In Silico Adoption of an Orphan Nuclear Receptor NR4A1
In Silico Adoption of an Orphan Nuclear Receptor NR4A1
Harald Lanig 0
Felix Reisen 0
David Whitley 0
Gisbert Schneider 0
Lee Banting 0
Timothy Clark 0
Irina U Agoulnik, Florida International
University, UNITED STATES
0 1 Computer-Chemie-Centrum der Friedrich-Alexander-Universität Erlangen-Nürnberg , Nägelsbachstraße 25, 91052, Erlangen, Germany, 2 ETH Zürich , Institute of Pharmaceutical Sciences , Wolfgang-Pauli-Straße 10, 8093, Zürich, Switzerland, 3 Centre for Molecular Design , School of Pharmacy and Biomolecular Sciences, University of Portsmouth , King Henry Building, Portsmouth, PO1 2DY , United Kingdom , 4 School of Pharmacy and Biomolecular Sciences, University of Portsmouth , St. Michael's Building, White Swan Road, Portsmouth, PO1 2DT , United Kingdom
A 4.1μs molecular dynamics simulation of the NR4A1 (hNur77) apo-protein has been undertaken and a previously undetected druggable pocket has become apparent that is located remotely from the 'traditional' nuclear receptor ligand-binding site. A NR4A1/bisindole ligand complex at this novel site has been found to be stable over 1 μs of simulation and to result in an interesting conformational transmission to a remote loop that has the capacity to communicate with a NBRE within a RXR-α/NR4A1 heterodimer. Several features of the simulations undertaken indicate how NR4A1 can be affected by alternate-site modulators.
Competing Interests: The authors have declared
that no competing interests exist.
“Orphan” nuclear receptors exhibit no obvious ligand-binding pocket in their X-ray crystal
structures; many of their biological functions are poorly understood. They thus represent
unique challenges for structural and mechanistic biology. We now describe a purely
computational approach to this problem that takes advantage of recent developments both in our
understanding of protein dynamics and in the technology of biological simulation.
The static “lock and key” view of protein-ligand binding has given way to a more dynamic
interpretation known variously as the Monod–Wyman–Changeux (MWC, concerted or
symmetry) model, [1–4] the two-state allosteric model,  the pre-equilibrium model,  or the
“new view” of allosteric binding. [6–9] Cui and Karplus  have pointed out the importance of
molecular-dynamics (MD) simulations in this picture. Indeed, apart from “slow” spectroscopic
techniques such as NMR-spectroscopy and technically difficult trapping experiments,  MD
simulations represent one of the very few tools able to provide a detailed description of protein
dynamics in solution.
This dynamic view suggests that the accessible conformations of the protein can be
characterized in MD simulations of a ligand-free protein provided (a) the force field used is accurate
enough and (b) the time scale of the conformational inter-conversion is short enough to be
accessible to MD simulations. Modern protein force fields, such as ff99SB  used here,
represent years of development that have made them capable of reproducing biological
conformations accurately and reliably. There is now ample indication in the literature that simulations of
100 ns and longer can reveal competing allosteric conformations. [11–15] A timely review of
protein flexibility and function has recently appeared. 
Here we report an extensive MD and cheminformatics investigation of the NR4A1 receptor,
an orphan receptor.
The nuclear receptors (NRs) are a sizable class of transcription factors. Lipophilic ligands
(steroids, fatty acids, retinoids and thyroid hormones, etc.) cause their translocation, normally
from the cellular cytoplasm to the nucleus, where they initiate transcription of cognate genes.
 NRs play essential roles in controlling cellular metabolism, division and proliferation in
many tissues [18, 19] and are part of the steroid receptor superfamily. NR4A1 is often
expressed in response to stress stimuli and cellular growth-factor signaling. NR4A1 and its
subfamily appear to be constitutively active, modulated at the cellular level by differing ratios of
their cognate NR coactivators and repressors and NR post-translational modification,
including phosphorylation  and acetylation  but are currently classed as ‘orphan’ receptors.
NR4A1 is expressed at low to moderate levels in many major physiological systems, including
the central nervous system, endocrine, reproductive, immune, gastrointestinal, cardiovascular,
respiratory and structural. [22, 23] It is known to play a vital role in tumor-cell apoptosis from
multiple tissue types and in the well-studied apoptotic signaling of thymocytes  and within
the hypothalmic-pituitary axis. 
