Using a structural and logics systems approach to
infer bHLH–DNA binding specificity determinants
Federico De Masi1, Christian A. Grove2, Anastasia Vedenko1, Andreu Alibe ´s3,
Stephen S. Gisselbrecht1, Luis Serrano3,4, Martha L. Bulyk1,5,6,* and
Albertha J. M. Walhout2,*
1Department of Medicine, Division of Genetics, Brigham & Women’s Hospital and Harvard Medical School,
Boston, MA 02115, USA,2Program in Gene Function and Expression and Program in Molecular Medicine,
University of Massachusetts Medical School, Worcester, MA 01605, USA,3EMBL-CRG Systems Biology
Research Unit, Center for Genomic Regulation, Universitat Pompeu Fabra, 08003 Barcelona, Spain,4Institucio ´
Catalana de Recerca i Estudis Avanc ¸ats (ICREA) Professor, Center for Genomic Regulation, Universitat Pompeu
Fabra, 08003 Barcelona, Spain,5Department of Pathology, Brigham & Women’s Hospital and6Harvard-MIT
Division of Health Sciences and Technology (HST), Harvard Medical School, Boston, MA 02115, USA
Received December 13, 2010; Revised January 23, 2011; Accepted January 24, 2011
Numerous efforts are underway to determine gene
regulatory networks that describe physical relation-
ships between transcription factors (TFs) and their
target DNA sequences. Members of paralogous TF
families typically recognize similar DNA sequences.
protein–DNA recognition by paralogous TFs is of
central importance for understanding how small dif-
ferences in DNA specificities can dictate target gene
selection. Previously, we determined the in vitro
DNA binding specificities of 19 Caenorhabditis
elegans basic helix-loop-helix (bHLH) dimers using
protein binding microarrays. These TFs bind E-box
(CANNTG) and E-box-like sequences. Here, we com-
bine these data with logics, bHLH–DNA co-crystal
structures and computational modeling to infer
which bHLH monomer can interact with which
CAN E-box half-site and we identify a critical
residue in the protein that dictates this specificity.
Validation experiments using mutant bHLH proteins
provide support for our inferences. Our study
provides insights into the mechanisms of DNA rec-
ognition by bHLH dimers as well as a blueprint for
system-level studies of the DNA binding deter-
minants of other TF families in different model
organisms and humans.
The regulation of gene expression is partially controlled by
transcription factors (TFs) that bind DNA in a sequence-
specific manner and that function in the context of intri-
cate gene regulatory networks (1,2). TFs can be grouped
into families according to the structural class of their
DNA binding domains. Members from some but not
all TF families typically bind to similar DNA sequences
(3–5). For instance, most homeodomain TFs prefer
AT-rich sequences (6), whereas basic helix-loop-helix
(bHLH) TFs bind E-box (CANNTG), or E-box-like
sequences that differ from E-boxes in 1 or 2nt (7).
A major question in the field of transcriptional regula-
tion is what determines the small differences in DNA
binding site specificity between different members of TF
families. In particular, how do differences in protein
sequence and structure result in differences in DNA
binding specificity and thereby target gene selection?
Many studies of protein–DNA recognition have focused
on C2H2 zinc fingers, for which relatively accurate predic-
tion of DNA binding specificities can be achieved (8–10).
*To whom correspondence should be addressed. Tel: +1 508 8564364; Fax: +1 508 8565460; Email: firstname.lastname@example.org
Correspondence may also be addressed to Martha L. Bulyk. Tel:+1 617 5254725; Fax:+1 617 5254705; Email: email@example.com
Federico De Masi, Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark, Building 208,
DK-2800 Kongens Lyngby, Denmark.
Christian A. Grove, California Institute of Technology, Pasadena, CA 91125, USA.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
Published online 18 February 2011Nucleic Acids Research, 2011, Vol. 39, No. 11 4553–4563
? The Author(s) 2011. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
However, the existence of a simple, amino acid to nucleo-
tide ‘recognition code’, in which certain amino acids
specify interactions with particular bases (11,12) has
been largely discounted as more intricate relationships
between the biochemical and biophysical properties of
TFs and their DNA binding sites have been revealed
The analysis of protein–DNA binding specificity is
greatly enhanced by the comprehensive determination of
DNA binding specificities, measured on a single experi-
mental platform, for a diverse set of proteins belonging
to a TF family. This approach permits even very small
differences in DNA binding specificity to be assessed.
Such data sets were recently generated on a large scale
and were used to gain insights into the DNA binding
specificity determinants of Drosophila and mouse homeo-
domain TFs (6,17). However, for the vast majority of
other TF families, such determinants remain largely
Many TFs-bind DNA as heterodimers that complicates
the analysis of protein–DNA recognition (2). This is
because each monomer contributes to different aspects
of binding site specificity. In heterodimers, particularly,
it is therefore important to first assess which monomer
contacts which part of the bound DNA sequence. Here,
we analyze the molecular mechanisms of DNA recogni-
tion by bHLH proteins which bind DNA as obligate
homo- or heterodimers (18). We chose this family for
several reasons. First, multiple co-crystal structures have
been obtained (19–21) that provide a high-resolution view
of bHLH dimerization and DNA binding. Dimerization
between bHLH proteins is mediated by two a-helices from
each of the bHLH monomers that face each other, and
DNA interactions occur mainly with residues in the basic
specificities of most C. elegans bHLH homo- and
heterodimers (7) using yeast two hybrid (Y2H) assays
(22) and universal protein binding microarrays (23).
