinoid, or fatty
acid; the ligand binds in a deep internal cavity, remodeling and
stabilizing the LBD’s conformation to generate a new binding
surface for coactivator proteins, which increase transcription of
nearby genes [5]. The second class of NRs are ligand-independent
transcription factors, often called ‘‘constitutive’’ receptors, the
LBDs of which can adopt the active conformation and activate
gene expression in the absence of a ligand or other modifications.
Some members of this class lack the internal cavity and are not
known to bind any ligands, whereas others do bind hydrophobic
PLoS Biology | www.plosbiology.org 1 October 2010 | Volume 8 | Issue 10 | e1000497
molecules, which up- or down-regulate their baseline activity
[6–9]. In the third class of NRs, the LBD lacks the capacity to
interact with coactivators, so these receptors function primarily as
transcriptional repressors that occupy NR response elements or
dimerize with and thereby silence other NRs [10–12].
It is widely believed that the NR superfamily evolved from a
ligand-independent transcriptional activator, with binding of
different ligands gained independently in numerous NR lineages
[13,14]. The alternate view—that NRs evolved from a liganded
ancestor, with ligand-dependence lost in the lineages leading to the
ligand-independent receptors—has received little attention. These
two hypotheses exemplify opposite views on the general nature of
molecular evolution and the origin of complex functions. The
hypothesis that the ancestral NR was ligand-independent implies
that a complex molecular function—allosteric regulation of
transcription by binding a ligand—evolved de novo many
independent times, requiring evolution to repeatedly create
novelty and complexity [15,16]. In contrast, the hypothesis of a
ligand-activated ancestor implies that evolution produced new
functions primarily by subtle tinkering with a conserved ancestral
mechanism [4,17], which allowed receptors to accommodate new
molecular partners or lose dependence on those partners because
of mutations that modified or degraded existing functions.
Several limitations have impeded rigorous inference about the
ancestral NR’s characteristics and the diversification of the
superfamily. First, the root of the gene family phylogeny has been
ambiguous, leaving unknown the location of the ancestor relative
to its descendants. Second, the topology of the NR phylogeny has
been uncertain, because of limited sequence sampling and/or use
of outdated phylogenetic methods. Third, the functions of NRs in
taxa branching near the root of the metazoan phylogeny have not
been characterized. Finally, whether distantly related NRs with
similar functions share homologous or convergent underlying
mechanisms has not been determined. Recently acquired
information—including genome sequences from basal metazoans
and extensive data on NR structures and functions—along with
improved algorithms for phylogenetic analysis of large datasets,
now allow these barriers to be overcome. Here we report on
biochemical, functional, structural, and phylogenetic analyses of
the NR superfamily, which allow us to reconstruct the character-
istics of the ancestral nuclear receptor and understand how the
functional diversity of NR LBDs evolved.
Results/Discussion
NRs in the Sponge Genome
The root of the NR phylogeny has been unknown because of
uncertainty about the relative ages of the various NR family
members. NRs appear to be a metazoan innovation, because they
are absent from the genomes of choanoflagellates, fungi, plants,
and prokaryotes. Until recently, however, all fully sequenced
animal genomes have come from protostomes and deuterostomes,
both of which contain virtually all the major NR subfamilies [18];
these data indicate only that most NR diversity was already
established by the time of the protostome-deuterostome ancestor.
To determine the most ancient NR lineages, we identified NRs in
the newly sequenced genome of the demosponge Amphimedon
queenslandica—a representative of the Porifera, the most anciently
branching metazoan phylum based on whole-genome phylogenies
[19]. We found that the A. queenslandica genome contains two NRs,
which we refer to as AqNR1 and AqNR2. We amplified
transcripts of each by polymerase chain reaction, verified their
sequences, and analyzed their developmental expression using in
situ hybridization. AqNR2 is expressed ubiquitously, whereas
AqNR1 is expressed in a range of cells that contact the external
environment and possess apico-basal polarity (Figure S1). We also
identified NRs in genomes from two other recently sequenced
early-branching lineages, the placozoan Trichoplax adhaerens and the
cnidarian Nematostella vectensis, which contain 4 and 17 NRs,
respectively (see also [20]). These results point to very limited NR
diversification before the origin of the Eumetazoa and indicate
that basal metazoan species have the potential to shed light on
early NR evolution.
Phylogeny of the NR Superfamily
To determine the phylogeny of the NR superfamily, we used
model-based phylogenetics to analyze a taxonomically diverse
database of 275 NR protein sequences (Figure 1, Table S1). The
alignment includes the DBDs and LBDs of the complete NR
complements in 11 sequenced genomes from eight broadly
sampled animal phyla, plus NRs from 30 other species strategically
chosen to maximize phylogenetic accuracy and minimize
redundant signal [21]. Unlike previous studies, which used sparser
sequence sampling and/or less powerful methods [14,18,22],
phylogenetic analysis of this alignment using maximum likelihood
yielded a well-resolved phylogeny (Figures 1, S2) with strong
support for the placement of the basal metazoan sequences and for
the relationships among most major NR families. (A few aspects of
the topology, however, had weak support, such as whether the SF-
1 class has a monophyletic or paraphyletic relationship to the
group containing the steroid hormone receptors.) We also
conducted Bayesian Markov Chain Monte Carlo (BMCMC)
methods using a slightly smaller 174-sequence dataset, assembled
Author Summary
Many protein families are so diverse that it is hard to
determine their ancestral functions and to understand
how their derived functions evolved. The existence of so
many different functions within protein families often
creates the impression that complex, novel functions must
have evolved repeatedly and independently. Nuclear
receptors (NRs) are a large family of related proteins that
regulate key biological processes in animals by binding to
specific DNA sequences and triggering expression of
nearby target genes. Many NRs are activated by a specific
hormone or other small molecule, but some do not require
a ligand, and still others are incapable of activating gene
expression and so act primarily as repressors of transcrip-
tion. To understand how the functional diversity of NRs
evolved, we reconstructed the structural and functional
characteristics of the ancient protein from which the entire
family evolved, using genomic, biochemical, functional,
and structural analyses in a phylogenetic framework. We
show, contrary to current belief, that the ancestral NR was
a ligand-activated transcriptional activator that existed in
the earliest period of animal evolution. Our analysis reveals
how the extraordinary functional diversity of modern
receptors was generated by subtle tinkering with this
ancestral template—slightly reshaping the ligand cavity,
stabilizing the protein’s active conformation so it no longer
required a ligand, or disabling the protein’s capacity to
activate transcription without affecting its other proper-
ties. We predict that, when sufficient data are gathered to
allow detailed evolutionary reconstructions in other
protein families, it will become apparent that most protein
functional diversity evolved by tinkering with ancient
functions; invoking the evolution of wholesale ‘‘novelty’’
will seldom be necessary.
Nuclear Receptor Evolution by Molecular Tinkering
PLoS Biology | www.plosbiology.org 2 October 2010 | Volume 8 | Issue 10 | e1000497
by removing sequences at the ends of very long branches and
multiple orthologs within the same phylum. This analysis
recovered a nearly-identical phylogeny to the maximum likelihood
analysis (Figures 1, S3).
The phylogeny is unlikely to be an artifact of the presence of
rapidly evolving sites or taxa. When the 40% of sites with the
fastest evolutionary rates were removed from the analysis, only the
placement of the RXR group was affected (Figure S4). Further,
maximum likelihood analysis of the reduced 174-sequence
dataset—from which the longest terminal branches were
removed—yielded the same phylogenetic relationships as the
unreduced analysis (Figure S5).
