REVIEW

The nuclear receptor superfamily:

A structural perspective

Emily R. Weikum†, Xu Liu †

, and Eric A. Ortlund *
Department of Biochemistry, Emory School of Medicine, Atlanta, 30322, Georgia

Received 19 July 2018; Accepted 6 August 2018

DOI: 10.1002/pro.3496
Published online 00 Month 2018 proteinscience.org

Abstract: Nuclear receptors (NRs) are a family of transcription factors that regulate numerous physiologi-
cal processes such as metabolism, reproduction, inflammation, as well as the circadian rhythm. NRs
sense changes in lipid metabolite levels to drive differential gene expression, producing distinct physio-
logic effects. This is an allosteric process whereby binding a cognate ligand and specific DNA sequences
drives the recruitment of diverse transcriptional co-regulators at chromatin and ultimately transactivation
or transrepression of target genes. Dysregulation of NR signaling leads to various malignances, metabolic
disorders, and inflammatory disease. Given their important role in physiology and ability to respond to
small lipophilic ligands, NRs have emerged as valuable therapeutic targets. Here, we summarize and dis-
cuss the recent progress on understanding the complex mechanism of action of NRs, primarily from a
structural perspective. Finally, we suggest future studies to improve our understanding of NR signaling
and better design drugs by integrating multiple structural and biophysical approaches.
Keywords: nuclear receptor; ligand binding domain; DNA binding domain; co-regulator; transactiva-
tion; transrepression

Introduction
The nuclear receptor (NR) superfamily is composed of
a family of transcription factors (TFs) that play an
important role in a number of biological processes
Abbreviations: AF, activation function; cryo-EM, cryo-electron including metabolism, reproduction, and inflamma-
microscopy; DBD, DNA binding domain; ER, estrogen recep-
tion.1,2 The first member of this family was cloned in
tor; FXR, farnesoid X receptor; GR, glucocorticoid receptor;
HDX-MS, hydrogen/ deuterium exchange coupled with mass 1985, but today the family has expanded to include
spectrometry; H12, helix 12; LBD, ligand binding domain; LXR, 48 members in humans.3,4 Most NRs are regulated
liver X receptor; NR, nuclear receptor; NTD, N-terminal endogenously by small lipophilic ligands such as ste-
domain; RE, response element; SMT, single-molecule tracking; roids, retinoids, and phospholipids, but this protein
SR, steroid receptor; TR, thyroid hormone receptor; TF, tran-
family also contains “orphan” members for which no
scription factor.
ligand has yet been identified.5 Ligand binding
Grant sponsor: American Heart Association17-
induces conformational changes within the receptor,
POST3366011014GRNT20460124; Grant sponsor: National
Institutes of Health1F31GM113397-01A1R01DK095750; Grant which in turn binds specific DNA sequences through-
sponsor: W.M. Keck Foundation. out the genome.6,7 Once DNA-bound, co-regulator
*Correspondence: Eric A. Ortlund, Department of Biochemis- proteins, chromatin remodelers, and the general tran-
try, Emory School of Medicine, Atlanta, GA. 30322. E-mail: scriptional machinery are recruited to the DNA in
[email protected] order to activate or repress target gene
†
Emily R. Weikum and Xu Liu contributed equally to this work. expression.8–10 Since NRs are responsible for

