, 189 (2012);
et al. Nian Huang
Transcriptional Activator Complex
Crystal Structure of the Heterodimeric CLOCK:BMAL1
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Crystal Structure of the Heterodimeric
Nian Huang,1* Yogarany Chelliah,2,3* Yongli Shan,2Clinton A. Taylor,1,4Seung-Hee Yoo,2
Carrie Partch,1,2,3† Carla B. Green,2Hong Zhang,1‡ Joseph S. Takahashi2,3‡
The circadian clock in mammals is driven by an autoregulatory transcriptional feedback mechanism
that takes approximately 24 hours to complete. A key component of this mechanism is a
heterodimeric transcriptional activator consisting of two basic helix-loop-helix PER-ARNT-SIM
(bHLH-PAS) domain protein subunits, CLOCK and BMAL1. Here, we report the crystal structure of
a complex containing the mouse CLOCK:BMAL1 bHLH-PAS domains at 2.3 Å resolution. The
structure reveals an unusual asymmetric heterodimer with the three domains in each of the two
subunits—bHLH, PAS-A, and PAS-B—tightly intertwined and involved in dimerization interactions,
resulting in three distinct protein interfaces. Mutations that perturb the observed heterodimer
interfaces affect the stability and activity of the CLOCK:BMAL1 complex as well as the periodicity
of the circadian oscillator. The structure of the CLOCK:BMAL1 complex is a starting point for
understanding at an atomic level the mechanism driving the mammalian circadian clock.
scriptional activators within the circadian clock
mechanism of mammals. Since the molecular
identification of the Clock gene 15 years ago
(1, 2), the transcriptional network that drives cir-
cadian oscillations has been systematically iden-
and interact with E-box regulatory elements in
the Period (Per1 and Per2) and Cryptochrome
(Cry1 and Cry2) genes to activate their transcrip-
tion during the daytime (6, 7). Their protein
products, PER and CRY, accumulate, dimerize,
and translocate into the nucleus at night, where
they interact directly with CLOCK:BMAL1 to
repress their own transcription (7–10). As the
PER:CRY repressor complex is targeted for deg-
(11–14), repression is relieved, and CLOCK:
tion to begin the circadian cycle anew. This
cell-autonomous, autoregulatory transcriptional
feedback loop takes ~24 hours to complete and
in mammals (5).
he basic helix-loop-helix PER-ARNT-
SIM (bHLH-PAS) proteins, CLOCK and
BMAL1 (ARNTL), are the primary tran-
CLOCK and BMAL1 belong to a family of
transcriptional regulators that contain bHLH and
PAS domains. In mammals, these bHLH-PAS
transcription factors participate in a wide array of
functions, including responses to environmental
hypoxia [Hypoxia inducible factor (HIF)], neu-
rogenesis (SIM1), synaptic plasticity (NPAS4),
and circadian regulation (CLOCK, NPAS2, and
BMAL1) (15), and most of them remain poorly
characterized at the structural level. In contrast,
the structures of individual PAS domains and
their interactions with small-molecule ligands
such as heme and flavin cofactors are well un-
derstood, especially among microorganisms and
plants, in which PAS domains serve important
roles in two-component signaling and blue-light
ly conserved, it has intrinsic flexibility and can
adapt to different conformations depending on
bound ligands or interacting partners (16). Here,
we present the three-dimensional (3D) struc-
ture of the bHLH-PAS domains from the mouse
Overall structure of CLOCK:BMAL1. To ob-
tain stable CLOCK:BMAL1 complexes suitable
structs containing the bHLH and the two tandem
His-tagged mouse CLOCK (residues 26 to 384)
and native mouse BMAL1 (residues 62 to 447)
constructs were coexpressed in Sf9 insect cells
and copurified (supplementary materials, mate-
rials and methods). To confirm that the resulting
affinity of binding to oligonucleotides containing
the canonical E-box sequence (CACGTG) from
the mPer1 and mPer2 promoters and observed dis-
sociation constants (Kds) of ~10 nM (Fig. 1B and
that diffracted to 2.3 Å at synchrotron sources.