These data sets provide high-confidence insights into the
sometimes subtle differences in DNA binding specificity
and target gene selection between members of this import-
ant TF family. We first infer the half-site specificities of
each of the bHLH monomers. Subsequently, we interro-
gate available bHLH-E-box co-crystal structures for the
amino acids that contact the DNA. We identify a residue
in the bHLH-basic region that partially explains the spe-
cificity for interacting with different E-box half-sites. PBM
assays on three HLH-1 mutant proteins provide support
for these determinants.
MATERIALS AND METHODS
bHLH-contact frequency plots
Co-crystal structures of the upstream stimulatory factor
(USF) (PDB:1AN4) (24), Max (PDB:1HLO) (25), Pho4
(PDB:1A0A) (26), MyoD (PDB:1MDY) (27) homodimers
and Max/Mad (PDB:1NLW), Max/Myc (PDB:1NKP)
(28) and E47/NeuroD (PDB:2QL2) (21) heterodimers
were analyzed for proximity of DNA-contacting residues
to each nucleotide base, deoxyribose or phosphate group.
Contacts within 3.5A˚ were considered ‘direct’ contacts,
and distances up to 5A˚ away were considered ‘partially’
contacting. All observed ‘direct’ contacts and ‘partial’
contacts (counted as 1 and 0.5, respectively) were
summed for each amino acid position and divided by
the total number of instances of that amino acid that
could be analyzed. This number depended on the total
number of bHLH monomers represented in the seven
crystal structures (22 total, see below) and whether or
not the amino acids were represented adequately (e.g.
alanine substitutions were not counted). There were
either two (1HLO:Max, 1AN4:USF, 1A0A:Pho4) or
1MDY:MyoD, 2QL2:E47/NeuroD) bHLH monomers in
each bHLH-crystal structure depending on the crystal unit
composition, resulting in 22 bHLH monomers in total.
The resulting ‘contact frequency score’ indicates the
normalized contacting frequency for each residue; a
score of 100 indicates a ‘direct’ contact in 100% of the
monomers observed. Note that a score of 50 could
indicate either a ‘partial’ contact in 100% of the
monomers observed, or a ‘direct’ contact in 50% of the
monomers observed, or some combination of such
In silico modeling
The change in interaction energy upon mutation of each
of the 10 DNA-contacting residues of the bHLH-PDB
structures listed above were calculated with the protein
design tool FoldX (29,30). The side chain positions were
first optimized with the RepairPDB command, to correct
residues with positive internal energy. Next, each of the
10 DNA-contacting residues was mutated to each of
20 amino acids and the resulting change in interaction
energy upon mutation (??Gint) was calculated. We
defined ??Gint as the difference in interaction energy
between the structures of the wild-type and mutated
proteins plus the difference (if >0.6kcal/mol) in intramo-
lecular clashes (both for DNA and for the protein) in
order to penalize potentially destabilizing mutations.
FoldX simulations were performed for each mutation
5?, to increase the conformational space explored, and
the results were averaged. We have increased the default
number of rotamers used in FoldX 20-fold, in order to
improve the accuracy of the results. In the case of homo-
dimers, the mutation was introduced in both monomers
and its ??Gint effect was divided in half, while for
heterodimers each of the individual mutations was
evaluated separately. Values of ??Gint>1.0kcal/mol
might significantly affect the DNA binding properties of
the mutant, while values >2.0kcal/mol may completely
disrupt the DNA binding capabilities of the mutant. All
FoldX calculations were performed assuming temperature
equal to 298K, pH 7.0 and 150mM ionic strength. The
relative binding strength of each pair of monomers was
calculated as the ratio of the monomers’ Kds, which were
calculated from their ??Gintvalues (Kdis proportional to
4554Nucleic Acids Research, 2011,Vol.39, No. 11
Cloning, protein expression and quantification
A wild-type C. elegans hlh-1 open reading frame was
synthesized by gene synthesis and cloned into the pGS21a
vector (GenScript USA, Inc). This clone was used to
generate the L13R, L13T and L13V mutants (GenScript
USA, Inc). All clones used to produce protein for PBM
experiments were full-length DNA sequence verified. All
protein constructs were expressed using the PURExpress
coupled in vitro transcription and translation system
(New England BioLabs, Inc.) following the manufacturer’s
instructions. Protein concentrations were estimated by
western blotting by comparison to a dilution series of
recombinant glutathione-S-transferase (Sigma).
PBM experiments and data analysis
Microarray design, double stranding of the oligonucleo-
tide arrays by primer extension, and PBM experiments
were performed essentially as described previously using
custom-designed ‘all 10-mer’ arrays synthesized in the
Agilent ‘4?44K’ array format (Agilent Technologies,
Inc.; AMADID #015681) (31). All PBM experiments
were performed in duplicate at a final concentration of
400nM wild-type or mutant HLH-1 protein, except for
the HLH-1 L13T mutant, which was assayed at 460nM
in one of the PBM experiments. Microarray scanning,
spot quantification, data filtering, normalization and
analysis were performed as described previously (31).