1876 PROTEIN SCIENCE 2018 | VOL 27:1876–1892 Published by Wiley © 2018 The Protein Society
regulating thousands of genes, their activity is tightly heterodimeric complexes with many NRs and is the
controlled.11,12 If left unchecked, aberrant NR activity only receptor in the group with a known activating
can underlie numerous diseases such as cancer, dia- ligand, 9-cis retinoic acid.42
betes, and chronic inflammation.13,14 Subgroup 3: This group comprises the steroid
Our knowledge of the NR family has drastically receptors (SRs), which are key regulators of a host of
expanded within the last decade due to advance- metabolic, reproductive, and developmental pro-
ments in genome-wide methodologies, structural cesses.43 The SR family includes the androgen recep-
studies of receptor domains and full-length com- tor (AR),44 progesterone receptor (PR),45
46
plexes, and identification of new co-regulator proteins glucocorticoid receptor (GR), mineralocorticoid
that modulate receptor activity.15 This work has laid receptor (MR),47 and two closely related estrogen
the foundation for pharmaceutical companies and receptors (ERα and ERβ).48 Cholesterol-derived hor-
academic researchers to develop synthetic ligands mones, like cortisol and estrogen, regulate SRs
that target these receptors.16,17 Yet, due to the through direct binding.
diverse array of genes regulated by these proteins, Subgroup 4: This group contains the orphan
along with the fact that many drugs are not explicitly nuclear receptors nerve growth Factor 1B (NGF1-B),
specific for one receptor, drugs that target NRs tend nurr-related Factor-1 (NURR1), and neuron-derived
to have unwanted side effects.16,18 For this reason, orphan Receptor-1 (NOR-1). These proteins are
more research is required to understand all the mech- required for neuron development and maintenance.49
anisms that guide NR regulation. Improving our Subgroup 5: This group contains steroidogenic
understanding of NR regulation could pave the way Factor 1 (SF-1)50 and liver receptor Homolog-1 (LRH-
for future therapeutics. Here, we introduce this pro- 1).51 Although generally still classified as orphan
tein family and focus on the structural mechanisms receptors, evidence suggests these proteins are regu-
governing NR action. lated by phospholipids.27,52 LRH-1 and SF-1 are vital
for development and metabolism.51,53
Nuclear Receptor Superfamily Classification Subgroup 6: This group contains only one recep-
NRs are divided into seven subfamilies.19,20 A list of tor, germ cell nuclear factor (GCNF),54 an orphan
receptors, subfamilies, and their ligands are shown in receptor that has a critical role in development.55
Table I. This protein remains in its own category due to a crit-
Subgroup 0: This group includes the atypical ical difference in its LBD; it does not contain an acti-
NRs, dosage-sensitive sex reversal-adrenal hypopla- vator function HR (AF-H) and is known to drive gene
sia congenital critical region on the X chromosome, silencing.56
Gene 1 (DAX) and small heterodimer partner
(SHP).21,22 These two proteins are unique in their Structural Insight into Nuclear Receptor Action
structures and contain only a ligand-binding domain X-ray crystal structures of nuclear receptors, both
(LBD) that folds in a manner consistent with the rest full-length and discrete domains, have provided criti-
of the family.23–25 Their LBDs also contain motifs cal information on how ligands and DNA response
that are commonly seen in NR coactivators.26 These elements are recognized, how they dimerize, and
motifs interact with other NR LBDs to regulate interact with co-regulators.
transcription.27–31
Subgroup 1: This large family is formed by thy- Overall architecture
roid hormone receptors (TR),32 retinoic acid receptors Despite diversity in the size, shape, and charges of
(RAR),33 peroxisome proliferator activated receptors activating ligands, almost all members of the nuclear
(PPAR),34 reverse-Erb receptors (REV-ERB,35 reti- receptor superfamily share a common modular
noic acid related receptors (ROR),35 farnesoid X domain structure.15,57 Except for the atypical recep-
receptors (FXR),36 liver X receptors (LXR),37 and vita- tors SHP and DAX, the overall architecture is com-
min D receptors (VDR).38 These receptors are regu- posed of five domains: A–E [Fig. 1(A)]. Each of these
lated by a variety of lipophilic signaling molecules subdomains plays a specific role in receptor biology.58
including thyroid hormone, fatty acids, bile acids, and The mass of NRs can vary but they are generally
sterols. between 66 and 100 kD [Fig. 1(B)].
Subgroup 2: This subfamily contains orphan A/B: N-terminal domain (NTD): The NTD is a
receptors such as the retinoid X receptors (RXR),39 highly disordered domain, which explains why the
chicken ovalbumin upstream promoter transcription NTD is not amenable to structural analysis. Addition-
factors (COUP-TF),40 and hepatocyte nuclear Factor ally, there is little sequence conservation between NR
4 (HNF4).41 All of these orphans have been shown to NTDs and there is a large disparity in the size of this
bind fatty acids via structural studies. However, it is domain [Fig. 1(B)].
unclear whether these ligands play a role in dynamic The NTD contains the activator Function-1
ligand-driven regulation, as seen in other NR classes. region (AF-1), which interacts with a variety of co-
RXR is of particular importance as it forms regulator proteins in a cell- and promoter-specific

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Table I. Nuclear Receptor Superfamily
Family Common name Abbreviation Gene name Ligand

0B Dosage-sensitive sex reversal-adrenal DAX1 NR0B1 Orphan

hypoplasia congenital critical region on the X
chromosome, Gene 1
Short heterodimeric partner SHP NR0B2 Orphan
1A Thyroid hormone receptor-α TRα THRA Thyroid hormones
Thyroid hormone receptor-β TRβ THRB Thyroid hormones
1B Retinoic acid receptor-α RARα RARA Retinoic acids
Retinoic acid receptor-β RARβ RARB Retinoic acids
Retinoic acid receptor-γ RARγ RARG Retinoic acids
1C Peroxisome proliferator-activated receptor-α PPARα PPARA Fatty acids
Peroxisome proliferator-activated receptor-β PPARβ PPARD Fatty acids
Peroxisome proliferator-activated receptor-γ PPARγ PPARG Fatty acids
1D Reverse-Erb-α REV-ERBα NR1D1 Heme
Reverse-Erb-β REV-ERBβ NR1D2 Heme
1F Retinoic acid-related orphan-α RORα RORA Sterols
Retinoic acid-related orphan-β RORβ RORB Sterols
Retinoic acid-related orphan-γ RORγ RORC Sterols
1H Farnesoid X receptor FXRα NR1H4 Bile Acids
Farnesoid X receptor-β FXRβ NR1H5P Orphan
Liver X receptor-α LXRα NR1H3 Oxysterols
Liver X receptor-β LXRβ NR1H2 Oxysterols
1I Vitamin D receptor VDR VDR 1α,25-dihydroxyvitamin D3
Pregnane X receptor PXR NR1I2 Endobiotics and xenobiotics
Constitutive androstane receptor NR1I3 Xenobiotics
2A Hepatocyte nuclear Factor-4-α HNF4α HNF4A Fatty acids
Hepatocyte nuclear Factor-4-γ HNF4γ HNF4G Fatty acids
2B Retinoid X receptor-α RXRα RXRA 9-Cis retinoic acid
Retinoid X receptor-β RXRβ RXRB 9-Cis retinoic acid
Retinoid X receptor-γ RXRγ RXRG 9-Cis retinoic acid
2C Testicular Receptor 2 TR2 NR2C1 Orphan
Testicular Receptor 4 TR4 NR2C2 Orphan
2E Tailless homolog orphan receptor TLX NR2E1 Orphan
Photoreceptor-cell-specific nuclear receptor PNR NR2E3 Orphan
2F Chicken ovalbumin upstream COUP-TFα NR2F1 Orphan
promoter-transcription factor α
Chicken ovalbumin upstream COUP-TFβ NR2F2 Orphan
promoter-transcription factor β
Chicken ovalbumin upstream COUP-TFγ NR2F6 Orphan
promoter-transcription factor γ
3A Estrogen receptor-α ERα ESR1 Estrogens
Estrogen receptor-β ERβ ESR2 Estrogens
3B Estrogen-related receptor-α ERRα ESRRA Orphan
Estrogen-related receptor-β ERRβ ESRRB Orphan
Estrogen-related receptor-γ ERRγ ESRRG Orphan
3C Androgen receptor AR AR Androgens
Glucocorticoid receptor GR NR3C1 Glucocorticoids
Mineralocorticoid receptor MR NR3C2 Mineralocorticoids and
glucocorticoids
Progesterone receptor PR PGR Progesterone
4A Nerve growth Factor 1B NGF1-B NR4A1 Orphan
Nurr-related Factor 1 NURR1 NR4A2 Unsaturated fatty acids
Neuron-derived orphan Receptor 1 NOR-1 NR4A3 Orphan
5A Steroidogenic Factor 1 SF-1 NR5A1 Phospholipids
Liver receptor Homolog-1 LRH-1 NR5A2 Phospholipids
6A Germ cell nuclear factor GCNF NR6A1 Orphan
Table of human nuclear receptors, gene name, and their activating ligands.