The structural biology of NR4A1 is inconclusive. A crystal structure (PDB-ID  2QW4)
 of human NR4A1 reveals that the region of the “normal” ligand-binding domain is
blocked by hydrophobic residues.  NMR studies have shown the closely related Nurr1 to
undergo a conformational shift between “in” and “out” states, in which a comparable sequence
of hydrophobic residues perform a “self-binding” function,  on the NMR time scale.
A hydrophobic co-regulator cleft comprised of helices 3, 5 and 12 is found in many
members of the family. It plays a vital role in recruiting co-activators and co-repressors during gene
transcription and its access is normally modulated by helix 12 within the non-orphan NRs,
which undergo a conformational change on ligand binding.  However, this cleft is
surprisingly hydrophilic in NR4A1, ruling out a similar role. Furthermore, partial denaturation
studies suggest that NR4A1 has an unusually flexible helix 12.
Our interest was aroused by reports [31–33] of small-molecule modulators of NR4A1 action
(1 and 2, see Fig 1). These may be viewed as Y-shaped hydrophobic ligands of the same general
type that bind to ‘normal’ ligand-binding domains of other nuclear receptors.
Structurally different small molecule modulators of NR4A1 and Nurr1 are also known, 
although their mode of binding and mechanism of action remain unknown. These
observations suggest that NR4A1 exhibits druggable binding regions for which effective modulators
can be developed analogously to those for the ERR receptors, which were originally thought
not to have accessible ligand-binding domains. In this context, it is important that
Katzenellenbogen et al.  have discussed Nuclear Receptor Alternate-Site Modulators (NRAMs), which
bind to alternative pockets than the classical ligand-binding domain.
Fig 1. The structures of known small-molecule modulators of NR4A1.
Results and Discussion
Our purely calculational characterization of the structural and mechanistic biology of NR4A1
draws on a wide variety of simulation, analysis and bioinformatics techniques. Briefly, the
questions that we have tried to answer are:
Can we identify a binding pocket by MD simulations on the apo-receptor?
If so, do the known modulators 1 and 2 bind to this pocket?
Can we identify candidates for the native ligand(s)?
What is the conformational effect of binding ligands on the receptor?
What is the mode of their modulation?
The apo-NR4A1 simulation system consisted of the 233-residue ligand binding domain of
NR4A1 reported (PDB-entry 2QW4, X-ray resolution: 2.8 Å).  Three amino acids (EPQ)
that comprise the surface-loop region of a helix-turn-helix motif are not structurally resolved
and were modeled into the structure. Details of the system and simulation setup are given in S1
Text and S2 Text. A single simulation of 4.1 μs was used to investigate the dynamics of the
The conformations found in the resulting trajectory were identified and analyzed using
both classical RMSD-based clustering and a DASH-analysis  based on 4,100 snapshots of
the backbone dihedral angles taken every nanosecond (see S3 Text for details). The results of
the two analyses are compared (see Fig 2). Transitions between conformations identified by
DASH correspond well to those indicated by the cluster analysis. DASH conformation 3D lies
between clustering conformations 6 and 7 and cluster conformations 13, 14 and 15, which
switch frequently in the last μs of the simulation; these are interpreted by DASH as three
sequential conformations (7D, 8D and 9D). The clustering analysis reveals a series of
essentially irreversible (in the context of this simulation) conformation changes between 15
conformations that eventually results in equilibrium between conformations 12–14 after
approximately 3.5 μs. The X-ray conformation itself, which corresponds to conformation 1
and 1D, is short-lived (although it would be considered stable by accepted MD standards as it
is observed for the first 83 ns of the simulation).
The apparently irreversible conformational change observed during the simulation is a
movement of the loop near the N-terminus (residues 175–185) from an “open” to a “closed”
conformation by approaching the N-terminal end of helix 1 (see S4 Text). This motion closes a
“novel” binding pocket discussed below. A second important feature of the simulation is that
helix 1 unfolds and refolds repeatedly.
Fig 2. Clustering (above) and DASH,  (below) analyses of the 4.1 μs simulation of NR4A1. The
clustering results are color-coded to indicate the population of the cluster in the given time period. The blue
vertical dashed lines indicate transitions detected by DASH. The red dashed boxes indicate the clusters/
conformations in which the binding pocket discussed below was found.
A PocketPicker  analysis was conducted for the 15 cluster-centers found by the classical
clustering of the 4,100 MD snapshots (the default settings described in reference  were
used). Large ( 100 Å3) pockets that are apparent in multiple cluster representatives but not
in the crystal structure were extracted for each structure, their pairwise overlaps calculated,
and the results organized in graphs (see Fig 3). Each vertex represents one pocket. The vertex
size is proportional to the volume of the pocket and the color encodes the MD-cluster
number. Vertices are connected if the pockets overlap by at least 30%. Vertices without emerging
edges are not shown. Highly connected clusters of large vertices indicate potential binding
sites that are not present in the crystal structure if they do not contain vertices that represent
pockets of the crystal.