Statistical analysis of HLH-1-mutant binding preferences
for E-box sequences
mutants), ungapped 8-mers were ranked by PBM E-score.
Statistical significance of preferential or disfavored binding
of 8-mers containing either all E-boxes or particular E-box
sequence variants was assessed by a Wilcoxon–Mann–
Whitney U-test. Preferential or disfavored binding of
each E-box variant by each of the three mutant proteins
was compared with wild-type HLH-1 by computing a
U-statistic within the context of all 8-mers and, separately,
of just E-box-containing 8-mers. The boxplots shown in
Figure 4d were created using the ‘boxplot’ function in the
‘graphics’ library of the R-project statistical software
(http://www.r-project.org). Significantly bound E-boxes
were identified as those E-boxesfor which the 75th percent-
ile of their E-score distribution is >0.4 and, as previously
described (7), their q-value is <0.001 and their area under
the curve (AUC) is >0.85.
DNA binding specificity of individual bHLH proteins
All C. elegans bHLH dimers, with the exception of
CANNTG. Here, we investigated the mechanisms of
DNA binding by 19 C. elegans bHLH proteins, using
PBM data that we recently generated (7). We focused
our analysis exclusively on E-boxes, since only E-boxes
were represented in available bHLH–DNA co-crystal
Within a bHLH dimer, each monomer contacts a CAN
half-site (Supplementary Figure S1). We reasoned that
the DNA binding specificities of each bHLH dimer
would enable the inference of the half-site preferences
of the participating monomers by the following logic
(Figure 1). When a bHLH homodimer binds a palindrom-
ic DNA sequence, each of the monomers binds to an iden-
tical half-site. We hypothesized that if only a single
palindrome is bound by a homodimer, then that half-site
should be optimal for its corresponding monomer. For
instance, HLH-30 homodimers bind only the CACGTG
palindrome, indicating that each HLH-30 monomer
prefers a CAC half-site over the other three possible
half-sites (Table 1). We first performed this type of
analysis for each of the nine C. elegans bHLH homo-
dimers’ PBM data (7). We subsequently extended this
analysisto the bHLH
10 heterodimers that we analyzed (7).
There are 10 non-redundant E-boxes when both the
forward and reverse complementary orientations are con-
sidered (e.g. CACCTG and CAGGTG are the same
E-box). These include four palindromes, one of which is
not bound by any bHLH dimer (CAATTG, Table 1). This
strongly suggests that bHLH proteins disfavor the CAA
proteins that formthe
Figure 1. Deduction of interactions between bHLH monomers and
CAN E-box half-sites. HLH-2/HLH-3 heterodimers are shown as an
Nucleic Acids Research, 2011,Vol.39, No. 114555
half-site. Only a single CAA-containing, non-palindromic
E-box was bound, and only by the HLH-1 homodimer (7).
This indicates that only HLH-1 monomers, and no other
bHLH proteins, tolerate a CAA half-site but only when
another, more optimal half-site is present. HLH-30,
MXL-3, HLH-25, HLH-26 and REF-1 homodimers can
bind to only one palindromic E-box, CACGTG, and so
each of these proteins must be able to recognize the CAC
half-site. Similarly, HLH-1 binds only the CAGCTG pal-
indrome, and so each HLH-1 monomer likely prefers
CAG. The fact that HLH-1 binds CAA only in the
context of the CAACTG E-box (7) (Figure 4d below)
provides further support that CAG is its preferred half-
site (i.e. CAACTG is composed of a CAA and a CAG
half-site). The observation that most bHLH homodimers
bind only a single palindrome further suggests that
these proteins indeed prefer the corresponding half-site.
In fact, in most cases these palindromes exhibited the
highest PBM-enrichment scores (E-scores) (7), a relative
measure of DNA binding preference (23). Moreover, most
of the non-palindromic bound E-boxes contain one
half-site from the preferred palindromic E-boxes and pal-
indromes of less preferred half-sites are typically not
strongly preferred. For example, the MXL-3 homodimer
significantly prefers the CACGTG E-box, but also binds
CACATG, composed of CAC and a CAT half-sites, but
not the CATATG palindrome (Table 1) (7). This strongly
suggests that MXL-3 monomers prefer CAC over CAT.
We used available co-crystallographic data for bHLH
dimers bound to an E-box to estimate, in silico, the
relative binding affinities (Kds) of each bHLH monomer
to DNA using the protein design tool FoldX (29,30,32).
The available co-crystallographic data are for the mam-
malian homodimers Max (19,25), USF (24) and MyoD
(27); the heterodimers Myc/Max, Mad/Max (28) and
E47/NeuroD (21); and the yeast homodimer Pho4 (26).