AqNR2
HNF4 (NR2a / 9)
AqNR1
2E35/1.00/1.00
TLL/TLX, DSF, FAX1, PNR (NR2e / 23)
4E14/1.00/1.00
3/0.89/0.74
COUP-TF, SVP (NR2f / 19)
TR2/4, HR78 (NR2c, d / 14)
1E6/1.00/1.00
4E9/1.00/1.00
RXR, USP (NR2b / 24)
2E8/1.00/1.00
SF-1, LRH-1, Ftz-F1, GCNF, GRF (NR5, 6 / 19)
2E5/1.00/1.00
ERR; Steroid Hormone
Receptors (NR3 / 40)
INR (10)
9/0.76/0.94
NURR, NOR1, NGFIB, HR38 (NR4 / 12)
2E33/1.00/1.00
ThR (NR1a / 12)
6E12/1.00/1.00
FXR, LXR, EcR
(NR1h / 20)
VDR, PXR, CAR, HR96
(NR1i, j / 11)
1387/1.00/0.97
8433/1.00/1.00
RAR (NR1b / 11)
27/0.84/0.98
Rev-Erb, E75 (NR1d / 12)
E78 (NR1e / 10)
129/0.99/1.00
1/0.82/0.42
ROR, HR3 (NR1f / 13)
PPAR (NR1c / 12)
462/1.00/1.00
18/0.57/0.97
3E11/1.00/1.00
9E5/1.00/1.00
4E14/1.00/1.00
7/0.84/0.93
112/0.98/1.00
1429/1.00/1.00
0.5
5E5/1.00/1.00
HNF4, hepatic nuclear factor-4
TLL/TLX, tailless proteins
DSF, dissatisfaction
FAX1, fasciculation of axons protein
PNR, photoreceptor-specific NR
COUP-TF, chicken ovalbumin upstream promoter transcription factor
SVP, seven-up
TR2/4, testicular receptors 2 and 4
RXR, retinoid X receptor
USP, ultraspiracle
SF-1, steroidogenic factor 1
LRH-1, liver receptor homolog 1
Ftz-F1, Ftz-factor 1
GCNF, germ cell nuclear factor
GRF, GCNF-related factor
ERRs, estrogen-related receptors
NURR, nuclear receptor-related protein
NOR1, neuron-derived orphan receptor
NGFIB, nerve-growth factor inducible receptor B
INRs, invertebrate-specific NRs
ThR, thyroid hormone receptor
FXR, farnesoid X receptor
LXR, liver X receptor
ECR, ecdysteroid receptor
VDR, vitamin D receptor
PXR, pregnane X receptor
CAR, constitutive androstane receptor
RAR, retinoic acid receptor
Rev-Erb, reverse Erb-receptor
ROR, retinoid orphan receptor
PPAR, peroxisome proliferator-activated receptor
471/1.00/1.00
471/1.00/1.00
4E6/1.00/1.00
9E13/1.00/1.00
3/ - /0.83
5E5/1.00/1.00
4E8/1.00/1.00
5E16/1.00/1.00
3515/0.67/1.00
7E5/1.00/1.00
3/1.00/0.77
Figure 1. Phylogeny of the NR superfamily. A reduced representation of the NR phylogeny inferred by maximum likelihood and Bayesian
method is shown. Colored circles indicate the presence of each clade in major metazoan taxa: sponges (light blue), placozoans (purple), Cnidaria
(green), protostomes (orange), and deuterostomes (dark blue). Clades are labeled with their common protein names; NR nomenclature codes are in
parentheses (see also Table S6), with the number of sequences analyzed in each group after the slash. Branch labels show support measured as
approximate likelihood ratios (the ratio of the likelihood of the best tree with that node to the best tree without it), Bayesian posterior probabilities,
and chi-square confidence estimates (the probability of a likelihood ratio at least as great as the observed ratio if the node is not resolved on the true
tree). INRs, clade of invertebrate-only nuclear receptors with no standard nomenclature. Scale bar, probability of substitutions per site. A key to
abbreviations of protein names is shown below the phylogeny. For unreduced phylogenies and a list of species, genes, accessions, and receptor
abbreviations, see Figures S2–S3 and Tables S1 and S6.
doi:10.1371/journal.pbio.1000497.g001
Nuclear Receptor Evolution by Molecular Tinkering
PLoS Biology | www.plosbiology.org 3 October 2010 | Volume 8 | Issue 10 | e1000497
Expansion of NRs Early in the Metazoa
The phylogeny can be rooted at a single most parsimonious
location between AqNR1 and AqNR2, allowing the ancestral NR
to be located on a specific branch of the phylogeny. This rooting
provides a coherent history of NR expansion by gene duplication
with few subsequent losses (Figure 2). All alternative rootings that
place other NR lineages in a basal position require many
additional duplications and losses (Figure 2A). For example,
placing the clade of ligand-independent estrogen related receptors
(ERRs) as the outgroup requires two additional duplications and
12 additional losses compared to the optimal root; placing the
ligand-independent NR4 class as the outgroup requires two
additional duplications and 15 additional losses.
The phylogeny indicates that AqNR2 is orthologous to the fatty
acid-binding HNF4 family and that AqNR1 is the unduplicated
ortholog of all other NRs. This phylogeny indicates that the last
common ancestor of all Metazoa contained two NRs—one
ortholog of HNF4 and one of AqNR1, which subsequently gave
rise to all other NR classes (Figure 2). After the divergence of
demosponges from other metazoans but before the split of
Cnidaria from the Bilateria, nine more duplications gave rise to
most of the major recognized NR types, except for those in the
named classes NR1 and NR4, which proliferated during the
interval between the cnidarian-bilaterian ancestor and the
protostome-deuterostome ancestor (Figure 2). Many NR subfami-
lies diversified further within the vertebrates.
Support for the placement of AqNR2 with the HNF4s and of
AqNR1 as sister to all other NRs is strong, with posterior
probabilities of 1.0 and 0.98, chi-square confidence values of 1.0
and 1.0, and approximate likelihood ratios of 471 and 112,
respectively (Figures 1, S2–S3). The next-best rearrangement of
the relationships between the sponge receptors and the rest of the
NRs has a likelihood several orders of magnitude lower than that
of the ML tree. This alternate phylogeny (Figure S6) would place
AqNR1 and AqNR2 as sister paralogs specific to the sponge
lineage. It would imply that the ancestral metazoan contained a
single NR; duplication of this ancestral gene in the sponges would
have yielded AqNR1 and AqNR2, and the first duplication that
separated the HNF4 group from the rest of the NR superfamily
would have occurred in the Eumetazoa after they diverged from
demosponges. The rest of the superfamily’s history would remain
unchanged.
Functional Characterization of Demosponge NRs
Hypotheses concerning the functions of the ancestral proteins
are strongly affected by the functions of proteins that branch off
the family phylogeny near its root. To understand how the
functions of the NR LBD evolved, we experimentally character-
ized the capacity of AqNR1 and AqNR2 LBDs to bind and be
regulated by ligands. In a reporter gene assay, AqNR1 had very
weak intrinsic activity—less than 2-fold activation—when incu-
bated with serum from which hydrophobic small molecules were
stripped using dextran-charcoal. When treated with complete
serum, however, AqNR1 transcription increased by 30-fold,
suggesting that the receptor is activated by a hydrophobic ligand
that is present in mammalian serum, such as a fatty acid or steroid
(Figure 3A).
To characterize AqNR1’s potential ligand, we expressed and
purified AqNR1-LBD in bacteria, extracted bound hydrophobic
molecules, and used mass spectrometry to identify the bound
compounds. Like mammalian HNF4 [23], AqNR1-LBD bound
an array of bacterial free fatty acids (FAs) with tail lengths ranging
from 16 to 19 carbons, with preference for 18:0 and 18:1 fatty
acids (Figures 3B, S7). When AqNR1 was incubated with complete
mammalian serum, palmitic acid was the dominant FA bound,
along with lower proportions of 18:0 and 18:1 FAs (Figure 3B). We
then confirmed and quantified FA binding by the purified
AqNR1-LBD using an enzymatic assay. As predicted, we found
that AqNR1-LBD binds both E. coli and mammalian FAs;
receptor occupancy by FAs approximately doubles when the
protein is treated with complete serum but does not increase upon
treatment with stripped serum (Figure 3C).