Visual inspection reveals that a potential binding pocket, present in the snapshots indicated
(see Fig 3), is flanked by the residues Leu6, Pro139, Cys172, Pro184; this pocket is shown in Fig
4. This clear druggable pocket is found in clusters 5, 6 and 7 and in DASH conformations 2D
and 3D and thus exists continuously for 500–800 nanoseconds of the simulation before the
Nterminal flap closes.
This pocket remained stable for at least 1 μs in an independent MD simulation starting with
the empty pocket found in the cluster analyses. A slight decrease in the pocket volume caused
by the flexibility of the surface loop around Val180 is found, but the pocket is still present.
A PoLiMorph  pocket graph description was then used to search for pockets structurally
related to this potential binding site in the scPDB.  Assuming that similar binding sites
bind to similar ligands, we extracted all bound ligands from the crystal structures of the most
similar binding sites (PoLiMorph score 0.15 using the default settings described in reference
). Four of these nine ligands (shown in Fig 5) are nucleotides; adenosine-50-β,γ-methylene
triphosphate (3), coenzyme A (4), FAD (5) and guanosine-50-monophosphate (6). We also
found a bis-aza-indole compound 7 among the top ranked molecules, which is remarkable
since one of the known NR4A1 activators is bis-indole 1.
Ligands 1 and 2 could both be docked well into the new pocket (for details see S5 Text) using
Autodock4.  The best docked poses were then used as starting points for 1 μs MD
Fig 3. Clustering of pockets that occur in the crystal structure and different snapshots of the MD
simulation. Each vertex represents one pocket. Vertices are connected if the pockets are located in similar
regions of the protein surface and have a mutual overlap of at least 30%. Vertex labels consist of the cluster
number (0–14, c = crystal) and the pocket number (as identified by PocketPicker,36 sorted by size in
ascending order). Vertex colors and sizes correspond to the number of the snapshots (red: crystal) and the
pocket sizes, respectively. Clusters 1–3 contain pockets that are present both in the crystal structure and in
some snapshots. Pockets of clusters 4–10 are not present or smaller than 100 Å3 in the crystal structure.
Cluster 4 (light green) represents a potential binding site. Clusters 5–10 represent pockets whose shapes or
sizes render ligand binding unlikely.
simulations to assess the stability of the ligands in the pocket and their effect on the
conformation of the protein.
RMSD plots of the protein α-carbon atoms and all ligand atoms (see S6 Text) show that the
protein conformation remains stable over the 1 μs simulation, with the Cα-RMSD from the
starting geometry varying between 2.5 and 3.5 Å after an initial 100 ns relaxation period. The
ligand-RMSD plot also shows an essentially stable 3 Å RMSD with frequent excursions to
structures with 7–8 Å RMSD from the starting structure. For ligand 1, closer examination
shows that the structure with 3 Å RMSD, which corresponds to the 100 ns snapshot shown
(see Fig 6b), involves movement of the ligand deeper into the pocket than is the case in the
docked structure (see Fig 6a).
Fig 4. The predicted binding site (blue/blue grey spheres) is located close to the N-terminus of
NR4A1. The flexible loop that is stabilized by ligand binding is indicated by the red ellipse.
Fig 5. Putative ligands for the newly found binding site in NR4A1.
The second conformation found periodically in the simulation with increasing frequency
after 200 ns corresponds to the 800 ns structure shown in Fig 6. In this structure, helix 1, which
unravels and reforms repeatedly during the last half of the simulation, is unraveled and the
ligand takes up a position deep in the pocket, but shifted towards the original position of helix
1. Thus, the simulation indicates that ligand 1 indeed binds tightly to the newly found pocket.
The RMSD plot of the α-carbon atoms for the simulation starting with docked ligand 2 also
shows that the protein maintains its secondary and tertiary structure for the whole 1 μs
simulation. The only exception is helix 1, which is again affected by the presence of the ligand, which
causes the helix to unwind and rewind repeatedly. For this reason, the highly flexible ligand is
able to move and reorient within the binding pocket, as can be shown by an overlay of
representative snapshots extracted from the trajectory (see S7 Text). The flexible alkyl chains of
ligand 2 explore the dynamically changing binding cleft more effectively than observed for
compound 1. This is supported by the considerably higher ligand RMSD values (8–9 Å).