This FoldX analysis predicted that the two monomers
within USF and Max homodimers exhibit ?100-fold
binding affinities. In other words, one USF monomer
binds to its half-site 100? more strongly than the other
USF monomer to the other half-site. Individual Pho4 and
MyoD monomers, on the other hand, appear to bind
to each half-site with similar affinities (Supplementary
Table S1). Similarly, within the three available hetero-
dimer structures bound to the CACGTG palindromic
E-box (Myc/Max, Mad/Max (28) and E47/NeuroD
(21)), the Kds of each monomer/half-site interaction
are predicted by FoldX to exhibit differences of two to
six orders of magnitude within the same structure
(Supplementary Table S1). These observations suggest
that the two monomers that comprise a bHLH dimer
can bind each E-box half-site with different affinities. In
non-palindromic E-boxes, the presence of a high-affinity
half-site could provide a platform for one bHLH
monomer and enable the other bHLH monomer to
tolerate a lower affinity half-site.
We next investigated the half-site preferences of
C. elegans bHLH proteins that bind DNA exclusively as
heterodimers. HLH-2-containing heterodimers collectively
Table 1. Deduced half-site preferences for 19 C. elegans bHLH proteins
Predicted binding sites
of bHLH dimer
Observed binding sites of bHLH dimer
CAG, CAC, CAT
CAG, CAC, CAT
CAGCTG, CACCTG, CATCTG
CAGCTG, CACCTG, CATCTG
CAGCTG, CACCTG, CATCTG,
CACGTG, CATGTG, CATATG
CAGCTG, CACCTG, CATCTG
CAGATG, CACATG, CATATG
CAGCTG, CACCTG, CATCTG,
CAGCTG, CACCTG, CATCTG,
CAGCTG, CACCTG, CATCTG,
CAGCTG, CACCTG, CATCTG,
CAGCTG, CATCTG, CATATG
Same as MDL-1
CAGCTG (0.96), CACCTG (0.98), CATCTG (0.91)
CAGCTG (0.98), CACCTG (0.99)
CAGCTG (1.00), CACCTG (1.00), CATCTG (0.95),
CACGTG (0.93), CATATG (0.94)
CAGCTG (0.96), CACCTG (0.95)
CATCTG (0.86), CACATG (0.88), CATATG (0.90)
CAGCTG (0.98), CACCTG (0.94), CATCTG (0.93),
CAGCTG (0.93), CACCTG (0.94), CATCTG (0.87),
CAGCTG (0.99), CACCTG (0.94), CATCTG (0.89),
CAGCTG (0.86), CATCTG (0.91), CATATG (0.98)
HLH-19CAG, CAT HLH-2
CND-1 CAG, CATHLH-2
CAGCTG (0.93), CATATG (0.96)
CAGCTG (0.95), CACCTG (0.92), CAACTG (0.86)
CACGTG (0.98), CACATG (0.88)
CACGTG (1.00), CACATG (0.93)
CACGTG (1.00), CACATG (1.00)
Same as MDL-1
The table indicates the bHLH TF, its (deduced) preferred E-box half-sites and dimerization partner(s). Additionally shown is a comparison of
E-boxes predicted to be bound based on these deduced half-site preferences (E-boxes predicted but not observed are indicated in bold text) versus
E-boxes observed to be bound based on actual experimentation (E-boxes observed but not predicted are indicated in bold text). Previously published
AUC values (7) for each observed E-box are indicated in parentheses to illustrate the differences in the relative binding affinities of each bHLH to
the different E-boxes bound. Note that the predictions are based only on observed palindromic E-box binding.
4556 Nucleic Acids Research, 2011,Vol.39, No. 11
bind all palindromes except CAATTG. Thus, the HLH-2
monomer must be able to bind CAT, CAC and CAG
half-sites (Figure 1 and Table 1). This is in agreement
with crystal structures of dimers containing E47, the
human HLH-2 ortholog, which collectively detected E47
binding to each of these half-sites (20,21). HLH-2 dimer-
izes with 14 other bHLH proteins, and the resulting
heterodimers have partially overlapping DNA-binding
specificities (Table 1) (7). The fact that the DNA binding
specificities of HLH-2 heterodimers are non-identical in-
dicates that each HLH-2 partner must, at least in part, be
responsible for the specification of particular E-boxes, pre-
sumably by preferring particular half-sites. For example,
the HLH-2/HLH-3 heterodimer binds only the CAGCTG
palindrome, indicating that HLH-3 prefers CAG (Figure 1
and Table 1). The non-palindromic sequences bound by
the HLH-2/HLH-3 heterodimer (CAGGTG and CAGAT
G) both contain a CAG half-site. We, therefore, propose
that HLH-3 specifies the CAG half-site, and that HLH-2
specifies the other half-site, although we cannot exclude
the possibility that HLH-3 half-site preference may be
E-box dependent. By similar reasoning, we propose that
HLH-8, which together with its partner HLH-2 binds the
CATATG palindrome and the CATCTG and CATGTG
non-palindromes, specifies a CAT half-site. All deduced
half-site preferences for HLH-2 and its partners are
provided in Table 1.