persion (SAD) method, using selenomethionine-
labeled CLOCK:BMAL1 crystals (fig. S2). Data
collection and refinement statistics are shown in
with all three domains—the N-terminal bHLH
domain and two tandem PAS domains (PAS-A
and PAS-B)—involved in dimerization inter-
actions. Each domain interacts primarily with
thecorresponding domain ofits partner subunit
so that CLOCK bHLH interacts with BMAL1
bHLH,andCLOCKPAS-A(or PAS-B) interacts
withBMAL1PAS-A (orPAS-B).Although the
primary sequences of these three domains are
the spatial arrangement of these domains with
respect to one another is strikingly different in
the two subunits (Fig. 1C). In BMAL1, the sec-
ond helix of the bHLH domain (a2) is nearly
continuous with the N-terminal flanking helix
(A’a) of the PAS-A domain despite insertion of
a ~15-residue flexible loop (L1) (Fig. 1C, right).
In contrast, in CLOCK there is a ~23 Å displace-
ment between the end of a2 and the beginning
of the PAS-A A’a helix (Fig. 1C, left). As a con-
contact with the a2 helix of its bHLH domain,
whereas there are no direct contacts between the
BMAL1 PAS-A and bHLH domains.
plex is also reflected in the divergent electrostatic
potential distributions on the two subunits. The
BMAL1 subunit has an overall positive electro-
static potential with an isoelectric point (pI) of
9.01 (and 8.55 for the PAS-A/B domains). The
CLOCK subunit, on the other hand, has an over-
all negative electrostatic potential, with a pI of
5.86 for the bHLH-PAS domains (and 5.28 for
thePAS-A/B domainonly). Inthe3DCLOCK:
BMAL1 complex structure, the exposed CLOCK
PAS domain surfaces have a largely negative
electrostatic potential, whereas the exposed
BMAL1 PAS domains are mostly positively
charged or neutral (Fig. 1D). These electrostatic
features of the CLOCK:BMAL1 heterodimer
produce an interesting dichotomy in the potential
interaction interfaces of the complex and are con-
PER2, CRY1, and CRY2 proteins differentially
interact with CLOCK and BMAL1 (8, 18–20).
The PAS-A domains. Although all three do-
mains of the CLOCK and BMAL1 subunits
are involved in intermolecular interactions, the
heterodimeric interfaces between the individu-
al PAS domains are of particular interest. The
CLOCK:BMAL1 heterodimer has long flexible
loops interspersed with canonical PAS secondary
structure elements in both PAS-A domains. In the
BMAL1 PAS-A domain, a total of ~60 residues
Medical Center, Dallas, TX 75390, USA.2Department of Neu-
roscience, University of Texas Southwestern Medical Center,
Dallas, TX 75390, USA.3Howard Hughes Medical Institute,
University of Texas Southwestern Medical Center, Dallas, TX
75390, USA.4Molecular Biophysics Graduate Program, Divi-
Center, Dallas, TX 75390, USA.
*These authors contributed equally to this work.
†Present address: Chemistry and Biochemistry Department,
‡To whom correspondence should be addressed. E-mail:
email@example.com (J.S.T.); zhang@
VOL 33713 JULY 2012
on July 31, 2012
in three loop regions are disordered in the crystal
this high degree of flexibility, the two PAS-A
square deviation (RMSD) of only 0.62 Å over
core of CLOCK and BMAL1 PAS-A domains
contains a five-stranded antiparallel b sheet (Ab,
Βb, Gb, Hb, and Ιb) and several a helices (Ca,
Da, Εa, and Fa) flanking the concave surface of
the sheet. In contrast to the PAS-B domains (Fig.