manner.59 For all NRs, the majority of the domain is post-translational modifications including phosphory-
disordered. However, the GR NTD can adopt a more lation, SUMOylation, and acetylation.61 These modifi-
alpha-helical structure when co-regulators are cations have varying effects, both driving and
bound.60 This region also gives rise to multiple iso- repressing transcription.
forms through alternative splicing, as seen in TR and C: DNA binding domain (DBD): This region is
GR.46 Finally, the NTD is the target for numerous the most conserved among all nuclear receptor

1878 PROTEINSCIENCE.ORG The Nuclear Receptor Superfamily

Figure 1. Modular domain structure of NRs. (A) Basic modular domain structure of NRs is composed of an unstructured NTD that
contains the Activation Function 1 (AF-1) surface, a zinc finger DBD, a flexible hinge region, and a LBD that binds to ligands and
interacts with co-regulator proteins through the Activation Function 2 (AF-2) surface. (B) General domain size and amino acid
length of a variety of NRs. The DBD and LBDs are the most conserved regions where as the other domains are more variable in
length and sequence composition. (C) Example of a full-length NR structure shows LXR-RXR heterodimer (PDB: 4NQA) (DBD
colored purple, hinge region in yellow, and LBD in green).

domains.62 The DBD has two subdomains that each this subdomain contains the distal box, or “D box,”
contains four cysteine residues that co-ordinate a zinc that contains residues for receptor dimerization.67–69
ion to create the canonical DNA-binding zinc finger Some NRs, like LRH-1 and GCNF, contain a DBD C-
motif [Fig. 2(A) and (B)].63 Each zinc finger is then terminal extension (CTE) that makes additional
followed by an amphipathic helix and a peptide base-specific contacts within the DNA minor
loop.64,65 The first subdomain contains the DNA- groove.70,71
reading helix, which interacts with the major groove D: Hinge Region: The hinge region is a short,
to make base-specific interactions with the DNA.66 flexible linker between the DBD and the LBD.58 This
The second subdomain helix makes non-specific con- region has the least sequence and size conservation
tacts with the DNA backbone. The peptide loop in between nuclear receptors. Like the NTD, this region

Figure 2. NR DNA binding domains. (A) Cartoon representation of NR DBDs indicating important motifs. This domain contains two
subdomains, each containing one zinc finger. The first subdomain residues interact with the DNA major groove to make base-
specific interactions on genomic response elements. The second subdomain participates in DBD dimerization and makes non-
specific contacts with the DNA backbone. Some NRs, like LRH-1 and GCNF, also contain C-terminal extensions (CTEs) that make
base-specific contacts with the minor groove. (B) Cartoon representation of folded GR DBD highlighting the important regions
(PDB: 3FYL). Zinc atoms are represented as spheres.

