From the Istituto di Biologia and Patologia
Molecolari Consiglio Nazionale delle Ricerche, Università La
Sapienza, 00185 Roma and ¶ Dipartimento di Biologia,
Università Tor Vergata, 00133 Roma, Italy
Received for publication, November 25, 2002, and in revised form, December 27, 2002
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ABSTRACT |
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Helix-loop-helix (HLH) and
helix-loop-helix-leucine zipper (HLHZip) are dimerization domains that
mediate selective pairing among members of a large transcription factor
family involved in cell fate determination. To investigate the
molecular rules underlying recognition specificity and to isolate
molecules interfering with cell proliferation and differentiation
control, we assembled two molecular repertoires obtained by directed
randomization of the binding surface in these two domains. For this
strategy we selected the Heb HLH and Max Zip regions as molecular
scaffolds for the randomization process and displayed the two resulting molecular repertoires on The helix-loop-helix
(HLH)1 proteins, with over
250 representatives in organisms ranging from yeast to man, are one of
the most important and versatile families of eukaryotic transcription factors and are involved in diverse processes such as lineage commitment and differentiation, angiogenesis, cell cycle, growth control, and apoptosis (1-3). They are characterized by a highly conserved structural motif organized in a DNA binding sequence, the
basic region, and a dimerization domain, either HLH (helix-loop-helix) or HLHZip (helix-loop-helix-leucine zipper). They associate in homo-
and heterodimeric complexes that recognize E-box sequences (CANNTG) on
DNA, recruit cofactors, and activate or repress transcription of many
genes (1-3). Selective dimerization is a regulatory mechanism that
allows the expansion of their functional repertoire and also a fine
tuning of gene expression by competition of different complexes able to
bind the same DNA target sequences. The bHLHZip protein Max,
constitutively expressed, is able to homodimerize as well as to
heterodimerize with the other bHLHZip factors of the Max network (Myc,
Mad1-4, Mnt/Rox), in which expression is regulated and which work only
in association with Max (2, 3). Myc, one of the most
frequently altered genes in human cancer, induces proliferation,
growth, and apoptosis but inhibits differentiation (2-5). Mad and
Mnt proteins, although possessing DNA binding specificities quite
similar to Myc, have only partially overlapping, and frequently
opposite, biological functions such as the ability to promote cell
survival and differentiation. Similar to Max, among the factors lacking
the Zip region, the omnipresent E-proteins (Heb, E47, E12, E2-2) also
bind DNA as homodimers (1). The numerous tissue-specific bHLH proteins
(MyoD, SCL/Tal, Mash, and many others) poorly homodimerize but require
the association with E-proteins to bind DNA and exert their biological
functions. HLH proteins lacking a basic region, such as the mammalian
Id1-Id4, impose another level of regulation by sequestering E-proteins in dimers that are unable to bind to DNA (1). Understanding molecular
recognition is a step toward a rational design of molecules that
interfere with HLH protein function. In this regard, we showed that it
is possible to inhibit Myc tumorigenic capacity by means of Omomyc, a
mutant bHLHZip domain, obtained by changing four residues in the Myc
Zip region (6). Omomyc sequesters Myc in complexes unable to bind DNA,
preventing transcriptional activation, enhancing repression,
potentiating apoptosis (7), and suppressing Myc-induced
papillomatosis.2
To gain insight into the rules of protein-protein recognition and to
isolate mutant domains capable of functional interference, repertoires
of HLH and HLHZip domains were designed, exposed on Phage, Plasmids, and GST Fusion Proteins--
DNA sequences
encoding Max bHLHZip (Ala22 to Leu102) and
repertoires of HLH and bHLHZip domains were PCR amplified and inserted into the Construction of HLH and bHLHZip Libraries--
A HLH domain
repertoire was obtained by PCR amplification of the heb gene
HLH domain sequence with two degenerate primers that contained
SpeI and NotI sites: HLH-SpeI,
5'-GAACGCACTAGTGTGCGGGATVTTAATSWMGCATTSRAMRMSCTTRRGCGADTSDBTCAG-3'; HLH-NotI,
5'GTTCCTGCGGCCGCCTTGCTGTKSTAGACTAAGGATGWMTGCTWYGGCTTGATGAAGARTGAGGABTTTTGDTWGGGG-3' (sequence symbols for degenerate oligonucleotides are:
V = ACG, S = GC, W = AT, M = AC, R = AG, D = ATG, B = GCT, K = TG, Y = CT). The reactions, containing 100 ng of template DNA, 2 µM oligonucleotide primers, and 4.5 Pfu
polymerase units, were cycled 35 times at two different annealing
temperatures (45 and 52 °C). The resulting products were mixed to
guarantee the highest level of variability.