HLH-2 half-site specification can depend on its
Our half-site model suggests that an HLH-2 monomer can
bind to CAC, CAG or CAT half-sites. Surprisingly,
however, we found that the half-site preference of
HLH-2 can depend on its dimerization partner. For
instance, when HLH-2 is dimerized with HLH-3, the
bound E-boxes are in order of higher to lower relative
affinity, CACCTG, CAGCTG and CATCTG (Table 1)
(7). Each of these E-boxes contains the CAG half-site
likely specified by HLH-3, and thus HLH-2 likely
prefers CAC, followed by CAG and then CAT in the
context of this heterodimer. When HLH-2 is dimerized
with HLH-8, however, CATATG, CATCTG and CACA
TG are bound, with the latter two E-boxes being bound
with similar PBM-enrichment scores (Table 1) (7). Since
we propose that HLH-8 specifies CAT, this suggests that
HLH-2 prefers CAT in the context of this heterodimer,
followed by CAG and CAC. Thus, HLH-2 may prefer
either a CAC or CAG half-site in the context of the
HLH-2/HLH-3 heterodimer, but prefers CAT when
dimerized with HLH-8. In sum, bHLH proteins that can
dimerize with multiple partners appear to prefer different
half-sites in the context of different heterodimers.
Analysis of bHLH monomer–DNA contacts
We next analyzed all available co-crystallographic data
for bHLH–DNA complexes, i.e. eight different bHLH
monomers bound to three different E-boxes. Structural align-
ment of these co-crystal structures indicated that all eight
bHLH dimers exhibit extremely similar DNA binding geom-
etry (mean RMSD=1.49±1A˚) (Supplementary Figure S2).
To inspect the protein–DNA interfaces of these bHLHs in
more detail, we devised a strategy to determine the relative
contribution of contacts between each amino acid in the
entire bHLH domain and the bound E-box. By inspecting
each bHLH–DNA co-crystal structure, we generated a
frequency map of molecular contacts made by bHLH
residues and each nucleotide position in and around the
E-box. From this frequency map, we inferred which
residues in the bHLH domain are most likely to partici-
pate in directing DNA binding specificity.
bHLH proteins contact DNA with residues in their
basic regions (indicated in green in Figure 2a). Although
each E-box half-site is contacted by a bHLH monomer,
there are also extensive contacts across the E-box, i.e. with
the other half-site, and most frequently these occur on the
Figure 2. Analysis
(a) Canonical structure of a bHLH monomer contacting the CANNTG
E-box. The E-box nucleotides on each DNA strand are indicated in blue,
the bHLH basic region is shown in green and bHLH helices 1 and 2 are
indicated in red and cyan, respectively. Residues 1, 15 and 50 indicate the
frequency of contacts between bHLH residues and the bases in or flanking
the E-box based on seven co-crystal structures. (c) As in (b) but indicating
contacts between bHLH residues and the DNA backbone. P, phosphate;
dR, deoxyribose. bHLH residue consensus numbering scheme based on
Atchley et al. (33).
of bHLH residue/DNA-contactfrequency.
Nucleic Acids Research, 2011,Vol.39, No. 114557
opposite strand of DNA (Figure 2b and c) (20,21). For
instance, residue 13 in the basic region often contacts the
base at position 4 of the CANNTG E-box (Figure 2b).
This suggests that residue 13 may contribute to half-site
specificity on the opposite side of the E-box.
The most critical residue for E-box binding is a nearly
invariant glutamate at position 9 that contacts both the C
and the A in the CAN half-site (33). Our analysis revealed
that a large number of base contacts are also made by
residues 5, 6 and 12, which contact either the C or the A
in the CAN half-site, the 50flanking nucleotide, or a com-
bination of these (Figure 2b). In addition to base contacts,
multiple amino acids make contacts with the DNA
backbone; each bHLH monomer contacts the backbone
of the entire E-box and a number of 50and 30flanking
nucleotides (Figure 2c). Our observations support the role
of extensive backbone contacts in stabilizing bHLH–DNA
bHLH half-site recognition determinants
To identify correlations between particular amino acid
residues and bound DNA sequences for our C. elegans
data set, we focused on the positions identified in the
above frequency map and coupled bHLH half-site speci-
ficity to the amino acids at each of those 17 DNA-
contacting positions (Figure 3a). In order to obtain a com-
prehensive understanding of the half-site recognition
mechanism of each bHLH protein, we included all half-
sites present at least once in each significantly bound
E-box (e.g. HLH-1 is associated with CAA, CAC and
CAG) (Table 1). As reported previously, bHLH proteins
containing an arginine at position 13 (Arg13) prefer CAC
(33–35). However, while HLH-30, HLH-26 and REF-1
exclusively specify CAC, MDL-1, MXL-1, MXL-3 and
HLH-25 also specify CAT (Figure 3a). Interestingly,
these proteins exhibit a difference at position 2 in the
basic region: proteins with an arginine (or a glutamine in
case of the first bHLH domain of HLH-25) at position 2
bind both CAC and CAT, whereas the remaining proteins
bind exclusively to CAC. This suggests that Arg2/Gln2
may enable CAT specification in the context of Arg13-
containing proteins. PBM data obtained for bHLH
UniPROBE database) (36) further support our model; of
the five high-quality PBM data sets for Arg13-containing
bHLH proteins (Saccharomyces cerevisiae bHLH TFs
Cbf1p, Tye7pand Pho4p;
bHLHb2), three exactly matched our residue/half-site
correlations. Tye7p has Arg13 but not Arg2 and show
specificity for CAC only, while Max and Pho4p, having
both Arg2 and Arg13, bind to both CAC and CAT
(Supplementary Figure S3).