Weikum et al. PROTEINSCIENCE | VOL 27:1876–1892 1879

 24 are classified as “orphans” or “adopted orphans,”
meaning that a likely ligand has been identified. In
the absence of ligand, NRs tend to be unstable,
explaining the dearth of apo-NR LBD struc-
tures.75,78,79 Ligand binding greatly increases the sta-
bility of the LBD, evidenced by changes in NMR
spectra between liganded and unliganded PPARs and
less proteolytic cleavage seen in the ER ligand-bound
versus apo state.77,78,80,81 This stabilization, among
other factors, facilitates co-regulator binding.82
Ligands bind the receptor within the LBP at the
base of the LBD. This pocket is composed of ~75%
hydrophobic residues, but also contains critical polar
residues that make key hydrogen bonding interac-
tions to the ligand.75 These hydrogen bonds help posi-
tion the ligand in the correct orientation. For
example, endogenous SR ligands are composed of a
rigid fused 4-ring scaffold that positions various H-
bond donors and acceptors to interact with the recep-
tor [Fig. 4(A) and (B)]. SRs use a conserved glutamine
Figure 3. NR ligand binding domains. Cartoon representation
on H3 and arginine on H5 to lock the ligand’s A ring
of the structurally conserved NR LBD. This domain is
in place [Fig. 4(A) and (B)].83,84 A striking example of
composed of 11 α-helices and 4 β-strands that fold into three
the importance of these hydrogen bond networks in
layers of a helical sandwich bundle. This fold creates a
hydrophobic ligand binding pocket at the bottom of the the LBP is seen in FXR and LXR ligands; although
receptor. This domain also contains the AF surface, composed similar, these ligands are bound in completely oppo-
of H3, H4, and the AF-H, which interacts with co-regulator site orientations due to the available hydrogen bond-
proteins (PDB: 1PZL). ing network within the LBP [Fig. 4(C) and (D)].85,86
These differences ensure the natural ligands are
is also a site for regulatory PTMs. The hinge can also bound by the correct receptor. Ligand selection is fur-
contain a nuclear localization signal.61,72 ther achieved by a dramatic difference in the size of
E: Ligand binding domain (LBD): The LBD is a ligand binding pockets across NRs. For example, SR
complex allosteric signaling domain that not only LBP pocket volumes tend to be 400–600 Å3 and
binds to ligands but also interacts directly with co- 700–850 Å3 for FXR and LXR, and almost 1300 Å3 for
regulator proteins.73,74 This structurally conserved PPARs [Fig. 4(E)].83,85,87 The volume of the pocket
domain commonly contains 11 α-helices and four generally corresponds to the size of the ligand sug-
β-strands that fold into three parallel layers to form gesting significant component of ligand selection
an alpha helical sandwich (Fig. 3).75 This folding cre- stems from steric selection.
ates a hydrophobic ligand-binding pocket (LBP) at
the base of the receptor.73,76,77 Superposition of NR
LBD structures reveals that the top part of the recep- NR–DNA interactions
tor is most similar whereas the base, which contains Nuclear receptor DBDs bind to a variety of DNA
the LBP, is more variable.15,75 This variability across response elements (REs) whose nucleotide sequences
NRs at the ligand-binding region allows NRs to recog- can take the form of a palindrome, direct repeat, or
nize a diverse cadre of ligands. extended monomeric sites (Fig. 5).63,67 The SRs bind
The LBD contains another activation function sur- palindromic repeats [Fig. 5(A)]. These palindromes
face (AF-2), which is composed of helices 3, 4, and 12. contain two AGGACA repeats that can be separated
Helix 12, or the activation function helix (AF-H) has by a spacer region that varies in length. The length of
been shown to be conformationally dynamic upon this spacer has been shown to allosterically modulate
ligand binding, altering the orientation of AF-2 to facili- SRs, resulting in varied transcriptional outputs.88–90
tate interaction with different co-regulator proteins.73,75 However, the most common spacer length is
3 bp.68,91,92 Receptors that bind direct repeats include
the RXR-RAR heterodimer, GCNF, and VDR [Fig. 5
NR–ligand interactions (B)].93–95 These sequences are composed of two
Nuclear receptors bind directly to a variety of small, AGGTCA sites separated by a spacer sequence from
lipophilic ligands such as steroids, thyroid hormone, 0 to 5 bp long. Finally, LRH-1 and SF-1 are examples
retinoids, and lipids that either diffuse or are trans- of receptors that bind extended monomeric sequences
ported across the cell membrane.5 Of the 48 human [Fig. 5(C)].71,96 These REs contain one AGGTCA site
NRs, 24 have known ligands and the remaining as well an A/T rich sequence directly upstream.

1880 PROTEINSCIENCE.ORG The Nuclear Receptor Superfamily

Figure 4. NR ligand interactions. Close up view of SR LBPs showing that (A) GR LBD-cortisol (PDB: 4P6X) and (B) ER LBD-
estradiol (PDB: 1ERE) use conserved Glu and Arg residues (blue sticks) to make hydrogen bonding interactions (red) with steroid
ligands. These interactions help orient the ligand within the pocket. (C) Close up views of FXR LBD-CDCA (PDB: 1OT7) and
(D) LXR LBD-epoxycholesterol (PDB: 1P8D) show, despite similar ligands, the receptors orient them in opposite directions. This
allows natural ligands to discriminate between NRs whose LBDs are highly conserved (E) Comparisons of ligand cavity sizes
between GR (PDB: 4P6X), FXR (PDB: 1OT7), and PPAR (PDB: 5AZV).