A bHLHZip repertoire was generated by two successive PCR amplifications
on a max bHLHZip template. A leucine zipper (Zip) repertoire was obtained in the first reaction with the two degenerate primers: Lz,
5'-ACAGAGTATATCCAGTATATGSRAAGGVAMRASCACACACWCMDACAAVWMRWAGACGAC-3'; and Lz-NotI:
5'-CAGTGAATTCCCGGGGCGGCCGCCCAGTGCACGAABTYKCTGCWBCAGAAGAGCSYKCYBCCGTYKGAG-3'. The Zip repertoire was used as 3'-primer for the second PCR reaction, whereas an oligonucleotide matching the max basic region
(Max-SpeI, 5'-TGGGTACTAGTGCTGACAAACGGGCT-3')
served as 5'-primer, creating a bHLHZip repertoire with degenerate
Zip regions linked to Max bHLH. Following hot start with Taq
polymerase (Sigma), the reaction was cycled 35 times (1 min at
95 °C, 1 min at 55 °C, 1 min at 72 °C) followed by a 7-min
elongation step.
DNA of both repertoires was digested with SpeI and
NotI restriction enzymes and gel-purified. 20-30 ng of
purified insert was ligated to 2 µg of
SpeI/NotI-digested Panning with GST Fusions to Target HLH Proteins--
Affinity
selection of phage libraries was performed with GST fusion Id2, MyoD,
Mad, and Rox proteins. Phage particles (1 × 109 pfu)
were incubated for 1 h at 4 °C with 10 µg of purified GST fusion protein, immobilized on glutathione-Sepharose beads, and preincubated for 2 h in PBS, 3% bovine serum albumin. The beads were washed repeatedly in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.5% Tween 20 and suspended in 100 µl of SM
buffer. Bound phage was recovered by infection of BB4 cells and plated
onto 143-mm dishes. Phage was eluted with SM, titered, and subjected to
two more biopanning rounds.
Filter Immunoscreening of Phage Clones--
Lysates were
prepared from single phage plaques, concentrated by polyethylene glycol
precipitation, and titered. 1 × 107 pfu from each
phage stock were spotted onto nitrocellulose membrane (Nitroplus,
Micron Separation Inc.), which was incubated at room temperature
for 2 h in blocking buffer (PBS, 5% milk, 0.1% Nonidet P-40) and
again for 2 h with 1 µg/ml GST target protein in the same
buffer. After washing in PBS, 0.1% Triton, membranes were incubated
for 1 h at room temperature with anti-GST goat serum (Amersham
Biosciences, 1:1000) and preadsorbed on bacterial lysate, followed by
horseradish peroxidase (HRP)-conjugated anti-goat IgG (1:10000),
washed, and developed with an enhanced chemiluminescence kit (ECL, from
Amersham Biosciences).
ELISA--
Multiwell plates (Nunc) were coated overnight at
4 °C with 100 µl of anti-GST goat serum (5 µg/ml in PBS), washed
in PBS, 0.05% Tween, and incubated in PBS, 0.05% Tween, 5% milk for
1 h at 37 °C. 0.5 µg of GST fusion protein was added to each
well, for 1 h at room temperature. After washing, phage
(108 pfu/well) was added and incubated for 1 h at room
temperature. The plates were washed with PBS, 0.05% Tween, incubated
for 1 h at room temperature with anti- DNA Sequencing--
Phage DNA inserts were PCR-amplified
from 1 µl of phage lysate with two primers flanking the
SpeI and NotI cloning sites: 5'-CACGTTCCGTTATGAGGATGT-3' and 5'-ATGTATCAGTGCCTAGC-3'. The PCR products were purified from agarose gel using the ConcertTM
Rapid PCR Purification system (Invitrogen), and their sequences were determined with an ABI-3700 automated sequencer.
Western Blotting--
Phage was lysed by boiling for 5 min in
2× SDS-gel sample buffer; proteins were separated by SDS-PAGE and
transferred to polyvinylidene difluoride membranes (Amersham
Biosciences). Blots were incubated for 1 h at room temperature
with anti-D-protein (1:1500, courtesy of R. Cortese) or anti-Max (Santa
Cruz C-124; 1:5000) antibodies followed by HRP-protein A (1:10000) and
developed with an Amersham Biosciences ECL kit.