2A), both PAS-A domains in the two subunits
contain an N-terminal flanking helix (A’a) exter-
nal to the canonical PAS-domain fold. The A’a
helices of the CLOCK and BMAL1 PAS-A do-
mains pack in between the b-sheet faces of the
two domains to mediate the heterodimericPAS-A
interactions (Fig. 2B). The A’a helix of CLOCK
face of BMAL1PAS-A,whereastheA’a helixof
PAS-A (Fig. 2B). This domain-swapped helical
domain proteins, such as the N-terminal PAS do-
main of the nitrogen fixation regulatory protein
NifL from Azotobacter vinelandii (Fig. 2C) (21)
and the N-terminal domain of the transcriptional
factor TyrR from Escherichia coli (22).
is largely mediated by conserved hydrophobic in-
on the A’a helix of CLOCK (corresponding res-
idues on BMAL1 PAS-A are Phe141, Leu142,
and Leu150) contact a hydrophobic region on the
b-sheet face of BMAL1 PAS-A composed of res-
idues Leu159on strand Ab, Thr285and Tyr287on
similar interface can be found between the A’a
helix of BMAL1 and CLOCK PAS-A domain
(Fig. 2E). As a result, the two PAS-A domains in
CLOCK:BMAL1 form a parallel dimer related
by anapproximate twofold symmetry, with an ex-
tensive buried surface area (~1950 Å2) and
topologically complex interface between the two
subunits. Many of the residues observed in the
served among bHLH-PAS transcription factors
(fig. S4), suggesting that these proteins may share
a common PAS-A domain dimerization mode.
The PAS-B domains. An ~15-residue linker
each of the CLOCK and BMAL1 subunits, al-
though the linker conformation and the relative
spatial arrangement of the two PAS domains
in the two subunits are different (Fig. 1C). In
CLOCK, a large portion of L2 is buried between
the interface of CLOCKand BMAL1 and is well
ordered (Fig. 1C). In contrast, in BMAL1, L2 be-
tween PAS-A and PAS-B is solvent-exposed and
very flexible, as indicated by high atomic dis-
placement parameters (B-factors). The PAS-B
domains of CLOCK and BMAL1 are related
predominantly by a ~26 Å translation and are
stacked in a roughly parallel fashion (Fig. 3A)
E-box DNA (nM)
Fig. 1. Overall structure of mouse CLOCK:BMAL1. (A) Domain or-
ganization of CLOCK and BMAL1. Crystals were obtained from the
truncated proteins (indicated by the amino acid residue number)
encompassing the bHLH-PAS-AB domains. (B) DNA-binding affinity of the
of the fluorescein-labeled mPer2 E2-box DNA was 59 T 7.3 nM by direct
the Kds of unlabeled 18-nucleotide oligomer mPer1 E1-box DNA (blue) and
mPer2 E2-box DNA (red) (40) were 9.0 T 2.3 nM and 13 T 2.0 nM, respec-
of CLOCK:BMAL1 heterodimer (center). The CLOCK subunit is green, and
BMAL1 is blue. Each individual domain is labeled. The CLOCK (left) and
BMAL1 (right) subunits are also shown separately in order to illustrate their
different spatial domain arrangements. The linker regions between domains
in the two subunits (L1 and L2) are highlighted in red or orange. Flexible
loops lacking density are indicated by dotted lines. (D) Electrostatic po-
tentials of CLOCK:BMAL1 heterodimer showing that the surfaces composed
of CLOCK PAS domains (right, red ovals) have mostly negative potentials,
the Boltzmann constant, T is the temperature, and q is the magnitude of the charge on an electron) (red) to positive 5 kBT/q (blue).
13 JULY 2012VOL 337
on July 31, 2012
different from the antiparallel b-sheet interface
seen forthe isolated PAS-B domaincomplex of
HIF-2a:ARNT (Fig. 3B) (23). The b sheet of
BMAL1 PAS-B makes contacts with the helical
face of CLOCK PAS-B, burying a patch of hy-
drophobic residues on both subunits. Several
surface-exposed hydrophobic residues on both
CLOCK and BMALl PAS-B become mostly or
partially buried upon dimerization, including
Tyr310, Val315, and Leu318of CLOCK and Phe423,
of buried surface area (Fig. 3, C and E). Most
prominently within these hydrophobic interac-
tions, BMAL1 Trp427, located on the short HI
loop (connecting the Hb and Ib strands), intrudes
into a hydrophobic cleft created between the Fa
D and F), and partially stacks against the indole
ring of CLOCK Trp284.