NRs function as monomers, homodimers, or commonly forms heterodimers with RXR.3,98 Similar
heterodimers to the ER structure, the dimer interface is formed
NRs are generally found as monomers in solution but among H7, H9, H10, H11, and Loops 8 and
upon DNA binding can form higher order complexes. 9 [Fig. 6(C)].99
NRs can be monomeric on DNA but are more often
found as homodimers or in heterodimeric complexes NR–co-regulator interactions
with RXR.3 This increases overall size and complexity After DNA binding, NRs recruit a variety of proteins
of NRs, allowing new surfaces to be accessed for collectively known as co-regulators.8,99 To date, there
PTMs or co-regulator binding.46 are approximately 200 different co-regulator proteins,
LRH-1, NGF1-B, and SF-1 are among the few which fall into two main categories: co-activators and
NRs that bind DNA as monomers.71,96 These receptors co-repressors.8,9 These interact directly with NRs at
utilize the CTE within their DBDs to facilitate addi- the AF-1 and AF-2 surfaces.59 Since the AF-1 lies
tional DNA contacts within the minor groove, expand- within the unstructured NTD, we have not been able
ing their DNA footprint. Members of the SR subfamily to obtain structural information about these interac-
commonly form homodimers. The ER LBD structure tions.58,60 However, almost all NR LBD structures
shows H8, H9, H10, and Loops 8–9 from each mono- are co-crystallized with fragments of co-regulator NR-
mer interact to form a homodimer [Fig. 6(A)].84 This is interaction domains at the AF-2 surface.59
in contrast with the GR dimer, which showed a unique Co-activator proteins interact with NRs via an
dimer interface not seen in other NR structures [Fig. 6 alpha-helix containing a short LXXLL motif (L- leu-
(B)].97 Finally, the rest of the NR superfamily cine, X- any amino acid).26,82 This motif interacts

Weikum et al. PROTEINSCIENCE | VOL 27:1876–1892 1881

Figure 5. Genomic response elements. Nuclear receptors bind to genomic response elements (RE) that come in a variety of
forms. (A) Members of the SR subfamily bind to palindromic repeats (shown as red DNA cartoon). These repeats are separated by
different spacer lengths (shown as yellow DNA cartoon). As examples, the ER DBD – estrogen response elements (ERE) and GR
DBD – glucocorticoid response element (GRE) crystal structures are shown. (B) Most other NRs bind to direct repeats, which can
also be separated by spacers from 0 to 5 bp. The structures of the RXR-RAR DBD heterodimer is shown in complex with a DR
with 1 bp spacer (DR1) and the VDR homodimer DBD is shown in complex with a DR with 3 bp spacer (DR3). (C) Although rare,
some NRs bind to DNA as a monomer to extended half site sequences. Examples include LRH-1 DBD and SF-1 DBD (PDBs, from
left to right: top row – 4AA6, 1DSZ, and 5L0M; bottom row – 3FYL, 1KB4, and 2FF0).

with the NR AF-2 surface. The co-regulator’s leucine which suggests that H12 is not in one fixed position,
residues lie within the hydrophobic groove of the AF- but rather is dynamic.79 Ligand binding stabilizes
2 surface and the ends of the helical peptide are gen- the helix into a more fixed conformation [Fig. 7
erally held in place by a charge clamp formed by a (E) and (F)]. Methods that measure dynamics of H12
lysine on the NR’s H3 and a glutamate on H12 that have been pivotal in providing evidence to support
cap the helix dipole [Fig. 7(A)].82 Co-repressors con- this model.77,81 In addition, other LBD surfaces are
tain conserved (L/I)XX(I/V)I or LXXX(I/L)XXX(I/L) stabilized upon ligand binding and appear to com-
motif (referred to as CoRNR box) (L- leucine, I- iso- municate with the AF-2 surface to modulate recep-
leucine, X- any amino acid).100,101 These extended tor activation. Examples include LRH-1, PPAR, ER,
motifs interact at the same hydrophobic AF surface and GR.108,109
but their length inhibits the canonical charge clamp
formation [Fig. 7(B)].102,103
Nuclear Receptor Signaling
The discrimination between either co-activator
or co-repressor binding has been linked to the con- Nuclear receptor mechanism of action
formational flexibility of H12.6,75 Originally, the NRs have been classified as into four mechanistic
“mouse-trap” model was proposed. This model was Subtypes I–IV (Fig. 8):
based on the structures of apo RXR and ligand- Type I Nuclear Receptors: Receptors of this group
bound RAR [Fig. 7(C) and (D)].104–106 It was posited are SRs and are activated by cholesterol-derived ste-
that upon agonist binding, there was a large struc- roidal hormones such as estrogens, androgens, pro-
tural rearrangement of H12, causing it to snap shut. gestagens, and corticoids.43 These receptors are
However, this phenomenon was only observed for a sequestered to the cytoplasm bound to chaperone pro-
few proteins.84 Other NR LBD structures, like LRH- teins but upon ligand activation, they exchange their
1 in both the apo and the ligand bound state, did not chaperone proteins and undergo nuclear transloca-
demonstrate large movements in H12.107 This sug- tion. In the nucleus, SRs generally bind as homodi-
gested another model was possible. The current mers to DNA REs that consist of two inverted repeats
favored model is the “dynamic stabilization model,” [Fig. 8(A)].110,111

1882 PROTEINSCIENCE.ORG The Nuclear Receptor Superfamily

Figure 6. NR dimerization interfaces. Many NRs utilize the H10/H11 surface to form homodimers or heterodimers. (A) ER LBD –
estrogen homodimeric complex shows dimerization occurs between H7, H9, H10/11 (PDB: 1ERE). (B) The LXR-RXR LBD heterodi-
mer shows a similar dimerization interface (PDB: 1UHL). (C) Unlike the other two, the GR LBD homodimer structure revealed a
novel dimerization interface (PDB: 1M2Z). The dimerization interface is colored blue, ligands are shown as sticks (green) and co-
regulator peptides are colored yellow.

