(Received for publication, October 19, 1995; and in revised form, November 21, 1995)
From the
Dimerization represents a key regulatory step in the function of basic helix-loop-helix transcriptional factors. In many instances tissue-specific basic helix-loop-helix proteins, such as the hematopoietic factor SCL/tal or the myogenic factor MyoD, interact with ubiquitously expressed basic helix-loop-helix proteins, such as E2A or E2-2. Such dimerization is necessary for high affinity, sequence-specific DNA binding. Previous biochemical and structural studies have shown the helix-loop-helix region to be necessary and sufficient for this interaction. In the present study, we analyzed the relative affinities of various helix-loop-helix interactions using the yeast two-hybrid system. The relative affinities of selected helix-loop-helix species for the partner protein E2-2 were as follows: Id2 > MyoD > SCL/tal. Mutants of SCL/tal with increased affinity for E2-2 were selected from a library of randomly mutated basic helix-loop-helix domains. The amino acid changes in these high affinity versions of SCL/tal introduced residues that resembled those in the corresponding positions of the Id proteins and MyoD. One of the mutants, SCL 12, also contained mutations in highly conserved residues previously thought to be necessary for dimerization. This mutant of SCL demonstrated diminished temperature sensitivity in in vitro interaction assays as compared with the wild type protein. Computational modeling of helix-loop-helix dimers provides an explanation for the increased dimerization affinity of SCL mutant 12.
Basic helix-loop-helix (bHLH) ()transcriptional
factors play a fundamental role in cell fate determination in
eukaryotic organisms ranging from Caenorhabditis elegans to
humans. This family of over 60 different proteins has been implicated
in processes such as lineage commitment, differentiation programming,
cell cycle regulation, and oncogenesis (1, 2, 3, 4, 5) . Many of
these activities arise from sequence-specific DNA binding by bHLH
factors, followed by transcriptional activation of target genes. The
structural motif shared by these proteins, the bHLH domain, mediates
dimer formation as well as direct DNA
contact(6, 7, 8) . The bHLH domain consists
of a
15-amino acid basic region, followed directly by two
amphipathic helices separated by a loop. The basic domain inserts into
the major groove of DNA to bind a specific half-site within the core
recognition sequence CANNTG, also known as an E box. Stable DNA binding
occurs only with dimeric complexes of bHLH proteins, each of which
contributes a basic domain to a specific half-site. Dimerization of
bHLH proteins occurs via the amphipathic helices of the
helix-loop-helix (HLH) portion.
Dimerization plays a central role in regulating the function of bHLH proteins. The composition of the bHLH dimeric complexes determines which specific DNA sites will be recognized. Because each basic domain within a complex contributes a half-site specificity, combinatorial diversity of bHLH complexes expands the array of potential target sequences. In general, tissue-specific bHLH proteins, such as MyoD, SCL/tal, and MASH, poorly homodimerize and preferentially heterodimerize with the broadly expressed E bHLH proteins: E2A, E2-2, and HEB(9, 10, 11) . The E proteins, in particular E2A, may either homodimerize or heterodimerize with tissue-specific bHLH proteins. These patterns of bHLH dimer formation, with an array of tissue-specific bHLH proteins vying for common E protein partners, may account for the capacity of cells to make mutually exclusive developmental decisions(12) . Another level of regulation is imposed by the dominant negative HLH proteins of the Id family(13) . Id proteins contain an HLH domain without a DNA binding basic domain and preferentially heterodimerize with E proteins to form inactive complexes(14) . By competing with tissue-specific bHLH proteins for limiting quantities of E protein partners, Id proteins may globally down-regulate bHLH mediated transcriptional activity(15, 16, 17) .