transactivation function. To probe the rela-
tionship between the observed conformation of
the CLOCK:BMAL1 heterodimer and its func-
domains (Fig. 4A). For the bHLH domains, the
the a2 helices of both CLOCK and BMAL1,
participate in the formation of a canonical four-
helical bHLH bundle in the heterodimer simi-
lar to that observed in USF1 and MYC:MAX
(fig. S3A) (24–26). As seen in other bHLH pro-
hydrophobic (26, 27), indicating that dimeriza-
tion of the bHLH domains should help stabilize
the CLOCK:BMAL1 complex. The proper con-
formation of the bHLH domain is also critical
for E-box DNA recognition because the DNA-
binding a1 helices need to be positioned precisely
DNAduplex (24, 26, 27). Indeed,when the bHLH
hydrophobic core residues Leu57and Leu74of
tated to glutamate, the transactivation activity of
these full-length CLOCK:BMAL1 mutants are
driven luciferase reporter gene activity in human
embryonic kidney (HEK) 293T cells with tran-
siently transfected CLOCK and BMAL1 mutant
proteins (Fig. 4B).
We examined the dimerization of these mu-
tants in living cells through a bimolecular fluo-
rescence complementation (BiFC) assay, in
which the N- and C-terminal fragments of the
fluorescent protein Venus (Ven-N and Ven-C)
were fused to the C-termini of truncated bHLH-
ization of CLOCK and BMAL1 brings the two
Venusfragments into close proximity to facilitate
interactions in living cells (28). The BiFC data
showed that mutation at the bHLH hydrophobic
core reduced formation of a stable heterodimeric
complex (Fig.4C andfig.S5).Furthermore,three
(C:L74E), BMAL1 L95E, and L115E (B:L95E
and B:L115E)—also destabilized the full-length
CLOCK:BMAL1 heterodimer,asshownwith co-
immunoprecipitation (co-IP) assays (Fig. 4D) (In
the mutants, other amino acids were substituted at
leucine at position 74 was replaced by glutamic
Glu; F, Phe; G,Gly; H, His; I, Ile; K,Lys; L, Leu;
M, Met;N, Asn; P,Pro;Q,Gln;R,Arg;S,Ser;T,
Thr; V,Val; W, Trp; and Y, Tyr.).
Next, we mutated residues involved in PAS-A
effects on transactivation activity and CLOCK:
BMAL1heterodimerformation.To examine the
PAS-A domain interface, we made the following
CLOCK and L150E (on A’a) and I317D (on Ib)
of BMAL1. We then performed transactivation,
Fig. 2. Structure and interaction of the PAS-A domains of CLOCK:BMAL1. (A) Ribbon representations of
CLOCK PAS-A domain. Secondary structures are color ramped from blue to red and labeled from the A’a
in CLOCK:BMAL1, looking down the approximate twofold symmetry axis. (C) Similar domain-swapped
structure of the redox-sensing PAS domain of NifL from A. vinelandii (PDB 2GJ3). (D) Detailed interface
between A’a helix of CLOCK PAS-A (green) and the b sheet face of BMAL1 PAS-A (blue). (E) The
corresponding interface between A’a helix of BMAL1 PAS-A and the b sheet face of CLOCK PAS-A.
VOL 33713 JULY 2012
on July 31, 2012
to reduce dimerization or transactivation activity
(Fig. 4, B to D). However, BMAL1 mutant I317D
had decreased transcriptional activity [~80% of
wild type (control)] (Fig. 4B) and decreased affin-
and co-IP experiments (Fig. 4, C and D, and fig.
S5). Furthermore, when opposing CLOCK and
BMAL1 PAS-A domain interface residues were
ciation between full-length CLOCK and BMAL1
subunits was not detectable under the assay con-
ditions, and transactivation activity was reduced
to ~25% of the control (Fig. 4, B to D).