Display of Max bHLHZip Domain on Design of HLH and HLHZip Repertoires--
Repertoires were
constructed by mutating only selected amino acids within the scaffold
domain sequences, because the library size necessary to fully represent
the diversity obtainable by random variations would rapidly saturate
the possibilities of phage display libraries. The sequences of Max
HLHZip and Heb E-protein HLH were taken as scaffolds for the two domain
families (Figs. 2 and 3) because of their
dimerization versatility and because of the availability of either
their high resolution crystallographic structure
(Max (17, 18)) or that of a close
relative (E47, an E-protein that shares a high degree of homology with
Heb (19)). The amino acid sequences of a large number of HLH and HLHZip
domains from different organisms were aligned and the occurrence of
different amino acids in each position determined. Strictly conserved
residues, likely to be essential for domain stability, were maintained
constant in the repertoire design, whereas the artificial repertoire
variation was directed at residues that presented natural variability
or were shown to be involved in contacts between subunits in the dimeric structures of Max, E47, MyoD, USF, PHO4, and SREBP
(17-23). Because a complete randomization of these residues could not
be represented fully in a phage display library, only the amino acids found in natural proteins were included in the design. In this way,
diversity was reduced to about 7 × 108 combinations,
representing a large fraction of the variability observed in natural
domains (Figs. 2B and 3B).
In more detail, in the bHLHZip repertoire the degeneration was
restricted to the 29-amino acid-long Zip region, which previously had
been shown to dictate recognition specificity among bHLHZip domains
(6, 24-26). We introduced variations at 13 amino acids occupying the a, d, e, and
g positions of the helical wheel (Fig. 3B). These
residues represent the interface between the two Zip monomers, whereas
the b, c, and f positions are
solvent-exposed and were therefore kept invariant (17, 20, 25, 27).
The 44-amino acid-long HLH domain has a more complex structure
(Fig. 2A). The helix-loop-helix dimerization motif is a
compact four-helix bundle, where the two
Degenerate DNA sequences encoding the designed HLH and bHLHZip domain
repertoires were synthesized by PCR and cloned in the display vector
Affinity Selection with GST-tagged HLH and HLHZip Domains--
GST
fusions to MyoD and Id2, or to Mad and Rox, were used as baits for
panning the HLH and the HLHZip libraries, respectively. For each
experiment, after three rounds of selection, ~100 phage clones were
amplified, and the interactions with the protein baits were tested by a
filter assay. Approximately 10% of the isolated phage clones could be
proved to display protein domains that consistently bound the bait.
Binding was specific because the clones did not bind GST alone or GST
fusions to unrelated protein domains, such as p75 neurotrophin receptor
and amyloid precursor protein cytoplasmic regions. We quantified the
interaction to MyoD, Id2, HEB, Rox, Mad, and Max by ELISA, revealing a
number of phage clones with high binding affinity (Figs.
4 and 5). The amino acid sequences of
HLH(Zip) inserts were deduced from the DNA sequences and aligned to
pinpoint the residues responsible for dimerization specificity and
affinity. A number of differences were
evident in the sequence alignment (Figs. 4B and
5B). The amino acid frequency profiles of the domains with
the highest and the lowest affinity for
Id2, MyoD, Mad, and Rox are shown in
Tables I and II.
The protein domains isolated from the HLH repertoire were shown in
ELISA experiments to bind MyoD, Id2, and Heb with different intensities, ranging from 1 to 8 on an arbitrary scale (Fig.
4B and Table I). Id2 was invariably bound more strongly than
MyoD, reflecting the different interaction strength between natural E-proteins and the two baits (1, 28). Amino acid alignment showed a
preference for many residues of the E-protein consensus sequence,
suggesting that these residues increase dimer stability (Fig.
4B and Table I). They include Ile1,
Gly9, Met11, and Cys12 in helix 1, Gln22 and Thr23 in the loop, and
Leu25 and Val34 in helix 2. The sequence
glycine, methionine, and cysteine at positions 9, 11, and 12 is a
specific motif of E-proteins, which precedes their extra helical turn
at the helix 1 C terminus (Fig. 2B (19)). At positions 11 and 12 only a few of the residues present in the repertoire were found
in the selected domains; the preference for Cys12 was
stronger than for Met11 (76 versus 53%).