To more directly test the hypothesis that AqNR1 binds and is
activated by FAs, we characterized the functional effects of
mutations in the predicted AqNR1 ligand pocket. We first
predicted the structure of AqNR1-LBD using a homology model
based on the X-ray crystal structure of mammalian HNF4, the NR
with the highest sequence similarity to AqNR1. The predicted
structure (Figure 4A) indicates that AqNR1 is likely to have a large
ligand pocket (835 A
˚
3
) with ample space to accommodate FAs. As
in the crystal structure of mammalian HNF4s, the FA in AqNR1 is
predicted to be coordinated by a hydrogen bond from a conserved
arginine (Arg226 in rat HNF4a, Arg492 in AqNR1) to the FA’s
carboxylate oxygen; further, packing interactions between hydro-
phobic amino acids that line the pocket and the ligand’s tail are
also conserved.
We then used directed mutagenesis and functional assays to
experimentally test the hypothesis that AqNR1 binds and is
activated by FAs in a manner conserved with mammalian HNF4.
As predicted, when the basic Arg492 was mutated to alanine,
reporter activation by AqNR1 in the presence of complete serum
was abolished (Figure 4B), and FA binding by the purified protein
was dramatically reduced (Figure 3C). Replacement of Arg492 by
several other amino acids, each of which would remove the
hydrogen bond to the FA’s carboxyl oxygen, also abolished
reporter activation by AqNR1 (Figure 4B). Although rat HNF4a is
a weaker activator in this cell line than AqNR1, mutations at this
site in HNF4a also reduced activation, consistent with a common
structural mode of ligand-binding (Figure 4B). Rat HNF4a, which
is thought to be activated by fatty acids produced endogenously in
liver cells [24], is not further activated by complete serum,
indicating that its specific ligand is different from that of AqNR1
(Figure 4B).
We also mutated several hydrophobic residues in AqNR1 that
are predicted to contact the FA’s tail; as expected, activation by
complete serum was dramatically reduced (Figure 4B). One bulky
mutation in the predicted pocket, I444W, conferred strong activity
on AqNR1 in the presence of stripped or full serum, implying that
this mutation stabilizes the active conformation without ligand or
allows binding of an unknown ligand that is present in the cultured
cells or medium. Taken together, these data indicate that
AqNR1’s transcriptional activity is affected by binding of a
hydrophobic ligand, that the ligand may be a FA, and that key
aspects of the AqNR1’s structure-function relations are largely
conserved with those of mammalian HNF4. Identification of the
specific natural ligand for AqNR1—like that of the ligand for
mammalian HNF4 [25]—requires further research, as does
determination of whether that ligand has an endogenous or
exogenous source.
Purified AqNR2 also bound fatty acids (Figure 3B). It did not,
however, activate transcription in the mammalian reporter assay
but acted as a very strong repressor of basal transcription,
irrespective of the type of serum used (Figure 3A). These results
indicate that AqNR2 can repress transcription and, like its
ortholog HNF4 and its paralog AqNR1, bind FAs. We cannot
rule out the possibility that AqNR2 may have the capacity to
activate transcription in the presence of some yet unknown
ligand.
Nuclear Receptor Evolution by Molecular Tinkering
PLoS Biology | www.plosbiology.org 4 October 2010 | Volume 8 | Issue 10 | e1000497
A
HNF4
NR2c-f
RXR
Steroid receptors/ERR**
SF-1/GCNF
SF-1/
GCNF
Steroid
receptors/
ERR
Choanoflagellates
Demosponges
Placozoans
Cnidarians
Protostomes
Deuterostomes
FXR/ LXR/EcR
VDR/PXR/CAR/HR96
ThR
RAR
Rev-Erb/E75
E78
ROR/HR3
PPAR
Origin of ancestral NR
NR4s
Eumetazoa
Bilateria
Metazoa
COUP-TF
FAX-1
TLX/PXR
B
TR2/TR4
INR
TR2/TR4
NR1/NR4/
INR*
HR39
+3,+16
+3,+16
+3,+17
+3,+17
+3,+15
+3,+15
+2,+12
+2,+12
porNR2
placHNF4
cHNF4
dHNF4/pHNF4
porNR1
placCOUP
cCOUP_1
cCOUP_2
dCOUP-TF/pCOUP_SVP
cNR2E2
dTLX/pTll
dDSF/pDSF
cNR2E1
dPNR/pPNR
cNR2E3
dFAX/pFAX
cTR24
dTR2_TR4/pHR78
placRXR
cRXR
dRXR/pRXR_USP
cSF1
dSF1_LRH1/pFtzF1
pHR39
dGCNF/pGRF
placNR3
dERR/pERR
dSteroid receptors/pER
cINRa
dINR/pINR
cINRb
dNURR_NGFIB_NOR/pHR38
dE78a/pE78a
dE78b/pE78b
dReverb/pE75
dPPAR/pPPAR
dROR/pHR3
dLXR_FXR/pECR
dVDR_CAR_PXR/pHR96
dTHR/pTHR
dRAR/pTHR
(plac)
(plac)
(c)
(plac)
X
X
X
X
+2,+5
+1,+2
+1,+2
+2,+5
+3,+9
+3,+9
+2,+7
+1,+2
+2,+9
+3,+17
+2,+13
+2,+11
+2,+11
+2,+13
+3,+17
+3,+21
+3,+21
+3,+13
+3,+13
+1,+3
+1,+4
+1,+4
+2,+7
+2,+9
+2,+8
+2,+7
+2,+7
+3,+11
+3,+11
+2,+8
+1,+2
+1,+3
+1,+4
+1,+5
+1,+7
+1,+5
+1,+7
+1,+4
+2,+12
+1,+8
+2,+15
+2,+18
+2,+9
+2,+12
+2,+9
+2,+12
+2,+15
+2,+15
+2,+12
+2,+9
+2,+9
+2,+12
+2,+18
+1,+8
+2,+12
+2,+15
+2, +27
+2,+24
+2,+21
+2, +27
+2, +24
+2,+21
+2, +24
+2, +24
+2, +27
+2,+24
+2,+21
+2, +27
+2,+24
+2,+21
NR2
NR5,6
NR3
NR1
NR4
+3,+17
Figure 2. Duplications and losses in NR evolution. (A) Reconciliation of NR gene family phylogeny with the species tree shown in panel B. The
phylogeny is rooted to minimize the total number of gene duplications and losses; each branch is labeled with the number of additional duplications
and losses required for the tree to be rooted on that branch. Gene duplications are shown as green circles, gene losses as Xs, and speciation events as
yellow shapes (circles, split of demosponges from eumetazoans; squares, split of placozoans from eumetazoans (s.s.); diamonds, split of cnidarians
from bilaterians). Colors of gene names indicate the taxon from which they are derived, using the color scheme in panel B. (B) Gene duplication
history implied by the reconciled tree in panel A. Green bars, duplications; red bars, losses. Duplications are labeled with the named NR lineages they
generated. The NR1/NR4/INR ortholog lost in the placozoans (marked *) was generated in the duplication marked **. The large bar comprises
numerous duplications that cannot be temporally ordered.
doi:10.1371/journal.pbio.1000497.g002
Nuclear Receptor Evolution by Molecular Tinkering
PLoS Biology | www.plosbiology.org 5 October 2010 | Volume 8 | Issue 10 | e1000497
Phylogenetic Reconstruction of the Ancestral NR’s
Functions
A robust rooted protein family phylogeny, functional data on
basally branching receptors, and recently gathered information on
the functions and structures of NRs from model and non-model
organisms allow us to infer the characteristics of the ancestral NR.
Although the sequences of the NR superfamily are too divergent to
allow unambiguous reconstruction of the ancestral NR LBD at the
amino acid level, there is substantial phylogenetic signal in the
structural and functional features of NR LBDs. We coded these
features as discrete phylogenetic characters and reconstructed the
best-supported ancestral states using phylogenetic methods
(Figure 5A). The ancestral NR (AncNR) is decisively reconstructed
as having had the capacity to activate transcription, bind a ligand,
and be activated by that ligand. The vast majority of extant NRs,
including those in the basal lineages, have these characteristics.