To examine the unusual interface between
the PAS-B domains of the CLOCK:BMAL1
heterodimer, we made the following PAS-B do-
main mutations: W284A and V315R on the
CLOCK helical face and W427A, F423R, and
V435R on the b-sheet face of BMAL1. Single
mutations in either CLOCK or BMAL1 PAS-B
domains had a limited effect on the transactiva-
tion activity by the full-length mutant protein
(Fig. 4B), although the activities of C:W284A,
C:V315R, and B:F423R were reduced by ~20 to
30% compared with the wild-type (WT) pro-
tein (Fig. 4B). Additionally, the BiFC signal of
mutants C:W284A, C:V315R, and B:V435R
decreased dramatically compared with the WT
protein (Fig. 4C and fig. S5), indicating that the
be altered. The effect of these single mutations
on the interactions of the full-length proteins,
as measured with co-IP, was more subtle, with
partially weakened interactions for mutants
C:W284A and B:W427A (Fig. 4D). The double
BMAL1 PAS-B domain mutant B:F423R/V435R
and the combined CLOCK:BMAL1 mutant C:
W284A+B:W427A showed a decreased inter-
action in both co-IP and BiFC assays as well as
a reduction in transactivation activity (Fig. 4, B
to D). These data support the unusual PAS-B
domain interface observed in the crystal structure
involving the helical face of CLOCK and the
b-sheet face of BMAL1 and specifically indicate
Trp427is important for PAS-B interaction.
in cells. To examine the functional consequences
of mutations that compromise CLOCK:BMAL1
heterodimer formation and transactivation po-
tential, we assessed circadian rhythmns in mouse
or BMAL1 constructs introduced by lentiviral
vectors (supplementary materials, materials and
methods, and fig. S6). On the basis of in vivo
levels are rate limiting and that overexpression
of CLOCK leads to a shortening of circadian
period in both constitutively expressed or con-
ditionally expressed transgenic mice (2, 29). In
contrast, overexpression of BMAL1 can have
no effect or can lengthen circadian period (30),
and these effects of BMAL1 overexpression are
consistent with the hypothesis that BMAL1
is normally in excess of CLOCK. Higher over-
expression ofBMAL1canleadtoperiod length-
ening, possibly by the sequestering of CLOCK
via a squelching mechanism (31). Thus, we can
assay the function of WT CLOCK and BMAL1
by overexpression in PER2::luciferase–cycling
cell assays (32) and, by extension, infer loss-of-
function mutations by their inability to mimic
WT function or, in contrast, dominant-negative
mutations by their disruption of normal rhythms.
Control Per2Lucfibroblasts overexpressing
green fluorescent protein (GFP) had robust lu-
ciferase rhythms, with a period of 23.1 hours
(fig. S6). Cells overexpressing WT CLOCK or
BMAL1 exhibited rhythms with either shorter
(~22.0 hours) or longer (24.6 hours) periods,
tested (C:L57E and C:W284A) failed to mimic
WT CLOCK and had period values similar to
those of the GFP control cells (~23 hours) and
therefore behaved as loss-of-function mutations.
The C:L57E mutant abolished transactivation
by full-length CLOCK:BMAL1 and reduced di-
merization ofthetruncated heterodimer(Fig.4,
B and C).Although the C:W284APAS-B mutant
had only a 20% reduction in activty in transacti-
vation assays (Fig. 4B), it weakened CLOCK:
BMAL1 dimerization significantly (Fig. 4, C
and D) and failed to mimic the function of WT
CLOCK on circadian periodicity. Overexpres-
sion of BMAL1 mutants within the bHLH and
of circadian rhythms (fig.S6). Overexpression of
CLOCK PAS-B:BMAL1 PAS-B
Fig. 3. Interface between CLOCK:BMAL1 PAS-B domains. (A) The spatial arrangement of the two PAS-B
domains in CLOCK:BMAL1. (B) Antiparallel orientation of b sheet–mediated interaction between isolated
HIF-2a:ARNT PAS-B domains (PDB 3F1P). (C) Detailed interface between CLOCK:BMAL1 PAS-B domains.