We found that bHLH proteins that do not have an
arginine at position 13, with the exception of HLH-8,
which has a threonine at that position (see below), can spe-
cify the CAG half-site either exclusively or in combination
with any of the other possible half-sites (Figure 3a). In
addition, we inferred that proteins with Val13 specify
CAG as their default half-site. These can also specify
CACand CATif they
(e.g. HLH-2 and HLH-10). Publicly available PBM
data (5,36) for another Arg1+Arg2+Val13 protein,
mouse Tcfe2a, shows specificity for CAG, CAC and
CAT (Supplementary Figure S3). Of all 560 bHLH
proteins listed in UniProt, only 17 contain the Arg1+
Arg2+Val13 combination. Alignment of these bHLH
proteins shows extremely strong conservation of their
basic regions (Supplementary Figure S4), supporting the
idea that these bHLHs share similar DNA binding
Two C. elegans bHLH proteins have unique DNA
binding specificities as well as unique amino acid residues
at position 13. The first is HLH-1, the C. elegans MyoD
ortholog, which can bind a CAACTG E-box. HLH-1 is
the only protein in our data set to have both Leu13 and
tolerate a CAA half-site. Support for the importance of
Leu13 in specifying CAA comes from homologs of
HLH-1, such as myogenin and MyoD, that also bind C
AACTG (37,38) and that also have a leucine at position
13 (Supplementary Figure S5). The mouse Myf6 bHLH
protein also possesses a leucine at position 13 and
also binds to CAA, CAC and CAG (Supplementary
Figure S3). The second C. elegans bHLH protein that
has unique DNA binding specificity as well as a unique
amino acid residue at position 13 is HLH-8, the C. elegans
twist ortholog, which has a threonine at position 13 and
specifies CAT (Table 1). Homologs of twist in other
species also bind CATATG (39,40) and also have Thr13
(Supplementary Figure S5). Thus, comparisons between
the amino acid identities at DNA-contacting positions
and the DNA binding specificities of C. elegans bHLH
proteins and their homologs in other species provide
support for our inference that Leu13 and Thr13 may
specify CAA and CAT half-sites, respectively.
In silico analysis of the structural determinants of
In order to comprehensively evaluate which protein
residues are energetically compatible with E-box binding,
we performed structural modeling of DNA-bound bHLH
proteins using FoldX. We mutated, in silico, all eight
base-contacting residues identified by our frequency map
(positions 1, 2, 5, 6, 8, 9, 12 and 13) to each of the 20
amino acids in the context of the seven available bHLH–
DNA co-crystal structures. To evaluate the impact of mu-
tations in the base-contacting residues, we used position 3,
which makes no DNA contact (data not shown), as a
negative control. We also compared mutation of the
base-contacting residues against mutation analysis of
position 10, which makes only phosphate backbone
contacts (Figure 3d and Supplementary Table S2) and
thus may not be a major specificity determinant.
As expected, a proline is not tolerated in any of the eight
base-contacting positions, likely because of the destab-
ilizing effect that proline has on a-helices (41). Most of
the in silico mutations predicted to result in a loss of
DNA binding introduce amino acids rarely observed at
that position in naturally occurring bHLH proteins
(Supplementary Table S3). However, two detrimental mu-
tations—Pro6 and Ala9—are observed in >5% of
4558Nucleic Acids Research, 2011,Vol.39, No. 11
Figure 3. bHLH domain amino acids correlate with E-box half-site recognition (a) Amino acid residues at each position in C. elegans bHLH
proteins that are involved in DNA contacts. The final column shows the list of recognized half-sites for each bHLH (7). ‘A’ and ‘B’ (e.g. HLH-26A
and HLH-26B) refer to the first and second bHLH domain of two within the same protein. (b) Diagram illustrating the distribution of bHLH
proteins according to their half-site specification. (c) Schematic representation of the rules inferred from (a) and illustrated in (b). Filled circles
represent residues at position 13; purple rectangles represent half-sites and open circles represent specific sequence determinants for linking residue 13
to a particular half-site. (d) Graphical representation of the average ??Gintper mutation for the seven bHLH structures analyzed. Mutations for
which ??Gintis larger than 1.0kcal/mol (indicated by the red line) may have considerably diminished, or even abolished, the binding capabilities of
the mutant. Position 13 is split between the average ??Gintfor the scaffold of bHLH proteins that normally do or do not have an arginine in that
Nucleic Acids Research, 2011,Vol.39, No. 11 4559
naturally occurring bHLH TFs (Supplementary Table S3).
Our results predict that Pro6 results in a combination of
loss of DNA binding and destabilization of the protein.
The fact that Pro6 is found in naturally occurring bHLH
proteins suggests that the conformation of the a-helix
may be different in those proteins than in the ones
whose co-crystal structures were available (i.e. structures
for Pro6-containing bHLH dimers were not available) for
our analysis. Some bHLH proteins that have Pro6 are
members of the Hairy class of bHLH proteins (34) and
these proteins can bind to the N-Box, which is an
E-box-like sequence (CACNAG) (42,43). Other Pro6-
containing bHLH proteins are from the HAND1 group
of bHLH TFs, have Thr13 and bind NRTCTG (44).