The handful of exceptions—ligand-independent activators and
pure repressors—are scattered across the tree and are in most
cases nested deep within groups of liganded activators, indicating
that these states are almost certainly derived. The fact that some
ligand-independent receptors bind ligands, which can up- or
down-regulate their baseline activity [6–8], further supports the
reconstruction of the ancestor as having possessed these features.
No ligand-independent activators are present in the basally
branching NR clades.
When the evolution of these functional characters is traced on
the NR phylogeny, the hypothesis of a ligand-binding and ligand-
activated AncNR is by far the most parsimonious reconstruction.
This scenario explains the characteristics of the entire NR
superfamily with only five losses of dependence on ligand. Three
of these losses were accompanied by a loss of ligand-binding; in the
other two instances (the ERRs and constitutive androstane
receptor, CAR), receptors evolved ‘‘constitutive’’ transcriptional
activity but retained the ancestral capacity to bind ligands, which
regulate that baseline activity (Figure 5A). In contrast, the
alternative hypothesis of a ligand-independent AncNR would
require both ligand-binding and dependence on the ligand for
activation to have been gained 12 independent times, plus a
subsequent reacquisition of ligand-independent activity in one
lineage (Figure S8). Reconstruction of these characters on the
alternate phylogeny that places AqNR1 and AqNR2 as sponge-
specific duplicates causes no change in the support for an ancestral
liganded-activated receptor vis-a`-vis the ‘‘constitutive ancestor’’
hypotheses (Figure S9).
It is also clear that AncNR had the capacity to activate
transcription rather than acting as a pure repressor. An ancestral
activator requires five losses of activity in the lineages leading to
the inactive repressor NRs (Figure 5A), whereas 11 independent
gains of transcriptional activity would be required if AncNR were
transcriptionally inactive.
Phylogenetic Reconstruction of the Ancestral NR’s
Structure
A key element of assessing homology is to determine whether
shared structures and mechanisms underlie apparently similar
features in different lineages. To further test the hypothesis that
ligand-binding and activation are homologous functions derived
from the ancestral NR—and that ligand-independent activation
was repeatedly derived—we analyzed the underlying structural
mechanisms for these functions in a phylogenetic framework. We
coded as discrete phylogenetic characters the relevant structural
features of NR LBDs and phylogenetically reconstructed the best
supported ancestral state for each (Figure 5A).
AncNR is decisively reconstructed as having had the shared
features of extant ligand-activated receptors that underlie ligand
binding and activation. Specifically, there is strong support for the
ancestor having possessed (1) the classic NR fold in the active
conformation consisting of three layers of helices in highly
conserved positions; (2) an open ligand pocket with volume of at
least 300 A
˚
3
, bordered by helices H3, H4-5, H7, H10, and H12;
and (3) a surface for binding coactivator proteins, composed of
residues in the ligand-stabilized helices H3, H4-5, and H12, with a
-30 -20 -10 0 10 20 30 40
AqNR1
MBP
AqNR1-R492A
B
A
C
MBP
fold activation
Receptor occupancy by FFAs (%), relative to control
FA AqNR1
AqNR1 +
full serum
AqNR1 +
stripped AqNR2 rHNF-4a
16:1 7.16 0 1.46 8.82 0.3
16:0 11.32 56.43 52.63 20.5 32.43
17:1 19.72 0 0 12.17 30.58
17:0 0.09 0 0 0 0.08
18:1 22.11 24.94 17.24 37.82 9.83
18:0 27.22 18.63 28.66 20.46 26.71
19:1 12.38 0 0 0.23 0.07
Total 100 100 100 100 100
0.01
0.10
1
10
100
control AqNR1
AqNR2
Figure 3. Ligand binding and transcriptional activation by
A.
queenslandica
nuclear receptors. (A) Transcriptional effects of
AqNR1 and AqNR2 LBDs in a luciferase reporter assay. Transfected
cells were treated with stripped or complete serum (white and black
bars, respectively). Activation is scaled relative to vector-only control;
error bars, SEM for 3 replicates. (B) Fatty acid (FA) binding profile by
AqNR1 and AqNR2, as determined by electrospray ionization/mass
spectrometry. Binding of each FA species is shown as a percentage of
the total FAs bound by each receptor. The most abundantly bound FA
for each receptor is in bold. FA binding by rat HNF4a is shown for
comparison. Measurements are shown relative to a labeled palmitic
acid standard and represent the average of two runs. (C) Quantitative
fatty acid binding by AqNR1 and mutant AqNR1-R492A as determined
in a colorimetric enzymatic assay. Receptors were expressed in E. coli,
purified, and treated with no serum (gray), complete serum (black), or
stripped serum (white). Errors bars, SEM for 3 replicates. The percentage
of receptor molecules occupied by FAs (after subtraction of background
binding by MBP/no serum control) is shown for each experimental
condition.
doi:10.1371/journal.pbio.1000497.g003
Nuclear Receptor Evolution by Molecular Tinkering
PLoS Biology | www.plosbiology.org 6 October 2010 | Volume 8 | Issue 10 | e1000497
conserved coactivator-recognition motif in the latter [5,26,27].
These features and a similar location of the ligand within a highly
conserved LBD structure are shared by even the most distantly
related ligand-activated NRs (Figure 5B); indeed, even the ligand-
independent receptors retain some or all of these features.
The identical structural basis for ligand-activation throughout
the superfamily provides strong evidence that this function is
derived from the common NR ancestor. It is plausible that the
ancestral ligand was an FA, because several of the most basal
lineages bind FAs. Further, the key hydrogen bond between the
FA’s carboxyl-group oxygen and the Arg side chain on helix 5 is
conserved in several basal lineages, including HNF4s, RXRs, and
AqNR1. The ligand that historically activated AncNR could have
been a ubiquitous endogenous molecule that served as a receptor
cofactor, a hormone-like regulatory compound endogenously
produced under specific conditions, or an exogenous nutrient or
other substance, such as fatty acids produced by bacteria or other
species.
In contrast, the structural elements that appear to confer ligand-
independence differ dramatically among the ligand-independent
activators (Figure 6). In Nurr1/DHR38, the mollusk estrogen
receptor, and the vertebrate ERRs, the pockets are filled with
multiple bulky hydrophobic side chains that mimic the presence of
ligand [7,8,28,29], but the sites and states involved in the three
receptor classes are all different, with a single convergent exception
in two of the three receptors (Figure 6A). In Drosophila Ftz-F1, the
H6/H7 region adopts an unprecedented loop conformation that
turns inward and fills the cavity (Figure 6A). In CAR, residues in
helix 12 (H12) form unique hydrogen bonds to H4-5, and a novel
helix, absent from other NRs, packs against H12, stabilizing the
active conformation (Figure 6B) [30–32]. Finally, in the crystal
structure of mouse LRH-1, the active conformation is stabilized
without ligand due to a unique salt bridge between residues in H7
and H10, which replaces a similar bridge between the ligand and
H10 in orthologs of the same protein of humans and other species,
and in SF-1, the closest paralogous NR (Figure 6C) [33].