(D) Front-facing view of CLOCK:BMAL1 PAS-B interface highlighting role of BMAL1 Trp427and CLOCK
Trp284interaction. (E) Side view of PAS-B interface displaying surface electrostatic potential of CLOCK
PAS-B. (F) Front-facing view of CLOCK surface electrostatic potential displaying the binding pocket for
BMAL1 Trp427. The color scheme used is the same as in Fig. 1D.
13 JULY 2012VOL 337
on July 31, 2012
period (25.1 hours) for the first 3 days, followed
by disruption of circadian rhythmicity, whereas
overexpression of the PAS-A mutant B:I317D
led to a shorterperiod (~23.8 hours) as compared
Discussion.Here, wepresentthex-ray struc-
al activator complex, which is a central regulator
complex structure in hand, it will now be pos-
sible to analyze the multiprotein complexes
involved in mammalian circadian clock mech-
anisms at an atomic level. Existing genetic and
biochemical data indicate that the negative reg-
ulators CRY and PER physically interact with
CLOCK:BMAL1 to form the major repressive
clock complex containing CLOCK:BMAL1 and
PER:CRY (9, 10, 18, 33). Although the struc-
tural details of these interactions have not been
elucidated, the binding of CRY and/or PER to
CLOCK:BMAL1 could affect DNA binding,
modulate transactivation potential, or modify in-
teractions with coactivators and corepressors.
Previous work suggests that CRY interacts with
the PAS-B domain of CLOCK near its b-sheet
(18, 19, 33). Specifically, mutations of residues
Gly332, His360, Gln361, Trp362, and Glu367of the
by CRY. In the crystal structure, these residues
are located on the HI loop of the solvent-exposed
b-sheetface ofthe CLOCKPAS-Bdomain,fully
accessible for interaction with CRY (Fig. 5). The
is also consistent with the idea that CLOCK is
positively charged protein (pI = 8.24 for CRY1)
and would complement the negative surface
charge on CLOCK (Fig. 1D). Thus, the unusual
spatial arrangement of the PAS-B domains of
CLOCK:BMAL1 observed in the crystal struc-
ture is consistent with the earlier biochemical
data on the PAS-B domain function. The tandem
PAS domains in BMAL1 have a spatial arrange-
ment similar to that observed in the crystal struc-
tures of the mouse and Drosophila PER tandem
the tandem PAS domains in BMAL1 and PER
may have a deeper degree of structural and/or
functional conservation than was previously ap-
preciated, which may have implications for how
the PAS-A and PAS-B domains of PER2 interact
with either CLOCK or BMAL1 (10).
Trp362of CLOCK—implicated in an interac-
which was shown here to interact with CLOCK
at the same position is also conserved in the
Drosophila and mouse PER proteins (Trp482of
crystal structures to be involved in the interaction
with a second PER protein to form homodimers
(35). These observations highlight a potentially
Fig. 4. Functional analysis
of CLOCK:BMAL1 mutants.
(A) Locations of domain in-
terface mutants in CLOCK
(green) and BMAL1 (blue).
reporter assays to evaluate
performed in duplicate. (C)
and mutant CLOCK:BMAL1
plementary materials, ma-
terials and methods) were
three independent experi-
ments. (D) co-IP experiments
assessing the association of
CLOCK and BMAL1 in full-
length WT and mutant pro-
teins. Anti-FLAG affinity gel
tagged CLOCK along with
ern blots using an antibody
to HA were then performed
to detect the association of
WT and mutant CLOCK and
C:W284A, which had stron-
experiments, but on average was weaker than WT CLOCK.
VOL 33713 JULY 2012
on July 31, 2012
conservedfunctionalroleforthetryptophanresidue Download full-text
located at the HI loop of the PAS-B domains of
these clock proteins.