Type II Nuclear Receptors: Receptors of this type, Transactivation and transrepression

such as RAR and LXR, are often retained in the NRs modulate transcription through many distinct
nucleus, regardless of the presence of activating mechanisms that ultimately result in either activa-
ligand.10 Upon ligand binding, the receptor is tion or repression of specific gene programs. As stated
released from a co-repressor complex and swapped above, transcriptional activation is achieved by ligand
for co-activators and the transcriptional machinery. binding stabilizing an active state.7 In this state, NRs
These receptors commonly form heterodimers with recruit co-activator proteins, which are typically scaf-
RXR on direct repeat DNA REs [Fig. 8(B)].3 folds that initiate the formation of large protein com-
Type III Nuclear Receptors: This type of NR, such plexes that harbor histone modifying enzymes such
as VDR, has a similar mechanism of action to Type II as histone acetyltransferases (HATs) or histone
NRs but instead form homodimers on their REs, methyltransferases (HMTs).112,113 These activities
which are direct repeat sequences [Fig. 8(C)].63 facilitate the opening of chromatin, making it accessi-
Type IV Nuclear Receptors: This type of NR has a ble to additional regulatory proteins. Finally, the gen-
similar mechanism of action to Type II NRs but eral transcriptional machinery and RNA Polymerase
instead bind to DNA as a monomer and recognize II are recruited to drive transcription [Fig. 9(A)].114
extended half-sites within REs [Fig. 8(D)].71,96 Exam- Conversely, NRs can repress transcription by two
ples of Type IV include LRH-1 and SF-1. different mechanisms.115 First, NRs can bind to co-

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Figure 7. NR co-regulator interactions. (A) Cartoon representation of the co-regulator LXXLL peptide (green) interacting with the AF
surface (purple). The peptide is held in place by a conserved charge clamp interaction formed by a glutamate on H12 and a lysine on
H3. (B) Cartoon representation of co-repressor peptides (pink) interacting with the AF surface (blue). Co-repressors contain extended
(L/I)XX(I/V)I or LXXX(I/L)XXX(I/L) motifs that do not allow for the charge clamp formation. The basis of the “mouse-trap” model was
made by comparing the apo (C) and ligand bound (D) structures of RXR. Upon ligand binding a large rearrangement of H12 is seen
(PDBs: 1LBD, 1MVC). (E,F) The more favored “dynamic stabilization” model of NR activation suggests H12 does not undergo such a
large conformational change, but instead H12 flexible and ligand binding simply stabilizes the helix. This model was proposed after
other apo NR structures, did not show H12 displaced and, upon ligand binding, there was little change in the location of this helix
(PDBs: 4DOR, 4PLE). Co-regulator peptides are colored blue and ligands are shown as sticks (green).

repressors in their apo state as shown in Type II–IV different conformations than when bound to “positive”
receptors.115 These co-repressor proteins recruit histone DNA response elements and favors co-repressor recruit-
modifying enzymes such as histone deacetylases ment to block transcription.119
(HDACs),8 which act in opposition of HATs to restrict
chromatin and block the transcriptional machinery from Nuclear Receptors as Critical Pharmaceutical
accessing the DNA [Fig. 9(B)].115,116 Second, NRs can Targets
interact with “negative DNA response elements.”117,118 Aberrant nuclear receptor signaling pathways con-
Binding to these elements results in NRs adopting tribute to numerous disease states such as cancer,

1884 PROTEINSCIENCE.ORG The Nuclear Receptor Superfamily

Figure 8. Schematic of NR signaling mechanisms. (A) Type I receptors reside in the cytoplasm (C) in complex with chaperone
proteins. Upon ligand binding (hexagon), the receptor is released from this complex and is trafficked into the nucleus (N) where
they typically bind to palindromic hormone response elements (HREs) as a homodimer to regulate transcription. (B) Type II
receptors are localized in the nucleus. In their unliganded state, they interact with co-repressor proteins, but upon ligand binding
are exchanged for co-activators. NRs in this group generally form heterodimeric complexes with RXR. (C) Similar to Type II
receptors, Type III receptors reside in the nucleus and exchange bound co-repressors and co-activators. These receptors bind to
direct repeat HREs as homodimers. (D) Type IV receptors are almost identical to Type III except they bind HREs that are extended
half sites as monomers.

diabetes, obesity, and others.14,17 For this reason, treating diabetes.123 These effects were later linked
NRs are major pharmaceutical targets. Initial ligand to PPARγ signaling.123 The newest generation of NR
design has been quite simple as NR LBPs are ligands are termed “selective nuclear receptor
enclosed and are amenable to binding a variety of modulators,” which are designed against a single NR
ligands.75 However, due to the breadth and complex- to partially or selectively activate a subset of signal-
ity of NR biology, designing ligands with limited ing pathways. These idea is to separate the beneficial
cross-reactivity or partial agonism has proven quite outcomes of treatment from the less desirable side
difficult. Despite these issues, NR-targeting ligands effects.124 Such ligands would be highly beneficial for
make up 10–20% of current FDA-approved drugs targeting ER, AR, and GR.125,126 Due to the complex-
have a worldwide market of 30 billion dollars per ity of NR signaling, these compounds have been
year.120 largely unsuccessful thus far.
Historically, there have been two main
approaches for identifying NR ligands. First, NR
Future Perspectives
ligands were isolated from human tissue extracts.121
For example, the study of the adrenal gland led to Insights into allostery
the discovery of a compound effective at blocking Significant advances in understanding the mecha-
inflammation. This compound was later discovered to nism of action of NR LBDs have been made by imag-
be cortisol, the endogenous ligand for GR.121 Later, ing static structural features of LBDs with distinct
synthesis of cortisol sparked the development of the ligands and relatively short peptides derived from co-
synthetic compounds dexamethasone and predniso- regulators. However, this approach does not capture
lone.122 Second, compounds were identified by con- conformational and allosteric effects driven by other
necting ligand effects with protein biology.17 For domains within the receptors (e.g. DNA binding
example, thiazolidinediones showed promise in domain) and other effectors (e.g. DNA). We also have