All possible amino acids were found at position 9, where glycine
occurred with a 65% frequency, and it was strongly preferred by high
affinity binders (domains 43M, 72I, 42I, 13I, 98M, 27M, 18I, and
43I). Gly9 was present whenever Ile27 was found
(domains 13I, 53I, 98M, 27M), an observation that suggests a possible
interaction between residues 9 and 27, two positions involved in
intrachain interactions according to HLH modeling studies (30). The
positive correlation between a Gly9 residue and
dimerization strength can be explained by structural similarity to the
E47 dimer (19), which shows an intrachain hydrogen bond between
Gly9 and Gln22, a loop residue present in all
selected clones. The four-helix bundle must be stabilized if this
interaction is preserved in the mutant domains. A similar argument can
also explain the preference for Thr23, which, in the E47
dimer, interacts with Leu26, a residue not mutated in the
repertoire. Thr23 was found in all domains but two (71I and
37M) that have a Ser residue and are not very strong binders, whereas
Pro was never selected. Unlike the majority of the residues, the three
negatively charged glutamates found in E-proteins at positions 3, 7, and 39 were either totally absent (Glu3, Glu39) or
present (Glu7) only in domains that did not strongly interact
with MyoD and Id2 (14I, 24I, 30I, 92M; Fig. 4B), whereas
hydrophobic or neutral amino acids (Leu, Val, Ala, Pro, Asn, Gln, Thr)
were preferred in the domains isolated by panning. This was not because
of under-representation, because the glutamates were present at the
expected frequency in the HLH repertoire, as indicated by sequencing of
random clones (Table I). The three glutamates are involved in
E47 dimerization; Glu3 and Glu7 are on the surface of
helix 1, nearest to helix 2', whereas Glu39, on helix 2, interacts with His15', on helix 1' (19). It is interesting
to remark the E39Q and V34Y substitutions in the 72I domain, a high
affinity binder to Id2 and MyoD, because Gln and Tyr are found at the
corresponding helix 2 positions in MyoD and Id2 and in the yeast bHLH,
Pho4. In the Pho4 dimer, in particular, the two residues form an
interhelical hydrogen bond, which is not possible in the E47 dimer
(22). Because of the presence of the same Gln39 and
Tyr34 residues, the hydrogen bond is possible instead in
heterodimers between Id2 or MyoD and the 72I domain. Thus, these two
residues contribute in specifying the dimerization partner.
Valine was also present at position 34 of the high affinity binders.
Hydrophobic residues (Ile or Val) were more frequent at position 32 in
the high affinity binders, whereas Lys occurred with similar frequency in low and high affinity binding domains. Usually, charged residues were found predominantly in low affinity domains at specific HLH positions (Asp6; Asp7, Glu7,
Lys7; Glu9, Arg9;
Glu32; Asp34, Phe34), indicating
that their presence weakens heterodimeric associations (Fig.
4B and Table I). The consensus sequences for high affinity binding to MyoD and Id2 did not show substantial differences, making it
hard to identify the criteria for dimerization selectivity. The pattern
LKAG at positions 5, 6, 7, and 9 was present in two clones (42I and
18I) with higher than average relative affinity for Id2.
Mad and Rox binding affinities to the protein domains isolated from the
bHLHZip repertoire ranged from 1 to 5, Mad consistently being a
stronger interactor than Rox. Rox and Mad at positions 2, 8, 11, 12, 16, 23, 25, and 26 favored the same amino acids. Surprisingly, Max
residues occurred at low frequency in the clones showing the highest
binding affinity for Mad and Rox (Table II), with the only exceptions
being Lys4 (46%) and Asn5 (53%), as if the
Max Zip amino acid sequence was tuned to guarantee dimerization
flexibility rather than strength (Fig. 5B and Table II). In
the Max dimer, the Asn5 residue is located in front of
Asn5' and destabilizes the complex (19, 31). Consistent
with the presence of negatively charged residues at position 5 in Mad
and Rox (Asp and Glu, respectively), Glu5, which
occurred with a 18% frequency, was correlated to low affinity binding
of the phage clones (m19, r10, y71, y25). The role of residues 8, 18, 19, and 23 in molecular recognition, suggested by the Max bHLHZip dimer
crystallographic structure and by the Myc/Max heterodimeric leucine
zipper solution structure (17, 26), was consistent with the amino acid
frequency profiles of Table II. Histidine at position 8 was present
mainly in clones with low binding affinity, whereas the hydrophobic
leucine was strongly preferred by domains with high affinity to Mad and
Rox. Position 8 is His in Max, Ala in Mad and Tyr in Rox. Max
His8 plays a role in Myc/Max recognition via specific
interactions with Myc Glu5 and Glu12 residues
(26). Only one of the two salt bridges observed in Myc/Max would be
possible in heterodimers with Mad and Rox, which have a negatively
charged residue at position 5 only (Asp and Glu,
respectively). In the Max Zip dimer, histidine 8 is close to residues 8 and 9 (histidine and glutamine, respectively) of the other monomer.
Glutamine 9, although present in the repertoire (Fig. 5B and
Table II), never occurred in the selected domains, where ILR
substituted it. The binding affinity to Mad and Rox was similar in the
presence of a hydrophobic residue (Ile or Leu) at position 9 (clones
r45, m50, r15, r32). Position 18 (Gln) is closest to 19' (Asn) in the
Max dimer; the Gln18-Asn19 tetrad is
involved in stabilization of the dimer (32). Residue 18 is a Glu in
both Mad and Rox, whereas residue 19 is Gln in Mad and Lys in Rox.
Amino acids 18 and 23 (Glu in Max, Lys in Mad, and Gln in Rox; Fig.