These radical differences in putative underlying mechanisms
indicate that ligand-independent activity is a convergent character
with independent evolutionary origins rather than a homologous
feature inherited from the common NR ancestor. The hypothesis
of a ligand-dependent AncNR explains the structure-function
relations of the vast majority of present-day receptors as due to
descent from an ancestor that possessed those same features. In
contrast, the hypothesis of an unliganded AncNR can explain the
structure-function relations of only a single NR as due to descent
from the ancestral NR; it requires the ancestral basis for ligand-
B
A
0102030405060
AqNR1 WT
rHNF4 WT
rR226A
L502W
L486N
V485W
I444W
R492D
R492E
R492A
vector
1.0
0.8
0.5
2.7
1.1
1.0
47.4
0.8
0.9
2.0
38.7
5.3
1.2
fold activation
38.0
2.0
2.5
0.9
0.6
1.8
0.8
1.0
1.2
Figure 4. Conserved architecture and function of the AqNR1 ligand pocket. (A) The ligand cavity in the predicted structure of AqNR1
(green, with palmitic acid) and in the crystallographic structure of rat HNF4a (blue, with lauric acid) are shown. Cavities (volumes of 835 and 450 A
˚
3
,
respectively) are shown as surfaces, and the ligands as sticks. Labeled side chains at homologous positions that contact the ligand were subject to
experimental characterization. Dashed lines show hydrogen bonds. (B) Effect of mutagenesis of ligand-contacting residues on transactivation in a
luciferase reporter assay by AqNR1 and rat HNF4a in stripped (white bars) and complete serum (black bars). Bar labels show fold activation relative to
vector-only control with stripped serum; error bars, SEM for 3 replicates.
doi:10.1371/journal.pbio.1000497.g004
Nuclear Receptor Evolution by Molecular Tinkering
PLoS Biology | www.plosbiology.org 7 October 2010 | Volume 8 | Issue 10 | e1000497
Figure 5. Ancient origin of ligand-activation in the NR superfamily. (A) Reconstruction of structural and functional characters on the NR
phylogeny. Ligand-regulated transcriptional activators are shown in green, with ligands in parentheses. Red, activators with no known ligand.
Underlined, receptors with transcriptional activity in the absence of ligand or other modifications. Black, repressors that do not activate transcription.
The ancestral NR (AncNR, green circle) is shown, with the most parsimonious reconstructions at that node for the characters in the table. Hash marks
on branches show gains of ligand-independent activity with or without loss of ligand binding (filled and empty red boxes, respectively). States of
protein structural characters are shown in the table. ‘‘3 layers’’: 10 to 12 helices arranged in conserved three-layer sandwich as in panel B. ‘‘Ligand
pocket open or filled’’: O, open internal cavity bounded by helices 3, 5, 7, 10, and 12 in crystal structure (cavity volume in A
˚
3
); O*, open pocket in
homology model; O**, open pocket inferred from experimental data on ligand-binding or ligand-regulation; F, filled cavity, ,50 A
˚
3
.‘H12 coactivator
Nuclear Receptor Evolution by Molecular Tinkering
PLoS Biology | www.plosbiology.org 8 October 2010 | Volume 8 | Issue 10 | e1000497
independent activity to have been independently lost and replaced
with different underlying mechanisms in all other lineages of
ligand-independent receptors and the shared mechanisms for
ligand-dependent activation to have been gained independently in
the many lineages of liganded receptors.
How ‘‘Novelty’’ Evolves
Our findings indicate that NR LBDs evolved their functional
diversity by tinkering with a ligand-dependent transcriptional
activator. Ligand-regulated NRs are thermodynamically tuned so
that in the appropriate contexts the active conformation is favored
in the presence of activating ligand but not its absence. The most
common functional shift during NR evolution was modification of
ligand specificity due to subtle changes in the shape and surface
properties of the ancestral ligand pocket. Both historical and
contemporary studies indicate that such shifts in ligand preference
can evolve through a relatively small number of mutations that
subtly alter the ligand cavity (e.g., [34–36]).
In a few lineages, ligand-independent activity evolved by
mutations that stabilized the active conformation in the absence
of ligand; in two such cases, the cavity remained open, yielding a
receptor whose baseline activity can be antagonized or super-
activated by ligands [6,8]. Laboratory and clinical data contain
many examples of ligand-independent activity evolving via single
point mutations that add sufficient stability to the active
conformation in the absence of ligand (Table S2). Historical
studies also document the evolution of constitutive activity with a
very simple genetic basis [33]. Such transitions tip the thermo-
dynamic balance so that the formerly switchable LBD becomes
stuck in the ‘‘on’’ position, irrespective of ligand. In contrast,
evolving a ligand-dependent receptor from a ligand-independent
ancestor would require mutations that (1) generate a ligand pocket
of the appropriate size and shape to accommodate some ligand
and (2) destabilize the active conformation just enough to abolish
ligand-independent activity but not so much that the capacity is
lost to activate transcription when ligand is present. We observed
no such transitions on the NR phylogeny, and we are aware of
only one NR mutation that accomplishes this end in the
laboratory; that example reflects a return to the ancestral amino
acid state in a receptor that binds ligand but also possesses ligand-
independent activity [37].
Finally, in a few other lineages, inactive repressor NRs evolved
by degradation of the activation function without loss of other
functions, such as DNA binding, dimerization, or corepressor
binding (see [11]). Indeed, most inactive NRs have simply lost the
co-activator interaction motif in H12 but retain the classic LBD
secondary and tertiary structure, and some even retain an open
pocket [12]. Inactive repressor NRs have been shown to evolve
from ligand-activated precursors via simple genetic mechanisms
that disable ligand or coactivator binding but leave intact other
functions of the receptors, such as DNA binding, dimerization,
and corepressor binding [11].
Most gene families, like the NRs, have some common conserved
core function—some catalytic activity, for example, or the capacity
to interact with DNA. Functional diversity within such families is
conferred by members’ binding to and carrying out that function
on different partners. Our observations in the NRs underscore the
capacity of evolution to produce dramatic functional diversity by
tinkering with a common ancestral template over long periods of
time. The varied and subtle nature of these tinkering events is
revealed only when densely sampled structural and functional data
are analyzed in a phylogenetic context. We predict that, when
sufficient data are gathered to allow detailed evolutionary
reconstructions, it will become apparent that most protein
superfamilies diversified by subtle modification and partial
degradation of ancient, deeply homologous functions. Invoking
the evolution of wholesale ‘‘novelty’’ will seldom be necessary.
Methods
Alignment and Phylogenetic Analyses
Nuclear receptor protein sequences were obtained by mining
the genomes of Amphimedon queenslandica, Trichoplax adhaerens,
Nematostella vectensis, Lottia gigantea, Capitella capitata, and Branchios-
toma floridae (Table S1). The assembled genomes and develop-
mental expressed sequence tags were screened using tBlastn with
LBD and DBD amino acid sequences from each known NR
family. Further analysis using PFAM domain analysis (PF00104
and PF00105) [38] and a hidden Markov model-based method
(PTHR11865) [39] confirmed the presence of only two NRs in the
A. queenslandica genome, which has been sequenced at approxi-
mately 9-fold coverage [19]. In some of the other genomes, gene
model sequences were modified to resolve gaps in the sequence by
performing a local assembly with gene traces or to correct the
predicted protein sequence based on alignment with other
conserved domain sequences. Complete NR complements from
the curated whole-genome databases of H. sapiens, D. melanogaster,
C. intestinalis, F. rubripes, and S. purpuratus were also included.
Additional nuclear receptors were identified by using the SMART
domain-based sequence annotation resource [40] to search the
UniPROTKB/TrEMBL database based on the amino acid
sequence of the ERR of Marisa cornuarietis. Receptors for which
only partial sequence was available (missing .20% of the DBD or
LBD) and those entirely lacking a DBD domain (e.g., human
DAX1 and SHP) or LBD domain (e.g., D. melanogaster Knirps and
Knrl) were excluded from the analysis.