Nevertheless, ligand 2 remains firmly in the binding pocket defined by the flexible loop 175–185.
Fig 6. Representative snapshots taken from the MD simulation of ligand 1 within the new binding
pocket (cyan, after 100 ns; magenta, after 800ns), fitted onto the starting geometry of the docked
Starting at 780 ns, the loop forming the new binding pocket shifts towards helix 1 and closes
the pocket, so that the ligand is completely trapped. This closing is only possible because the
ligand adopts a position above helices 8 and 9, flanked by the C-terminus of helix 2,
additionally covered by the very flexible N-terminus of the protein. The highly flexible side chains of
compound 2 intercalate between the helix bundles of the protein, making binding even tighter.
As a further check of the relevance of the newly found binding site, we docked five known
[40–42] active NR4A1 bisindole ligands into this site (details of the docking runs and the
results are given in S5 Text). All five give good binding poses with high scores and adopt the
same binding mode as found for 1 and 2 above.
Effects of Ligand Binding
What are the consequences of the above results in identifying a biological role and mechanism
for NR4A1? Ligand binding at the newly identified site induces conformational changes both
adjacent to the pocket and in the remote flexible loop area highlighted in Fig 4. The more
remarkable of these two effects is a conformationally transmitted stabilization of the flexible
loop (25FQELVLPHFGKEDAGD40), even though it is quite remote from the binding site.
The pocket itself is close to the ‘hinge’ region of the ligand-binding domain. As outlined
above, helix 1 becomes very labile on ligand binding. Fig 7 shows that helix 9 is also partially
unraveled (color-coded green) compared to the apo-structure (color-coded blue).
The newly formed loop region adjacent to the binding site (173LKEHVAAVAGEPQPAS188,
red ellipse in Fig 7) is close to a to a well-recognized hinge region between the ligand- and
DNA-binding domains, as indicated by a modeled alignment with the PPAR-γ receptor bound
to a cognate Peroxisome Proliferator Response Element (PPRE)  (see S8 Text). This
comparison is equivalent to the known hetero-dimerization of NR4A1 with RXR-α.  Changes
in this hinge region can result in alterations in signaling because the hinge region of NRs can
contain elements of the nuclear localization signal.  NR4A1 has a predicted  nuclear
localization signal (358RRGRLPS365K) upstream of our modeled protein extremely close to the
N-terminus of the predicted ligand-binding domain. Thus, changes adjacent to the newly
Fig 7. Apo-NR4A1 (blue, PDB:2QW4) compared to a snapshot of NR4A1 after 45ns simulation in the
presence of 1 (green). The blue double headed arrows indicate the axes of the helices 1 and 9 in the
apostructure. The regions of the protein that rearrange on ligand binding are denoted by the ellipses (Red;
adjacent to the binding site. Violet; remote loop 25F FQELVLPHFGKEDAGD-D40).
identified binding site may switch NR4A1 localization, which in turn is intimately correlated to
its apoptotic function.  An identified nuclear export sequence under the control of
MEK-ERK-RSK cascade also lies close to the cognate nuclear localization sequence, with
phosphorylation being effected by RSK.  This region also attracts the attention of an additional
serine kinase MSK that operates in fibroblast cellular stress pathways.  Structural changes
in this vital region are highly likely to affect subcellular location of NR4A1. 
A less direct, but fascinating possibility is that the allosteric rearrangement observed for the
remote loop 25FQELVLPHFGKEDAGD40 (the violet ellipse in Fig 7 and violet loop in Fig 8) is
involved in biological regulation by modifying heterodimer stability and binding at a cognate
NuRE. Fig 8 shows a hypothetical alignment of a response element with a heterodimer of a
bisindole complexed NR4A1 and RXR-α based on the PPRE PPAR-γ and RXR-α X-ray structure
 and informed by SAXS data for the VDR/RXR-α dimer at its response element. The region
of NR4A1 highlighted by our simulations to change (labeled blue A) on ligand binding is close
to a similar loop region in the RXR monomer (labeled red B). This suggests that
protein/protein communication between heterodimer partners in this region of the complex can lead to
modulation of DNA binding. It is known that rexinoid AHPN has influence on both the RXR/
NR4A1 dimer’s transcription processes and on localization because the dimer is transported
from the nucleus to the mitochondria. 