Proteins with an Ala9 mainly belong to the bHLH–Per-
Arnt-Sim class (bHLH-PAS) and include HIF-1a and
SIM. The bHLH–PAS protein ARNT has a glutamate
at position 9 and binds the CAC half-site. HIF-1a and
non-canonical half-sites-like (A/G)C and GT (A/G)C,
respectively [JASPAR database (45)]. Thus, bHLH–PAS
heterodimers bind DNA sequences comprising a conven-
tional E-box half-site and a non-canonical half-site.
We postulate that proteins with Ala9 specify the
‘non-canonical’ half-site in the context of bHLH–PAS
dimers. Altogether, these observations suggest that the
DNA recognition rules we present here pertain only to
E-box binding bHLH proteins.
Our in silico mutation analysis indicates that positions
6, 9 and 13 are the least tolerant to mutations
(Supplementary Table S2). These residues all directly
contact the core NN sequence of the E-box; residues six
and nine also have a direct physical interaction with the
CA sequence (Figure 2b). Our analysis supports previous
experimental observations for mutant Max homodimers
(46) and predictions provided by computational studies
The energy calculations using the scaffold of bHLH
proteins that have Arg13 show that the majority of the
mutations in position 13 result in significant decreases in
binding energy. Only mutations to the aromatic amino
acids phenylalanine, tryptophan and tyrosine do not sig-
nificantly affect binding energy. The arginine at position
13 makes an H-bond with the guanine opposite from the
cytosine in the CAN half-site. Mutations at position 13 in
the bHLH protein’s basic region disrupt this H-bond,
leading to a decrease in binding affinity and specificity.
This may explain why phenylalanine, tryptophan and
tyrosine do not occur naturally at position 13 in any
bHLH protein (Supplementary Table S3), as these
residues are not able to form an H-bond to stabilize the
TF–DNA interaction and therefore will not discriminate
among the four bases since the binding energy will come
mainly from desolvation upon burial of these bulky side
chains. Interestingly, the scaffold of bHLH proteins that
do not have Arg13 tolerate mutations at positions 13–15
other amino acids in the FoldX analysis. This could be
because the amino acid at position 13 that is normally
present in these bHLH proteins is smaller than an
arginine and does not make specific contacts with DNA,
allowing for mutations to occur without negatively
impacting the overall bHLH–DNA binding properties.
However, although these mutations do not affect the
overall DNA binding propensity, they may affect DNA
binding specificity (see below).
Experimental confirmation of bHLH-E-box recognition
In order to experimentally test our inferred bHLH–E-box
recognition determinants, focusing position 13 because it
appears to play a crucial role in bHLH–E-box inter-
actions. We created three mutant versions of HLH-1
and determined each of their DNA binding specificities.
We selected this bHLH dimer because it is a homodimer
(which simplifies interpretations of DNA binding) and it
has broad DNA-binding specificity, as it is able to bind to
the CAA, CAC and CAG half-sites Figure 3b). We
created three mutants at position 13—in particular, we
mutated Leu13 to arginine (L13R), threonine (L13T)
and valine (L13V)—and measured their DNA binding
specificities by universal PBM assays (23). For each of
these three mutants, we predicted changes in their half-
site preferences based on the determinants we derived
above (Figure 4a and b). Briefly, we expected all three
mutants to lose specificity for CAA. Furthermore, we pre-
dicted that the L13R mutant would lose its specificity for
CAG, gain specificity for CAT and strongly favor CAC.
We also predicted that the L13T mutant would lose its
specificity for both CAG, gain binding to CAT, and also
lose specificity for CAC, and that the L13V mutant would
gain specificity for CAT while maintaining specificity for
both CAC and CAG (Figure 4a and b).
The PBM data obtained for each mutant HLH-1
protein are consistent with our predictions for their
altered half-site binding specificities (Figure 4c). As
expected, none of the HLH-1 mutants binds a CAA
half-site-containing E-box (Figure 4d). The L13T mutant
gained specificity for the CAT half-site (as indicated by
binding to CATATG); however, unexpectedly, the L13T
mutant also retained the ability to bind CAG, but with a
lower specificity compared to the wild-type protein, and
gained binding capacity for CAC (Figure 4d). These
results suggest that HLH-8 (which also has a threonine
at position 13) specifies CAT, CAG and/or CAC half-sites
when bound to HLH-2 as a heterodimer (while HLH-2 is,
in fact, restricted to the CAT half-site). The L13V mutant
retained specificity for both CAG and CAC half-sites (as
indicated by binding to CAGCTG and CACCTG
E-boxes, Figure 4d), and gained weak specificity for
CAT (as indicated by weak binding to CATATG and C
ATCTG, Figure 4d); all these observations agree with our
predictions. The L13R mutant gained specificity for the
CAC half-site (as indicated by strong binding to the CAC
GTG E-box, Figure 4d), and gained some specificity for
CAT (as indicated by binding to CACATG, Figure 4d),
while losing specificity for the CAG half-site (as indicated
by complete loss of binding to CAGCTG, Figure 4d).
Finally, this L13R mutant strongly preferred CACATG
and CACGTG over any of the other E-boxes (Figure 4d),
which is in agreement with our observation that Arg13-
containing bHLH proteins strongly prefer CACGTG and
CACATG (7) (see above).