A total of 275 nuclear receptor sequences were aligned. Full-
length sequences containing the DBD, highly variable hinge
region, and LBD were aligned using Multiple Sequence Alignment
by Log-Expectation (MUSCLE) v. 3.6 [41] in order to identify the
boundaries of the conserved regions. After removal of the variable
(non-alignable) hinge region, sequence blocks corresponding to the
DBD and LBD were then aligned separately using MUSCLE. The
DBD and LBD alignments were checked manually to remove
lineage-specific indels, and the LBD alignment was checked to
sequence’’: presence (+) or absence (2) of canonical co-activator interface ww*kww motif in helix 12 (w, hydrophobic; *, any residue; k, charged). ‘‘L6-11
salt bridge’’: presence (+) or absence (2) of salt bridge from the L6-7 loop to helix H10-11. ‘‘Rigid HX and H10-H12 h-bonds’’: presence (+) or absence
(2) of short additional helix N-terminal to H12 and hydrogen bonds between H10-11 and H12. ‘‘Bulky residues in pocket’’: presence (1,2,3) or absence
(2) of Phe or Trp residues in space occupied by ligand in other receptors; 1, 2, 3 indicate bulky residues at different sets of sequence sites (see
Figure 6). Blank cells, no data available. Rodent LRH-1 has been coded as a ligand-independent activator based on its crystal structure, but functional
assays have not established its ligand-independence. (B) Shared structural basis for ligand-dependent activation in NR LBDs. The peptide backbones
and ligands are superimposed for five distantly related NRs: human HNF4a, mouse RXRa, human ERa, mouse RARb, and human PPARa. Gray spheres
show ligands. Helices (shown as cylinders) are numbered, and the coactivator peptide (Co-Ac) is a magenta ribbon. The beta-sheet between H5 and
H6 is pink. The region between H1 and H3, which is structurally variable, has been removed to show the ligands more clearly. For details and PDB
identifiers, see Tables S4, S5, and S7.
doi:10.1371/journal.pbio.1000497.g005
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PLoS Biology | www.plosbiology.org 9 October 2010 | Volume 8 | Issue 10 | e1000497
ensure correct alignment of the conserved AF-2 core sequence
(ww*kww motif; w, hydrophobic; *, any residue; k, charged).
Amino acids C-terminal to this AF-2 core sequence could not be
reliably aligned among all nuclear receptors and the LBD
alignment was therefore truncated after the AF-2 core sequence.
The DBD and LBD alignments were then concatenated in
MacClade 4 (Sinauer Associates, Inc., MA, USA) for subsequent
phylogenetic analyses. We then used APDB software [42] to
characterize the quality of our alignment with reference to the 26
NR LBDs in the alignment for which X-ray crystallographic
structures are available; the average iRMSD (the root mean
square difference of the intramolecular distances between aligned
pairs of alpha-carbons) over the entire LBD alignment was 0.82
angstroms, well under the resolution of the structures themselves,
indicating that the alignment has high structural plausibility.
Phylogenetic analyses were performed using maximum like-
lihood in PhyML v. 2.4.5 [43] and Bayesian analysis using
MrBayes v. 3.1 [44]. The Jones-Taylor-Thornton model with a
four-category discrete gamma distribution of among-site rate
variation (ASRV) and a proportion of invariant sites was used. For
ML, all model parameters were optimized by maximum like-
lihood. Support was evaluated by obtaining the approximate
Figure 6. Independent structural mechanisms for ligand-independent activation in nuclear receptors. (A) Receptors with filled ligand
cavities. Bulky side chains (shown as sticks) that fill the cavity are shown for rat Nurr1 (cyan), human ERRa (magenta), and oyster ER (blue), with a-
carbons as balls. Green ribbon, backbone of the loop with side chains that fill the pocket in D. melanogaster Ftz-F1. The van der Waals space occupied
by estradiol in human ERa is in white. (B) In human CAR (bronze), a network of hydrogen bonds (blue) stabilizes the activation-function helix H12 and
a novel adjacent helix (orange). These interactions are absent from other NRs, such as the close paralog VDR (white), shown with its ligand vitamin D
(blue spheres). (C) In the LRH-1 protein of rodents (left, blue), a salt bridge and hydrogen bond between side chains on helices 7 and 10 replaces
similar interactions in human LRH-1 (right, green) between each helix and the ligand (white). The ligand-mediated interactions and the amino acids
involved in human LRH-1 are also present in the closest NR paralog SF-1 [33]. For PDB identifiers, see Table S7.
doi:10.1371/journal.pbio.1000497.g006
Nuclear Receptor Evolution by Molecular Tinkering
PLoS Biology | www.plosbiology.org 10 October 2010 | Volume 8 | Issue 10 | e1000497
likelihood ratio for each node—the estimated ratio of the
likelihood of the best tree with the split to the best tree without
the split [45]—as well as the chi-square confidence metric, which
approximates 12 p, where p is the probability that an approximate
likelihood ratio as great or greater than that observed at a resolved
node would occur if the null hypothesis of an unresolved node is
true [45]. To identify the next best alternative tree for the basal
split between the AqNR1 and AqNR2-containing groups, we used
Phyml to optimize the branch lengths and model parameters on
each of the two possible rearrangements of the ML tree around
this internal branch and then report their likelihoods. To
determine the effect of fast-evolving sites on the inference of
phylogeny, we used PAML software to identify sites in the top two
octiles of the gamma distribution (Table S3) and repeated the
analysis with those 113 sites removed. To facilitate adequate
sampling of tree space in Bayesian analysis [46], we used a 174-
sequence taxon-trimmed MUSCLE-aligned dataset including
nuclear receptors from the following taxa representative of the
major metazoan lineages: Acropora millepora, Nematostella vectensis,
and Tripedalia cystophora (cnidarians); Amphimedon queenslandica and
Suberites domuncula (poriferans); Branchiostoma floridae (cephalochor-
date); Capitella capitata and Lottia gigantea (lophotrochozoans); Ciona
intestinalis (urochordate); Drosophila melanogaster (ecdysozoan); Homo
sapiens (vertebrate); Saccoglossus kowalevskii (hemichordate); Strongylo-
centrotus purpuratus (echindoderm); and Trichoplax adhaerens (pla-
cozoan). Terminal branches of length $0.76 in the PhyML
analysis (except for AqNR2) were removed. Four heated chains
were run for 8 million generations with temperature 0.3; the cold
chain was sampled every 100 generations. Priors were uniform on
topologies, uniform (0, 5) on branch lengths, and uniform (0.1, 10)
on the alpha shape parameter. The first 6,694,000 generations
were discarded as burn-in, because at this point in the chain the
standard deviation of posterior probabilities over all splits was
,0.01 and the two chains had converged as evaluated using the
‘‘compare’’ option of AWTY software [47]. We also repeated ML
analysis on this reduced 174-sequence dataset and found no
change in the relationships among NR types (Figure S5).
The phylogeny shows that AqNR1 is the ortholog of the
previously identified but misnamed ‘‘RXR’’ gene identified in the
sponge Suberites domuncula [48] and is identical to the ‘‘HNF4’’ gene
previously reported in A. queenslandica [49]. To determine the
minimum number of gene duplications and losses, we used SDI
software [50]. The 275-sequence ML phylogeny was reduced by
collapsing sets of orthologous NRs within major taxa—Porifera,
Placozoa, Cnidaria, Protostomia, Deuterostomia—into single
clades. To avoid spurious inference of duplication/loss due to
phylogenetic error, nodes with likelihood-ratio support ,10 that
conflicted with the accepted taxonomic phylogeny (Porifera,
(Placozoa, (Cnidaria, (Protostomia, Deuterostomia)))) were treated
as unresolved and reordered to be congruent with the taxonomic
phylogeny. SDI software was then used to reconcile this gene family
tree with the taxonomic tree and identify the root with the lowest
possible mapping cost (duplications plus losses). The mapping cost
was also calculated for all possible roots of the gene family
phylogeny, except for rootings on branches after the Cnidaria/
Bilateria divergence, which have higher mapping costs and were
judged to be implausible. Reconstructions of ancestral structural and
functional states were performed manually using Fitch parsimony.
Isolation of Amphimedon Nuclear Receptor Genes
Demosponge Amphimedon queenslandica were collected from Heron
Island Reef, Great Barrier Reef, and total RNA was isolated from
larvae using RNeasy Mini kit (Qiagen, Valencia, CA). The coding
regions of AqNR1 and AqNR2 were obtained using BD SMART
RACE cDNA Amplification kit (Clontech, Mountain View, CA),
and the full reading frames were amplified by RT-PCR, cloned into
pGEM-T EASY vector (Promega, Madison, WI), and verified by
sequencing. In situ hybridization analysis of RNA expression was
conducted as previously described [49].