Construction of a NR4A1 / RXR heterodimeric complex model located
on a NBS at a cognate NRBE
In an attempt to understand more fully the effect of the allosteric binding of the NR4A1
bisindole ligand, a model of a NR4A1/RXR heterodimer at a NBS (hNurr77 binding sequence) on
a recognized NBRE, the RAR β2  was constructed. The RAR β2 sequence was obtained
 and modelled  to obtain ‘raw’ structural coordinates. The coordinates of the DBDs of
RXR-α and NR4A1 were obtained (PDB: 3DZY, 1CIT respectively) and positioned with their
preferred ‘polarity’ at their cognate binding sequences by a process of coordinate mapping via
the MacPymol align routine working with the crystallographic base pair coordinates of the
respective DBD/DNA complexes onto a modelled RARβ2 followed by editing. The monomer
coordinates of the LBDs of RXR-α and NR4A1 (PDB: 3DZY, 2QW4) were mapped onto those
Fig 8. Model of the apo-NR4A1 (green cylinders) in dimerization with apo-RXR-α (blue cylinders) at
the NBS of a cognate NBRE, DNA (CPK colors) indicates the NBS sequences (Red) and the location of
the bis-indole 1 (CPK spheres) post-superimposition of the NR4A1 45ns complex.
of the PPAR/RXR-α heterodimer crystallographic coordinates (PDB: 3DZY) in the absence of
the linking sequences that bridge the respective LBDs and DBDs. In doing so there is an
implicit assumption that the dimer interface of the modelled complex adopts the orientation of
the PPAR/RXR-α heterodimer. The modelled heterodimer was posed, using the translational
tools within MacPymol, approximately 23 Å above the RARβ2 (roughly equidistant from each
DBDs). The linking sequences were modelled using the loop-building functionality of
SwissPdbViewer, followed by energy minimization of the new protein fragments. The final model
was checked for sequence and backbone dihedral angle consistency.
Following an 85.2 ns MD simulation the resultant complex showed a considerably different
configuration than its starting point. It was pleasing to see that, during the course of the
simulation, the complex had reoriented to adopt a configuration reminiscent of that observed
(SAXS/SANS/FRET) for the solution structure of the RXR/RAR heterodimer at a DR5
response element with the RXR binding at the 5’ and the NR4A1 at the 3’ half sites respectively,
[54, 55] (see Fig 8).
The bis-indole/NR4A1 modelled complex coordinates were posed against the resultant
coordinates of the NR4A1 component of the heterodimer complex at the NBRE using
MacPymol align. This pose highlights the close proximity of the bis-indole to a critical ‘hinge’ region
of the NR4A1 complex (see Fig 8) and to the DNA binding interface, the detail of which will be
the subject of further study.
The key reviews, [33,40] covering the targeting of NR4A1 for cancer treatment, reveal a
high degree of complexity in the potential modes of action of 1, and its analogues, which
appear to act differently on varying tumor types and via NR4A1 partner protein complexes. It
is suggested that the p-hydroxy analogue of 1 acts on the A/B domain of NR4A1 whereas 1 on
the LBD within the E/F domain, fluorescence studies  confirm this. X-ray crystallography
of the NR4A1 homodimeric LBD show that small molecule Cytosporone B-like ligands of
NR4A1 such as ethyl 2-[2,3,4-trimethoxy-6-(1-octanoyl)phenyl]acetate (TMPA) are also able
to bind promiscuously. [32, 57–58] Taken together, this evidence suggests strongly that
NR4A1 may be modulated by small molecules in a non-classical way, at multiple sites, for a
member of the steroid receptor superfamily.
What is emerging is that allosteric modulation of the NRs is evident and that these
approaches have predictive potential when considering ‘transcriptional machinery’ stability
[59, 60] and that this aspect may be influenced by small molecules.
Although many of the biological functions of NR4A1 are still unclear, the simulations have
provided answers to the questions posed in the introduction and have revealed fascinating
features that point to new research directions. The simulations reveal a pronounced pocket that
binds the known ligands and is similar to known nucleotide-binding sites. Binding ligands in
this pocket induces two changes; a new loop adjacent to the binding site that is well placed to
switch DNA binding and a stable loop in a remote region of the protein that has the capacity to
affect localization at the NBRE. These are remarkable conclusions for a protein that has resisted
characterization experimentally. MD simulations combined with detailed analyses of the
trajectories have proven to be a powerful tool for exploratory research of protein structure and
Conceived and designed the experiments: LB TC. Performed the experiments: HL FR.
Analyzed the data: DW. Contributed reagents/materials/analysis tools: DW. Wrote the paper: HL
GS LB TC.
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