Weikum et al. PROTEINSCIENCE | VOL 27:1876–1892 1885

 were observed by HDX-MS, consistent with solution
NMR results. HDX-MS analysis also detected differ-
ent dynamical patterns in PPARγ between full and
partial agonist-bound states.109 Molecular dynamics
(MD) simulations are also powerful tools used to
characterize LBD conformational dynamics, espe-
cially when structural information is available. A MD
study revealed that the distinct allosteric communica-
tions in LRH-1 drive differential co-activator recruit-
ment preferences (i.e. Tif2 and PCG1α), despite the
same agonist being present. Moreover, these simula-
tion data agreed with experimental HDX-MS data,
providing cross-platform confirmation of different co-
regulator recruitment in LRH-1.108
Different biophysical techniques may also be
integrated to fully understand the conformational
plasticity and intrinsic allosteric/dynamic communi-
cation pathway utilized by NR LBDs. For example,
RXRα is known to form a heterodimer with either
PPARγ or most of the Type II NRs, such as
Figure 9. NRs both activate and repress transcription. (A) To TR. Intriguingly, the RXRα-PPARγ heterodimer, but
activate gene expression, NRs (blue) interact with their DNA not the RXRα-TR heterodimer, can be activated by
response elements. DNA-bound NRs recruit co-activator retinoic acid. This TR-mediated allosteric silencing
proteins (magenta), which in turn recruit histone-modifying signal is, therefore, critical for controlling the RXRα-
enzymes. These histone-modifying enzymes are commonly driven response. Integrative studies using x-ray crys-
histone acetylases (green), which acetylate histone tails. This tallography, NMR, and HDX-MS showed the alloste-
modification is a mark of active chromatin. Ultimately, the ric pathway initiated from the middle of dimer
general transcriptional machinery and RNA Polymerase Pol II interface, then propagated to the core of LBD, ulti-
(gray) are recruited to drive gene expression. (B) To repress
mately to Helix 12 and AF-2 to control ligand bind-
transcription, NRs recruit co-repressor proteins (orange).
ing.129 Therefore, LBD dynamics are an important
These proteins recruit other histone deacetylases (red) that
component in defining the complex NR signaling
reverse histone acetylation and restrict chromatin accessibility.
This condensation prevents the transcriptional machinery from code. Moreover, understanding dynamical differences
accessing the DNA, thus repressing gene expression. within the same structural ensemble strengthens a
structure–activity relationship pipeline in drug devel-
opment. This combined approach has been used in
a limited number of apo NR structures, although typ- drug discovery for PPARγ and should be used to bet-
ically only a few conformational populations are cap- ter guide design of ‘selective nuclear receptor modula-
tured in a crystalline lattice. tors’ targeting specific LBDs in the future.130,131
Solution NMR techniques are ideal for quantita-
tively dissecting the dynamic motions of proteins in Full length structures
distinct time scales, however this technique has seen Nuclear receptors contain no intrinsic activity;
limited use in studying NR LBDs. Since intrinsic rather, they nucleate the formation of large transcrip-
dynamics has been proposed as the “carrier” for allo- tional complexes that modulate gene expression.
steric communication,127,128 solution studies would Imaging these complexes, which contain dozens to
greatly further our understanding of NR activation. hundreds of individual proteins, would shed tremen-
For example, NMR studies of the PPARγ LBD dous light on NR function.
showed half of the expected resonances in the spec- So far, there are only three such crystal struc-
trum.77 These missing resonances stem from line tures available: PPARγ–RXRα heterodimer, HNF-4α,
broadening of specific regions, including the AF-2, and RXRα–LXRβ heterodimer.132–134 These struc-
suggesting microsecond (μs) to millisecond tures provide information about the inter-domain
(ms) timescale dynamics in these regions. Ligand interactions between NR dimers and organization of
binding rigidified these motions, rendering their reso- each domain in full-length NRs when bound to DNA
nances observable. Hydrogen/ deuterium exchange response elements. The small number of available
coupled with mass spectrometry (HDX-MS) is crystal structures reflects the challenge of obtaining
another powerful technique used to experimentally crystal structures. Inspection of these structures
characterize the conformational dynamics of NR shows that HNF-4α, PPARγ, RXRα, and LXRβ all
LBDs. Similar patterns of conformational dynamics have relatively short A/B and hinge regions [Fig. 1
in apo and various ligand-bound states in PPARγ (B)], which are known to be highly disordered and