3B) are in the g and e positions of the coiled-coil, flanking the dimer interface, and have the possibility of forming favorable electrostatic or hydrophobic interactions (24,
26). Positively charged residues (Arg, Lys) were prevalent at position
18 in the domains with lowest affinity, whereas
Glu18, which has the potential to establish a salt
bridge with Mad Lys23, occurred frequently in the Mad high
affinity binders (domains r45, r27, r10). No preference at position 18 was instead apparent for Rox binding. At position 19 all residues
allowed by the repertoire design were accepted. A glutamic acid at
position 23, as in Max, was correlated to low binding affinity to Mad
and Rox. This is consistent with the presence of a glutamic acid
residue at position 18 in Mad and Rox, which would lead to a
repulsive electrostatic interaction. Accordingly, high affinity
binders preferred a hydrophobic leucine or a basic lysine at position 23.
In this work, we have shown that it is possible to display HLH and
bHLHZip domain repertoires as fusion to the C terminus of protein D on
In the HLH domain as well as in the Zip region, several charged
residues at the dimer interface appear to represent discontinuity points that are critical for molecular recognition. In the
domains isolated from the HLH repertoire, hydrophobic or neutral amino acids were preferred to the charged glutamic acid residues occurring at
positions 3, 7, and 39, allowing the formation of stable heterodimers with MyoD and Id2 in the absence of all three Glu residues. Thus, they
appear to destabilize the dimers. Previous work suggested that
heterodimers of MyoD with the E12 E-protein are stabilized by
attractive pairs formed by Glu3, Glu7, and
Glu39 residues of E12 with MyoD residues Arg29,
Arg33, and Gln39, respectively (34). Because
more stable dimers can be obtained with noncharged amino acids, it
seems that the role of the charged Glu residues in the E-protein is to
prevent an excessively strong interaction with MyoD or Id2, allowing
the physiological partner exchange. Similarly, the presence of
histidine at Zip position 8 appears to destabilize dimers and promote
partner exchange, because this residue was counter-selected in the high
affinity binders to Mad and Rox (Fig. 5B, Table II).
Consistent with our findings, Max homodimers were strongly
stabilized by the replacement of His8 with a leucine and to
a lower extent by alanine and tyrosine (31). Leu8 is also
present in the bHLHZip protein USF, which forms homodimers that
are topologically indistinguishable from Max but does not form
heterodimers (17). The two e-g salt bridges, Myc
Glu11-Max Lys16 and Myc Arg18-Max
Glu23, contribute to Myc/Max heterodimerization (24, 26).
The residues found at positions 16 and 23 in the highest affinity
binders to Mad and Rox (e.g. domains r27, m52, r45, m20)
make either one or both of these electrostatic interactions impossible.
Thus they are dispensable for heterodimerization with Mad and Rox,
which is consistent with findings on bZip proteins showing that
interhelical salt bridges in heterodimers do not necessarily contribute
favorably to dimerization specificity and may indeed be unfavorable,
when compared with alternative neutral charge interactions
(35).
The consensus sequences for high affinity binding to MyoD and Id2 were
quite similar. Likewise, the amino acids in many Zip region positions
(2, 8, 11, 12, 16, 23, 25, and 26) showed the same preference
for Rox or Mad binding, indicating that these positions per
se are unable to determine specificity. Actually, it was shown
previously that it is necessary to mutate four residues (residues 5, 12, 18, and 19) in the Myc Zip to overcome its inability to dimerize
(6), that Id1 dimerization specificity can be conferred to E47 by
replacing four amino acids at the helix 1/loop junction (36), and that
a 6-fold increase in MyoD bHLH dimer stability is obtained by
substituting 18 amino acids from the loop and the adjacent regions of
E47 (33). Most of the mutants identified as binders show affinity for
more than one protein. Thus, a domain recognition code, if it exists,
must be rather tolerant. A strategy to increase specific
binding to a particular partner would be to assemble and
screen secondary libraries containing a larger number of mutations at a
more restricted set of sites, such as those that we found most critical
for molecular recognition. Altogether, these findings indicate that
natural selection did not operate to maximize specific recognition
between E-proteins and tissue-specific HLH, or between Max and the
other bHLHZip of the network, but rather to guarantee that these
proteins have a broad recognition spectrum to ensure effective binding
to their HLH or HLHZip partners. Unnecessarily high affinity for a
partner may represent an undesirable property, from an evolutionary
standpoint, since it may diminish the reversibility of HLH(Zip) complex
formation essential for cellular and developmental plasticity. The
charged residues (e.g. the three Glu residues in the HLH and
His8 in the Zip) may be critical for providing such function.
On the other hand, a mutant domain with a higher affinity for a partner
can be exploited for functional interference (6, 7). Therefore the
phage libraries described in this work represent a valuable collection
of reagents and can be used for the selection of HLH and bHLHZip
domains with novel recognition properties, to be employed for molecular
dissection of the pathways involving HLH transcriptional regulators.