Transcriptional Activation Assays
AqNR1, AqNR2, and rat HNF4a receptor LBDs, including the
hinge region and carboxy-terminal extension, were amplified by
high-fidelity PCR using Phusion DNA polymerase (New England
Biolabs, Ipswich, MA) and cloned into a GAL4-DBD-pSG5
expression vector (gift of D. Furlow). AqNR1-LBD (gi ACA04755)
consisted of amino acids 263636, and AqNR2-LBD (GU811658)
included amino acids 118–852. Rat HNF-4a template was a gift
from Frances Sladek; the LBD used consisted of amino acids 116–
465 (NP_071516). Site-directed mutagenesis was performed using
QuickChange II (Stratagene, La Jolla, CA) and verified by
sequencing.
Chinese hamster ovary (CHO-K1) cells were grown in a 96-well
plate and transfected with1 ng of receptor plasmid, 100 ng of a
UAS-driven firefly luciferase reporter (pFRluc), and 0.1 ng of the
constitutive phRLtk Renilla luciferase reporter plasmid, using
Lipofectamine and Plus Reagent in OPTIMEM (Invitrogen,
Carlsbad, CA). After 4 h, transfection medium was replaced with
phenol-red-free aMEM supplemented with 10% dextran-char-
coal-stripped fetal bovine serum (Hyclone, Logan, UT). Cells were
allowed to recover and express protein for 48 h, and then assayed
by luminometry using the Dual-Glo luciferase system (Promega,
Madison, WI). Firefly luciferase activity was normalized by Renilla
luciferase activity.
Protein Expression and Purification
AqNR1 LBD (residues 415–636), AqNR1 mutant proteins,
AqNR2 LBD (residues 616–852), and rHNF4a (residues 133–382)
were expressed as N-terminal hexahistidine maltose binding
protein fusions with a TEV cleavable linker in pLIC-MBP (a gift
from J. Sondek) and grown in E. coli BL21(DE3) pLysS cells using
standard methods. Protein was purified using affinity chromato-
graphy using standard methods. Following TEV cleavage, the
resulting 6xHis-tagged MBP was removed using an additional
nickel affinity column and the AqNR1 or AqNR2 was polished via
gel filtration. Pure AqNR1 or AqNR2 was dialyzed against
150 mM ammonium acetate (pH 7.4) prior to lipid extraction.
Mass Spectrometry
Organic solvent extraction was performed on purified LBDs
from bacteria to facilitate detailed characterization of bound
ligands in the absence of protein. Before extraction, 0.1 mg of C13
labeled palimitic acid was added as an internal standard. Lipid
from approximately 4 mg of wild-type or mutant forms of AqNR1
LBD, AqNR2, or rHNF-4 LBD were extracted with a 2:1
chloroform/methanol (v/v) solution and then analyzed by
negative ion ESI/MS. All extractions were performed in duplicate.
Mass spectra were acquired on a LTQ FT Hybrid Mass
spectrometer (Thermo Finnigan LTQ-FTMS, Somerset, NJ)
equipped with an electrospray source. Typically, 10
mL of the
aforementioned lipid solution was diluted into 10
mL water/
acetonitrile (2:1 v/v) and subjected to ESI/MS in the negative ion
mode. In addition to the fatty acids shown in Figure 2B, an
additional unidentified substance at ,421 m/z was also bound
when AqNR1 was incubated with either complete or stripped
serum. All samples were run in triplicate. Data acquisition and
analysis were performed using the instrument’s xcalibur software.
Nuclear Receptor Evolution by Molecular Tinkering
PLoS Biology | www.plosbiology.org 11 October 2010 | Volume 8 | Issue 10 | e1000497
Free Fatty Acid Binding
Purified wild-type or mutant hexahistidine maltose binding
protein fused AqNR1 was incubated with undiluted complete
(Invitrogen - 26010) or cyclodextran/charcoal stripped (HyClone -
SH30068.03, Waltham, MA) serum at a ratio of 20 mg protein to
5 ml undiluted serum. The protein/serum mixture was incubated
overnight at 4uC followed by re-purification over a nickel affinity
column. Protein purity was assessed by SDS-PAGE and fractions
containing pure wt or mutant AqNR1 were pooled. Bound lipids
were then quantified using the free fatty acid quantification kit
from BioVison Inc. (Mountain View, CA). 0.5 mg of each purified
LBD was subject to chloroform/detergent extraction to isolate the
long chain free fatty acids. Extracted fatty acids were enzymati-
cally converted to their CoA derivatives and oxidized, allowing
quantitation in a colorimetric assay (l = 570) relative to a standard
curve generated using palmitic acid.
Homology Modeling and Calculations of Cavity Volumes
Efforts to determine the crystal structure of AqNR1-LBD were
unsuccessful, so its structure was predicted by homology modeling
and energy minimization. The AqNR1 LBD amino acid sequence
was aligned to and threaded on human HNF-4a (PDB 1M7W)
and then energy minimized with palmitic acid—the most
abundant experimentally bound ligand—using the Homology
module in InsightII (Accelrys, Inc., San Diego, CA).
To calculate ligand pocket volumes of receptors with X-ray
crystal structures, we used VOIDOO [51] in probe-occupied
mode. We assigned the centroid of the bound ligand or a manually
defined point as a starting locus for cavity searches. Cavity
volumes were calculated using 10 random orientations of the
protein using 10 different ‘‘van der Waals growth factors’’ ranging
from 1.1–1.3. Mean and mode cavity volumes with standard
deviation are listed in Table S4.
To calculate ligand pocket volumes of receptors whose
structures have not yet been determined by x-ray crystallography,
we inferred homology models. Specifically, we created homology
models of the LBDs of AqNR1 (gi 167859601, residues 404–534),
annelid ER (186908731, residues 231–479), Branchiostoma SR
(170178459, residues 298–532), and Branchiostoma ER
(170178461, residues 250–504). In each case, we used as templates
crystal structures of several NR LBDs with a variety of cavity
volumes, including human ERa with estradiol (PDB 1ERE:A,
cavity volume 447 A
˚
3
), human ERR3 apo form (1KV6:A, cavity
volume 262 A
˚
3
), human ERR1 apo form (3D24:A, cavity volume
42 A
˚
3
), and AqNR1 as modeled on template HNF4A with DAO
(1MV7:A, cavity volume 680 A
˚
3
). We generated 10 models for
every protein with Modeller 9.7 using the default parameters [52].
Models were visually inspected for artifacts (e.g., knotting) and
further assessed using RamPage in CCP4i software; only models
with 95% of residues in the preferred region and ,1% of residues
in the outlier region of the Ramachandran map were accepted.
We then used Voidoo software as described above to calculate
cavity volumes, which are listed in Table S5.
Supporting Information
Figure S1 Expression of AqNR1 and AqNR2 during sponge
development, visualized using in situ hybridization.
Found at: doi:10.1371/journal.pbio.1000497.s001 (0.49 MB PDF)
Figure S2 Maximum likelihood phylogeny of the nu-
clear receptor superfamily.
Found at: doi:10.1371/journal.pbio.1000497.s002 (0.33 MB PDF)
Figure S3 Phylogeny of the nuclear receptor family
inferred using Bayesian MCMC.
Found at: doi:10.1371/journal.pbio.1000497.s003 (1.24 MB PDF)
Figure S4 Maximum likelihood NR phylogeny with fast-
evolving sites removed.
Found at: doi:10.1371/journal.pbio.1000497.s004 (0.81 MB PDF)
Figure S5 Maximum likelihood NR phylogeny on a
dataset with reduced taxon sampling.
Found at: doi:10.1371/journal.pbio.1000497.s005 (0.68 MB PDF)
Figure S6 Next-best phylogenetic arrangement of the
basal split between sponge NR paralogs.
Found at: doi:10.1371/journal.pbio.1000497.s006 (0.61 MB PDF)
Figure S7 Profile of lipids associated with AqNR1,
detected using mass spectrometry.
Found at: doi:10.1371/journal.pbio.1000497.s007 (0.65 MB PDF)
Figure S8 Reconstruction of NR ligand-binding and
evolution if AncNR is assumed to have been a ligand-
independent activator.