1886 PROTEINSCIENCE.ORG The Nuclear Receptor Superfamily

disturb crystal packing. For this reason, crystal struc- Recent technological advances in live-cell micros-
tures of intact NRs with longer A/B region (such as copy and fluorescent labeling are now being leveraged
MR and GR) or hinge region (such as SF-1 and LRH- to study NRs as TFs in real-time.141 By combining
1) would be extremely challenging. Indeed, the A/B fluorescence correlation spectroscopy, fluorescence
region in PPARγ is highly dynamic based on HDX- recovery after photobleaching, and single-molecule
MS analysis and cannot be visualized in any three microscopes, two unique binding events were found
solved structures with different ligands.132 The A/B in the AR-DNA recognition process.142 The first bind-
regions were not included in the construct design for ing event spans only hundreds of μs and is character-
HNF-4α, RXRα, and LXRβ used in the crystallization ized by brief, stochastic DNA interaction, whereas
study. the second event spans several seconds indicating
To bypass the crystallization hurdles associated longer, sequence-specific DNA association. This study
with full-length structures, cryo-electron microscopy provided the first glimpse of NR action, following
(cryo-EM) and small angle X-ray scattering (SAXS) ligand activation, dynamically associating and disso-
have been used. With recent advances in the direct ciating with DNA to search for the target sequence.
electron detection devices, single particle cryo-EM Rather than integrating different complementary
can achieve atomic resolution and is currently well- methods, single-molecule tracking (SMT)-based direct
poised to determine large complex structures.135,136 measurement permits the quantification of both the
To date, three cryo-EM studies of human NRs, focus- dwell time and the fraction of NR molecules on target
ing on ER137,138 and RXR/VDR heterodimer,139 have DNA in live cells.143,144 By utilizing GFP-labeled
been reported. To obtain a large complex for cryo-EM polymerase II, only a small fraction of GR (~10%) was
studies, full-length co-regulators rather than short found to reside at sites with active transcription. The
peptides can be utilized. Therefore, conformation of dwell time of GR at these sites were ~10 s.143 SMT
full-length co-regulators with NRs will be visualized microscopy also permits characterizing highly
in atomic detail.137 Using different co-activators, a dynamic interactions of ER, GR and their pioneer fac-
recent cryo-EM study revealed the recruitment order tors, such as FoxA1, with chromatin.145 Interestingly,
of co-activators and how this controlled epigenetic FoxA1 does not present a DNAase footprint, reinfor-
regulation on histones.138 Likewise, orthologous pro- cing the advantage of monitoring fast and transient
teins of human NRs have also been studied. For interactions by SMT. A recent SMT report focusing
instance, cryo-EM structure of USP and EcR hetero- on GR and various co-factors further corroborated
dimer, the insect orthologs of the human RXR and these studies and showed that GR-chromatin associa-
FXR, respectively, has been determined providing tion was dominated by transient interactions charac-
first insight into the orientation of LBD on an terized by low populations (5–10%) of the receptor on
inverted repeat DNA sequence.140 Interestingly, the chromatin for only short times (<ms).146
A/B regions and most of the hinge regions are omitted SMT is technically challenging as there is a
in the constructs used in these cryo-EM studies due trade-off between delivering enough photons over
to their intrinsic disorder.139,140 Given that most time to permit accurate measurements and capturing
human transcription factors contain a significant inherently fast (μs) binding events.141 Even with this
fraction of unstructured regions, this remains a major challenge, single-molecule experiments hold the
hurdle in their structural characterization. This fur- potential to revolutionize how we define TF–
ther reinforces the importance of including co- chromatin interactions. For example, conventional
regulatory proteins to help order otherwise disor- studies performed by ensemble biochemistry (such as
dered structural elements in the context of transcrip- in vivo ChIP-seq collected via millions of cells), give
tional complexes. the impression of widespread NR-chromatin occu-
pancy with long residence time (min–hr timescale).
Single-molecule studies Single-molecule experiments revealed that only a
Another central question is how to combine high- small fraction of NRs are functionally bound to their
resolution structural and dynamical information to response elements in a given cell with rather short
advance our understanding of the biophysical basis residence time (μs–s timescale). Therefore, single-
that permits NRs (and other TFs) to control gene molecule studies support the notion of dynamic and
expression. Studies have linked DNA affinity or stochastic assembly of transcriptional complexes and
receptor dimerization to transcriptional output but offers a new paradigm of our mechanistic under-
given the complex landscape of a transcribing pro- standing of transcription initiation mediated by
moter it is still challenging to link these in vitro NRs.147 One important question that remains to be
observations to the direction and magnitude of gene addressed is what portion of sequence-specific DNA
expression. Can we have a more continuous picture of binding results in transcriptional activation. This
NR function in vivo, capturing both association with requires imaging multiple factors at a single-copy of a
DNA and recruitment of co-regulators, beyond the specific promoter. With advanced super resolution
discrete structural snapshots we currently have? microscopes, improved image acquisition techniques

Weikum et al. PROTEINSCIENCE | VOL 27:1876–1892 1887

and better statistical algorithms, single-molecule 14. Resche-Rigon M, Gronemeyer H (1998) Therapeutic
studies in live cells will simultaneously track the 3D potential of selective modulators of nuclear receptor
action. Curr Opin Chem Biol 2:501–507.
spatial distribution of NRs over time and monitor 3D
15. Huang P, Chandra V, Rastinejad F (2010) Structural
enhancer organization. This requires multi- overview of the nuclear receptor superfamily: insights
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Acta Pharmacol Sin 36:1–2.
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Acknowledgments Chem 1:504–523.
This work was supported by National Institutes of 19. Germain P, Staels B, Dacquet C, Spedding M,
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supported by a predoctoral F31 fellowship awarded Mol Life Sci 57:809–827.
by National Institutes of Health General Medical Sci- 21. Suntharalingham JP, Buonocore F, Duncan AJ,
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