This possibility is made more appealing by recent findings that
implicate HLH and HLHZip domains in direct interaction not only with
proteins of the HLH family but also with other transcriptional
regulators such as Miz-1 and JLP, which interact with Myc and
Max, or GRIPE and Pip, which interacts with the E-proteins (37-40).
Such interactions are biologically relevant and enrich the functional
plasticity of HLH proteins. Furthermore, mutant domains may be valuable
for designing therapeutic approaches to diseases in which cell
differentiation or proliferation is perturbed as a consequence of a
deregulated HLH protein function. In this context, the HLH domain may
represent a target for antiangiogenic drug design, because the
naturally occurring HLH proteins Id1 and Id3, as well as Myc, appear to
be required for tumor-induced angiogenesis (41, 42). The domains that
showed increased affinity for Id2 versus MyoD, such as 13I
and others, are intriguing in view of the role of Id2 as an antagonist
of multiple tumor suppressor proteins (43). More particularly, Id2 and
Myc were shown to collaborate in overriding the tumor suppressor
function of Rb in neuroblastomas, and it was suggested that it might be
possible to restore Rb control on cell proliferation in tumor cells, by sequestering Id2 (44). As the 13I domain is able to bind
intracellular Id2 (data not shown), it would be tempting to investigate
its in vivo function or that of other domains with altered
binding properties.
phage capsids. By affinity selection, many
domains were isolated that bound to the proteins Mad, Rox, MyoD, and
Id2 with different levels of affinity. Although several residues along
an extended surface within each domain appeared to contribute to
dimerization, some key residues critically involved in molecular
recognition could be identified. Furthermore, a number of charged
residues appeared to act as switch points facilitating partner
exchange. By successfully selecting ligands for four of four HLH or
HLHZip proteins, we have shown that the repertoires assembled are
rather general and possibly contain elements that bind with sufficient
affinity to any natural HLH or HLHZip molecule. Thus they
represent a valuable source of ligands that could be used as reagents
for molecular dissection of functional regulatory pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phage head, and
screened by in vitro panning. Several domains that bound
with different affinity to MyoD, Id2, Mad-1, and Rox were isolated;
their comparison allowed us to elucidate the contribution of different
amino acid residues to the stability and specificity of monomer-monomer
interactions. These repertoires are a source of potential competitive
inhibitors, useful for functional dissection and for drug design.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
D4 vector DNA, between SpeI and NotI
restriction sites at the 3'-end of a second copy of the D-gene (8).
pGEX-2T (Amersham Biosciences) expression plasmids containing GST
fusions to human Id2, mouse MyoD, human Max, baboon Mad (amino acids
36-221) and mouse Rox (amino acids 197-346) were introduced into BL21
E. coli cells. Cells were grown at 37 °C to an
A600 ~ 0.5 and induced with 0.1 mM
isopropyl-
-D-thiogalactopyranoside for 3 h at
37 °C (MyoD, Id2) or at room temperature (Max, Mad, Rox). After
lysis in the presence of 1% Triton X-100, fusion proteins were
affinity-purified on glutathione-Sepharose beads (Amersham Biosciences)
and analyzed by PAGE.
D4 vector DNA, purified by
isopropanol precipitation. The ligation products were
phenol/chloroform-extracted, isopropanol-precipitated, and in
vitro packaged with a Gigapack III Gold kit (Stratagene). The
libraries were amplified once by infection of Escherichia
coli BB4 cells, plated onto LB-agarose plates, and grown for 6-8
h at 37 °C. Phage was eluted overnight at 4 °C with SM buffer
(100 mM NaCl, 10 mM MgSO4, 35 mM Tris-HCl, pH 7.5), precipitated with polyethylene
glycol, and suspended at 1 × 1010 pfu/ml.
phage rabbit IgG
(1:1000, courtesy of R. Cortese, Istituto di Richerche di Biologia
Moleculare, Pomezia (Rome)), and then incubated with
HRP-conjugated protein A (1:10000, Sigma). Reactions were revealed by
adding 100 µl/well tetramethylbenzidine solution (Promega), and the
absorbance (A) values were recorded by an automated
ELISA reader set at 450 nm. All assays were repeated at
least three times. The reported values are in arbitrary
units, calculated by normalization to the background interaction
with GST and to the interaction of empty vector phage to
GST, according to the following formula:
[Aphage clone-GST
fusion
(Avector-GST
fusion
Avector-GST)]/Aphage
clone-GST.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Phage--
To identify the
most appropriate vector for the display of HLH and HLHZip domain
repertoires, we tested both filamentous phage vectors, successfully
exploited for the construction of peptide or antibody repertoires (9,
10), and
phage, reported to be generally more suitable for
exposing large polypeptides (11-13). The DNA sequence encoding Max
bHLHZip was cloned into the three filamentous phage vectors pC89,
pC178, and pHEN
, to obtain N-terminal fusions to pVIII or pIII coat
proteins (14, 15) and into the
display vector 4 (
D4) to display
fusions to the D-protein C terminus (8, 16). We asked which vector
would efficiently display Max bHLHZip and allow its binding to a
natural dimerization partner, the GST fusion protein Mad (2). We found
that only the
vector particles were able to incorporate the
D-Max chimeric capsid protein in an amount sufficient for
immunological detection in Western blots (Fig.