Found at: doi:10.1371/journal.pbio.1000497.s008 (0.54 MB PDF)
Figure S9 Reconstruction of ancestral structural and
functional characters on an alternate NR phylogeny.
Found at: doi:10.1371/journal.pbio.1000497.s009 (0.58 MB PDF)
Table S1 Species, taxonomic classification, abbrevia-
tions, and accession numbers of NR sequences used in
phylogenetic analyses.
Found at: doi:10.1371/journal.pbio.1000497.s010 (2.00 MB PDF)
Table S2 Mutations known to cause constitutive activity
in liganded NRs or ligand-activation in constitutive
receptors.
Found at: doi:10.1371/journal.pbio.1000497.s011 (0.18 MB PDF)
Table S3 Sites in NR alignment classified by evolu-
tionary rate.
Found at: doi:10.1371/journal.pbio.1000497.s012 (1.10 MB PDF)
Table S4 Protein structures and ligands used for
calculating cavity volumes.
Found at: doi:10.1371/journal.pbio.1000497.s013 (0.53 MB PDF)
Table S5 Cavity volumes of modeled nuclear receptors
using different templates
Found at: doi:10.1371/journal.pbio.1000497.s014 (0.75 MB PDF)
Table S6 Key to nuclear receptor nomenclature.
Found at: doi:10.1371/journal.pbio.1000497.s015 (0.44 MB PDF)
Table S7 Protein data bank accession numbers for
structures referred to in the text.
Found at: doi:10.1371/journal.pbio.1000497.s016 (0.30 MB PDF)
Acknowledgments
We are grateful to Vincent Laudet, who proposed the first testable
hypothesis about the functions of the ancestral NR. We thank Jesse
Zaneveld and Victor Hanson-Smith for technical assistance. We are
grateful to Bill Cresko, Patrick Phillips, and members of the Thornton lab
for suggestions on the manuscript.
Author Contributions
The author(s) have made the following declarations about their contribu-
tions: Conceived and designed the experiments: JTB EO BMD JWT.
Performed the experiments: JTB GNE CL KD MJH MG EO JWT.
Analyzed the data: JTB GNE CL KD MJH MG EO BMD JWT. Wrote
the paper: JWT.
Nuclear Receptor Evolution by Molecular Tinkering
PLoS Biology | www.plosbiology.org 12 October 2010 | Volume 8 | Issue 10 | e1000497
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PLoS Biology | www.plosbiology.org 13 October 2010 | Volume 8 | Issue 10 | e1000497
    • "In the last few years, there has been significant progress towards understanding the evolution of steroid hormone receptors (Bertrand et al., 2004; Bridgham et al., 2008; Markov et al., 2009). These receptors, including the estrogen, androgen, progesterone, mineralocorticoid and glucocorticoid receptors, belong to the nuclear receptor family of transcription factors that mediate the physiological actions of their cognate steroids (Bridgham et al., 2010; Gronemeyer et al., 2004; Huang et al., 2010). Phylogenetic analyses indicate that the vertebrate ERs are in a separate clade from the other steroid receptors (SRs), which includes the receptors for androgen (AR), progestogens (PR), corticosteroids (GR and MR) (Baker et al., 2015; Bridgham et al., 2008; Markov et al., 2009; Thornton, 2001). "
    [Show abstract] [Hide abstract] ABSTRACT: Estrogens regulate many physiological responses in vertebrates by binding to the estrogen receptor (ER), a ligand-activated transcription factor. To understand the evolution of vertebrate ERs and to investigate how estrogen acts in a jawless vertebrate, we used degenerate primer sets and PCR to isolate DNA fragments encoding two distinct ER subtypes, Esr1a and Esr1b from the Japanese lamprey, Lethenteron japonicum. Phylogenetic analysis indicates that these two ERs are the result of lineage-specific gene duplication within the jawless fishes, different from the previous duplication event of Esr1 (ERα) and Esr2 (ERβ) within the jawed vertebrates. Reporter gene assays show that lamprey Esr1a displays both constitutive and estrogen-dependent activation of gene transcription. Domain swapping experiments indicate that constitutive activity resides in the A/B domain of lamprey Esr1a. Unexpectedly, lamprey Esr1b does not bind estradiol and is not stimulated by other estrogens, androgens or corticosteroids. A 3D model of lamprey Esr1b suggests that although estradiol fits into the steroid binding site, some stabilizing contacts between the ligand and side chains that are found in human Esr1 and Esr2 are missing in lamprey Esr1b.
    Full-text · Article · Jul 2016
    • "Glucocorticoids (Fig. 1 ) regulate a variety of physiological functions including carbohydrate and protein metabolism, blood pressure , immune function and the body's anti-inflammatory processes via transcriptional activation of the glucocorticoid receptor (GR) [1][2][3][4][5]. The GR and other steroid receptors belong to the nuclear receptor family, a large family of transcription factors, which includes receptors for thyroid hormone, retinoids and other small lipophilic molecules [6][7][8][9][10]. The GR and other steroid receptors have a characteristic modular structure consisting of an N-terminal domain (NTD) (domains A and B), a central DNA-binding domain (DBD) (domain C), a hinge domain (D) and a C-terminal ligandbinding domain (LBD) (domain E) [9,11–13] (Fig. 2). "
    [Show abstract] [Hide abstract] ABSTRACT: We investigated the evolution of the response of human, chicken, alligator and frog glucocorticoid receptors (GRs) to dexamethasone, cortisol, cortisone, corticosterone, 11-deoxycorticosterone, 11-deoxycortisol and aldosterone. We find significant differences among these vertebrates in the transcriptional activation of their full length GRs by these steroids, indicating that there were changes in the specificity of the GR for steroids during the evolution of terrestrial vertebrates. To begin to study the role of interactions between different domains on the GR in steroid sensitivity and specificity for terrestrial GRs, we investigated transcriptional activation of truncated GRs containing their hinge domain and ligand binding domain (LBD) fused to a GAL4 DNA binding domain (GAL4-DBD). Compared to corresponding full length GRs, transcriptional activation of GAL4-DBD_GR-hinge/LBD constructs required higher steroid concentrations and displayed altered steroid specificity, indicating that interactions between the hinge/LBD and other domains are important in glucocorticoid activation of these terrestrial GRs.
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    • "ing of endocrine systems and their exploitation by xenobiotics. For example, Retinoic Acid receptors (RAR) and Estrogen receptors (ER), albeit absent from ecdysozoans, are present in annelids and molluscs with variable ligand specificities (Bridgham et al., 2010; Keay and Thornton, 2009; Thornton et al., 2003). These examples further emphasize the need to consider an appropriate phylogenetic sampling to recognize NR function in invertebrate phyla (Castro and Santos, 2014). "
    [Show abstract] [Hide abstract] ABSTRACT: The origin and diversification of the metazoan endocrine systems represents a fundamental research issue in biology. Nuclear receptors are critical components of these systems. A particular group named VDR/PXR/CAR (NR1I/J) is central in the mediation of detoxification responses. While orthologues have been thoroughly characterized in vertebrates, a sparse representation is currently available for invertebrates. Here, we provide the first isolation and characterization of a lophotrochozoan protostome VDR/PXR/CAR nuclear receptor (NR1J), in the estuarine bivalve the peppery furrow shell (Scrobicularia plana). Using a reporter gene assay, we evaluated the xenobiotic receptor plasticity comparing the human PXR with the S. plana NR1Jβ. Our results show that the molluscan receptor responds to a natural toxin (okadaic acid) in a similar fashion to that reported for other invertebrates. In contrast, the pesticide esfenvalerate displayed a unique response, since it down regulated transactivation at higher concentrations, while for triclosan no response was observed. Additionally, we uncovered lineage specific gene duplications and gene loss in the gene group encoding NRs in protostomes with likely impacts on the complexity of detoxification mechanisms across different phyla. Our findings pave the way for the development of multi-specific sensor tools to screen xenobiotic compounds acting via the NR1I/J group.
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