4560 Nucleic Acids Research, 2011,Vol.39, No. 11
Altogether, these experimental results confirm the role
of position 13 in the determination of bHLH E-box spe-
cificity. Moreover, these results confirm that bHLH
proteins containing Arg13 exhibit clear preferences for
the CACGTG E-box and CAC half-sites in general.
Importantly, we demonstrate that mutating the residue
at position 13 in the bHLH basic region does not signifi-
cantly disrupt overall DNA binding but can alter the
DNA-binding specificities of bHLH proteins in a predict-
In this study, we used a systems-level protein–DNA inter-
action network, together with logics, computational
modeling and co-crystal structures to investigate the
Figure 4. Prediction and experimental validation of HLH-1 Leu13 mutant E-box half-site specificities. (a) Classification of the three HLH-1 mutant
proteins based on the E-box half-site specificity diagram in Figure 3b. (b) Predictions of the half-site specificities for each of the HLH-1 Leu13
mutants. (c) Summary of the statistical analysis of the observed half-site specificities determined from PBM data for each of the HLH-1 mutants.
(d) Boxplot representation of the PBM-derived E-box specificities of HLH-1 wild-type and mutant proteins. Significantly bound E-boxes are colored
in black (see ‘Materials and Methods’ section). Eight-mers that do not contain an E-box are marked as ‘other’. For each box, the central horizontal
bar shows the median of the distribution, the box’s edges mark the 25th and 75th percentile and the whiskers represent the most extreme points of
the distribution, which were not determined as being outliers. The horizontal bar of ES value of 0.4 shows our significance ES threshold, as
previously determined (7).
Nucleic Acids Research, 2011,Vol.39, No. 114561
mechanisms of E-box recognition by bHLH TFs. We first
determined the CAN half-site preferences of individual
bHLH monomers that bind E-boxes either as homodimers
or as heterodimers. We then identified residue 13 in the
bHLH basic region as a main selector of half-site specifi-
city. Interestingly, when an arginine is present at position
13 in the bHLH protein, different specificities can be
attained when a glutamine is present at position 1 and
an arginine at position 2, compared with other residues
at these positions. These amino acids, however, do not
directly contact the bases within the CANNTG E-box,
but rather makes contacts with the DNA backbone
(Figure 2) and in silico modeling indicates that positions
1 and 2 are not of critical importance for E-box binding
(Figure 3). Together, this suggests that residue 13 func-
tionally interacts with other amino acids in the basic
region. It is also possible that the residues that make pri-
marily phosphate backbone contacts with the DNA could
contribute to DNA binding affinity and/or specificity via
indirect readout (47,48).
Our data overall agree that there is no straightforward
protein–DNA recognition code in which one amino acid
always specifies a particular base. Indeed, the ‘recognition
rules’, we have described in this manuscript are not abso-
lute, but rather have exceptions; for example, HLH-1 has
Leu13 yet can tolerate a CAA half-site. Various features
of protein–DNA interfaces contribute to complexity in
recognition rules. Our analyses treated the DNA-
contacting residues essentially independently. Position
interdependence among amino acid residues involved in
DNA recognition has been observed for EGR zinc-finger
proteins (49) and likely also plays a role in other struc-
tural classes of TFs. In addition, structural studies of
C2H2 zinc-finger proteins have shown that even modest
rearrangements of protein side chains’ docking geometries
upon binding DNA can make it difficult to predict DNA
binding specificities with high accuracy consistently
(50,51). Future studies involving determination of DNA
binding specificities for proteins with greater coverage
of combinations of amino acid variants at DNA-
contacting positions may permit higher order statistical
models of DNA binding specificity determinants to be
learned that capture the context dependence of specificity
determinants, both for bHLH proteins and for TFs of
other structural classes.
The determinants we identified here are not the sole
factors that dictate target gene selection by bHLH
dimers. Other factors that are important include the
specific spatiotemporal co-expression of bHLH dimeriza-
tion partners, interactions with TFs or cofactors and, im-
portantly, accessibility to different E-boxes that can
depend on the chromatin state. Further, DNA binding
by TFs occurs along a spectrum of affinities and, there-
fore, the concentration of dimerization partners, as well as
their affinity and specificity for different recognition se-
quences will ultimately dictate which genes they regulate
under different developmental or physiological conditions.
Our study demonstrates that comprehensive DNA
binding specificities that were determined on a single
experimental platform can be integrated with available
co-crystal structures to gain insight into the molecular
approach will likely be powerful for similar analyses of
other important TF families such as nuclear hormone re-
ceptors and bZip proteins and in a variety of model or-
ganisms and humans. In the future, the derivation of
predictive recognition rules may further facilitate the
design of synthetic TFs with precisely engineered DNA
Supplementary Data are available at NAR Online.
We thank members of the A.J.M.W. and M.L.B.
laboratories and Job Dekker for advice and critical
reading of the manuscript.
A.J.M.W. and HG003985 to M.L.B.); European Union’s
Funding for open access charge: The National Institutes
of Health (DK068429).
National Institutes ofHealth (DK068429to
Conflict of interest statement. None declared.
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