1A). Furthermore, in a
simulated panning experiment, we were able to selectively enrich
phages displaying Max by 1000-fold after three cycles of affinity
purification over glutathione resin containing GST-Mad (Fig.
1B). Thus,
D4 was selected for the display of domain
repertoires.
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Fig. 1.
Display on phage
coat and affinity selection of Max bHLHZip domain. A,
Western blots. Phage (105 pfu/lane) proteins were probed
with Max (left) and phage head D-protein (right)
antisera.
, empty D4 vector phage;
-Max,
phage containing a D coat protein-Max bHLHZip domain fusion (27 kDa);
GST-Max, GST-Max fusion protein (47 kDa). B,
plaque immunoscreening.
-Max, at a 1:10000 ratio with empty vector
phage, was subjected to three panning cycles with immobilized GST-Mad.
Plaques were screened with Max antibodies. The amount of phage exposing
Max bHLHZip was enriched by ~1000-fold after the third cycle.
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Fig. 2.
Design of an HLH domain repertoire.
A, structural overview of E47 bHLH dimers complexed
with DNA (19). The first and last residues of the E47 HLH region
(Ile352 and Gln392) are indicated. The
subdomains of one of the two monomers are highlighted in
different colors: basic region (BR) in green,
helix 1 (H1) in fuchsia, loop in gray,
and helix 2 (H2) in blue. The amino acid residues mutated in
the repertoire are in lighter tones. The arrows denote three
mutated helix 1 residues at positions f, b, and
c of the helical wheel. They correspond, respectively, to
residues Glu354, Arg357, and Glu358
of the E47 sequence, which are on the surface of helix 1, nearest to
helix 2' (Glu356, Glu358) or helix 2 (Arg357) (19). B, outline of the HLH repertoire.
Sequence alignments of the most representative HLH domains, grouped in
subfamilies, are shown below the Heb scaffold domain. The most
conserved residues are highlighted with the same color
scheme that was used for the subdomains. Positions degenerated in the
repertoire were numbered as shown above the sequence alignment.
Nucleotide composition and encoded amino acids for each degenerate
position are shown at the top; the classical
a-b-c-d-e-f-g heptad repeat of helical structures is
indicated.
View larger version (27K):
[in a new window]
Fig. 3.
Design of an HLHZip domain repertoire.
A, overview of Max bHLHZip dimers complexed with DNA (17).
The first and last residues of Max bHLHZip domain (A22 and L104) are
indicated. The subdomains are highlighted with different
colors in one monomer; the bHLH has the same color code as described in
the legend for Fig. 2, and the leucine zipper is red. The
positions mutated in the repertoires are in lighter tones.
B, outline of the Zip region repertoire. Zip region sequence
alignments of the most representative bHLHZip proteins, grouped in
subfamilies, are shown underneath the Max sequence, which is used as
scaffold. The most conserved residues are highlighted.
Degenerate position numbers are shown
above these sequences. Nucleotide composition and encoded
amino acids for each degenerate position are shown at the
top; the classical a-b-c-d-e-f-g heptad repeat of
helical structures is indicated.
-helices package in a
coiled-coil only near the carboxyl terminus of the dimer (19). In this
case, also residues at b, c, and f
positions significantly contribute to the four-helix bundle. Moreover,
loop residues, such as Gln22 and Thr23
in the E-proteins, are involved in intermolecular bonds (19). On the
basis of these observations, the 15 positions illustrated in Fig.
2B were degenerated in the designed repertoire. Among the
residues that were left unchanged there are those at positions 8, 24, 28, 35, 38 in which mutation had previously been shown to impair
dimerization (28).
D4 as fusions to the D capsid protein C terminus (8). Following
in vitro packaging, ~2 × 106 and
~1 × 106 pfu were obtained for the HLH and bHLHZip
libraries, respectively. By PCR amplification and sequencing of DNA
inserts from randomly chosen phage plaques, we found that ~80% of
the phages in each library were recombinant, and that each one
contained an insert incorporating from 5 to 10 amino acid changes when
compared with the natural scaffold sequence (data not shown).
View larger version (27K):
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Fig. 4.