The CLOCK:BMAL1 PAS-B domain inter-
face reveals details of a mode of PAS protein-
protein interaction involving the a-helical face of
CLOCK PAS-B and the b-sheet face of BMAL1
PAS-B (Fig. 3, C to F, and fig. S8A). The same
region (between Fa and the AB loop) on the heli-
cal face of PER PAS-B is used for intramolecular
interactions with a C-terminal a helix (aE) con-
taining nuclear exporting signal residues (helix
aE is equivalent to Ja in canonical PAS nomen-
shown by means of nuclear magnetic resonance
studies to use the same helical region for inter-
that are required for transactivation by the hetero-
dimeric HIF:ARNTcomplex (38, 39). Moreover,
the same region of many bacterial and plant PAS
proteins binds to small-molecule ligands such as
flavin cofactors, flavin adenine dinucleotide, and
flavin mononucleotide (fig. S8C) (16). Overall,
these data highlight the remarkable structural
plasticity and adaptability of PAS domains. Be-
cause CLOCK:BMAL1 is a prototypical bHLH-
of the CLOCK:BMAL1 complex may be shared
by other bHLH-PAS proteins. It will be important
in future work to determine the structures of addi-
tional heterodimeric bHLH-PAS proteins such as
tural basis by which these homologous proteins
confer their distinct and pathway-specific functions.
The structure of CLOCK:BMAL1 has re-
vealed the locations of previously identified sites
on these proteins that affect their inhibition by
CRY. It has also revealed an unexpected simi-
larity in the orientation of the tandem PAS-A and
PAS-B domains of BMAL1 to that found in the
PERIOD proteins. These observations provide a
starting point for the determination of how the
CRYand PER proteins interact with and repress
CLOCK:BMAL1, which in turn should yield in-
which this transcriptional feedback loop drives
the circadian clock.
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Acknowledgments: This work was supported by the Howard
Hughes Medical Institute (J.S.T.), American Heart Association
grant 10GRNT4310090 (H.Z.), NIH grant R01 GM090247
(C.B.G.), and R01 GM081875 (K. Gardner). We thank N. Grishin
and K. Gardner for helpful discussions; S. Padrick for help with
the fluorescence polarization assay; M. Rosen for use of his
fluorometer; D. Tomchick and H. Aronovich for technical
assistance; and C. Ralston, L. Steinhour, and C. Brautigam for
help with data collection. The Berkeley Center for Structural
Biology is supported in part by NIH, the National Institute of
General Medical Sciences, and the Howard Hughes Medical
Institute. The Advanced Light Source is supported by the Director,
Office of Science, Office of Basic Energy Sciences, of the U.S.
Department of Energy under contract DE-AC02-05CH11231.
Results shown in this report are derived from work performed at
Argonne National Laboratory, Structural Biology Center at the
Advanced Photon Source. Argonne is operated by UChicago
Argonne, LLC, for the U.S. Department of Energy, Office of
Biological and Environmental Research under contract DE-AC02-
06CH11357. J.S.T. is an investigator, Y.C. is a research specialist
3, and C.P. was an associate in the Howard Hughes Medical
Institute. J.S.T. is a cofounder of, a Scientific Advisory Board
member of, and a paid consultant for Reset Therapeutics, a
biotechnology company aimed at discovering small-molecule
therapies that modulate circadian activity for a variety of
disease indications. C.B.G. is on the Scientific Advisory Board
of, is a paid consultant for, and owns stock in ReSet
Therapeutics. Atomic coordinates for the reported crystal
structures have been deposited with the Protein Data Bank
(PDB) under accession code 4F3L.
Materials and Methods
Figs. S1 to S8
Tables S1 and S2
3 April 2012; accepted 15 May 2012
Published online 31 May 2012;
Fig. 5. Mutationsthatre-
duce repression of CLOCK:
BMAL1 transactivation by
mutations arising from a
or directed mutagenesis
study Q361P/W362R (19)
are predominantly found
on the b sheet face of
CLOCK PAS-B domain and
are fully solvent-accessible.
Residues mutated in these
studies are in orange. The
locations of the SUMOyla-
tion site on BMAL1 PAS-A
nase 2 phosphorylation
site on BMAL1 (S90) (42),
and the phosphorylation
site on CLOCK (S42) (43)
strand DNA is modeled
on the basis of the super-
position with USF-DNA
complex structure (25).
CRY binding site
CRY binding site
13 JULY 2012 VOL 337
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