Sequence and binding affinity of selected HLH
domains. A, ribbon representation of the E47 HLH (19)
depicting the residues that were mutated in the repertoire. E47
residues, in the same color code as described in the legend for Fig. 2,
are connected to the amino acid substitutions introduced in the
repertoires (yellow). B, amino acid sequences and
relative binding strengths. Phage clones were affinity selected from
the HLH repertoire using GST-Id2 and GST-MyoD as baits. Dimerization
with Id2, MyoD, and Heb was measured by ELISA. Relative binding
strengths, normalized and expressed in arbitrary units (average
values ± S.D. from five independent experiments), are indicated
at the left of each clone. The Heb HLH amino acid sequence,
used as scaffold in the repertoire design, is underlined.
The residues introduced in the repertoire at each degenerate
position are indicated above the Heb sequence, and the
sequences of each selected clone are indicated below the E47
sequence.
View larger version (29K):
[in a new window]
Fig. 5.
Sequence and binding affinity of selected
bHLHZip domains. A, ribbon representation of
Max Zip region (17) depicting the residues that were mutated in the
repertoire. Residues, in the same color code as described in Fig. 3
legend, are connected to the amino acid substitutions introduced in the
repertoires (yellow). B, amino acid sequences and
relative binding strengths. Phage clones were affinity selected from
the bHLHZip repertoire using GST-Mad and GST-Rox as baits. Dimerization
of phage clones and a -Max control with Max, Mad, and Rox bHLHZip
domains was measured by ELISA. Relative binding strengths, normalized
and expressed in arbitrary units (average values ± S.D. from five
independent experiments), are indicated on the left of each
clone. The amino acid sequence of Max Zip region, used as
scaffold in the repertoire design, is underlined; the
residues introduced in each degenerate position are indicated
above the Max sequence.
Amino acid frequency profile of affinity-selected HLH domains
Zip region amino acid frequency profile of affinity-selected bHLHZip
domains
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phage head, a system that in our hands proved to be better suited
than filamentous phage. The repertoires contained different
combinations of amino acids found in naturally occurring proteins,
grafted into a limited number of positions involved in partner
recognition by Heb HLH and Max Zip. Using this approach, it was
possible to assemble in an artificial repertoire a large fraction of
the binding surfaces of HLH and HLHZip domains explored by natural
evolution. To identify patterns of recognition specificity, domains
that bind to some natural proteins (MyoD, Id2, Mad1, Rox) with
different affinities were isolated by in vitro screening. Overall, it proved difficult to explain the changes in binding affinity
by single amino acid substitutions. It appears that the complexity due
to multiple amino acid changes produced many alternative combinations
of similar binding strength. This is compatible with a view of
dimerization as a distributed property of the amino acids in the domain
and is consistent with the E47 dimer structure, in which conserved
hydrophobic residues at the interior of the HLH form an extensive van
der Waals surface that provides most of the favorable dimer
interactions (19). However, several correlations were uncovered in our
experiments. The presence of hydrophobic residues correlated to
stronger interaction of HLH domains, confirming the importance of a
hydrophobic core at the dimerization interface for the helix-loop-helix
dimerization affinity (29). The presence of a number of residues that
were found at high frequency in the HLH domains (Gln22 and
Thr23; Ile1, Leu5,
Met11/Val11, and Cys12) did not
correlate to either greater affinity or specificity to any of the
targets, suggesting that these residues have a role in proper
folding of the domain and its display on phage coat. The strong bias
for the two loop residues Gln22 and Thr23 is in
agreement with previous work describing the loop as a key determinant
of bHLH stability (33). This role is particularly evident for
Gln22, which occurred in all domains; its structural role
is visible in the E47 dimer structure, where it participates, together
with Gln13 and Gln30, in a hydrogen bond
network that connects the loop with helices 1 and 2, stabilizing the
four helix bundle (19).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Nicola Rizzo for technical
assistance, Robert Eisenman, Germana Meroni, and Armando Felsani for
GST fusion plasmids, Simona Panni and Giovanna Vaccarello for help with
phage display technology, Barbara Brannetti and Richard Jucker for
thoughtful advice, and Laura Soucek for discussions and for critical
reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was funded by a grant from the Associazione Italiana Ricerca sul Cancro.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These two authors contributed equally to the work.
To whom correspondence should be addressed: Università
La Sapienza, Istituto di Biologia e Patologia Molecolari CNR,
Piazzale Aldo Moro 5, 00185 Roma, Italy. Tel.: 39-0649912241;
Fax: 39-0649912500; E-mail: sergio.nasi@uniroma1.it.
Published, JBC Papers in Press, January 3, 2003, DOI 10.1074/jbc.M211991200
2 L. Soucek, S. Nasi, and G. Evan, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HLH, helix-loop-helix; bHLH, basic helix-loop-helix region; Zip, leucine zipper; GST, glutathione S-transferase; HRP, horseradish peroxidase; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline.
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