The structural and biochemical features of HLH dimerization have not been thoroughly characterized. X-ray crystallographic studies indicate that the HLH regions dimerize in the form of a parallel four-bundle left-handed helix, with many of the dimerization contacts occurring within a hydrophobic core region(6, 7) . Biochemical studies indicate that for some dimers, e.g. E2A homodimers, intermolecular disulfide bond formation may be important in stabilizing interactions (18) . Biochemical studies of MyoD heterodimerization with E12, using site-directed mutagenesis, indicate a role for non-conserved charged residues in helix 2 of MyoD forming ionic bonds with charged residues in helix 1 of E12(19) . Similar studies have identified a homodimerization inhibitory domain, amino-terminal to the basic domain in E12, which directs preferential heterodimerization with MyoD (20) .
To further characterize the determinants of helix-loop-helix dimerization, we have employed the yeast two-hybrid system to analyze the heterodimerization of the hematopoietic bHLH protein SCL/tal with the E protein E2-2. E2-2 was chosen as the E protein partner because of its preferential formation of heterodimers with SCL as compared with E2-2 homodimer formation(11) . In addition the E2-2 bHLH domain is highly homologous (95%) to that of E47, for which a crystal structure is available(6) . The yeast two-hybrid system permits direct and accurate quantitation of protein-protein interactions (21) and has previously been applied toward analyzing helix-loop-helix dimerization (22) . In our studies Id and MyoD proteins, as compared with SCL/tal, displayed significantly higher affinity for E2-2. The bHLH domain of SCL/tal was subjected to random mutagenesis, and mutants with normal or increased affinity for E2-2 were selected. Several of the changes in the high affinity SCL/tal mutants introduced amino acids identical or similar to those in the corresponding positions of the Id and MyoD proteins. One of the mutants, SCL/tal 12, contained non-conservative amino acid changes in the two most highly conserved positions in the bHLH family. Using an in vitro interaction assay, we found that the increased affinity of SCL 12 for E2-2 displayed a temperature dependence, manifesting at 30 °C but not at 4 °C. Computational modeling of SCL/tal binding to E proteins provides an explanation for the properties of SCL/tal mutant 12.
HLH preys, consisting of fusions of MyoD,
Id2, and SCL/tal with the B42 transcriptional activation peptide, were
coexpressed via yeast mating with the LexA-E2-2 bait protein.
Quantitative -galactosidase assays were performed on equivalent
numbers of yeast for each interaction. The relative affinities for E2-2
are shown in Fig. 1A. Using SCL/tal as a standard of
comparison, MyoD has a slightly increased affinity for E2-2 (8.5-fold),
and Id2 has considerably increased affinity for E2-2 (65.2- fold).
These results correlate with previously published data using the Far
Western blotting system, in which Id1 had greater affinity than SCL/tal
for E2-2(11) . To further confirm that the results of the
-galactosidase assays reflected the affinities of the various
preys for the E2-2 bait, as opposed to differential expression levels
of the various preys, prey expression in the various strains was
assayed by Western blot. Using a monoclonal antibody (12CA5) that
recognizes an epitope tag common to all preys, Western blot shows
roughly equivalent levels of expression of all the preys (Fig. 1B).
Figure 1:
A, relative quantitation of HLH
interactions using the yeast two-hybrid technique. The preys were
expressed as fusions of the B42 transcriptional activator domain with
the respective HLH domains (wild type SCL/tal, Id2, and MyoD). The bait
consisted of the LexA DNA binding domain fused to the bHLH domain of
the E2-2 protein. Prey and bait plasmids, transformed into separate
yeast strains (YPH-499 and EGY48-pSH18-34, respectively), were
brought together through mating. Within the resultant diploid strains
activation of the lacZ reporter plasmid, pSH18-34, was
quantitated with liquid -galactosidase assays, which were repeated
on three different occasions. B, Western blot analysis of prey
expression in yeast. Equivalent quantities of yeast from A were subjected to SDS-PAGE followed by Western blot with the 12CA5
monoclonal antibody. 12CA5 recognizes an epitope (hemagglutinin)
present in all of the prey proteins.
The relative affinities for E2-2 and the amino acid sequences of the SCL/tal mutants are displayed in Fig. 2A. SCL/tal mutant 4, with similar affinity for E2-2 as wild type SCL/tal, contained a single amino acid change, K234E in helix 2. SCL/tal mutants 12 and 36, both with significantly increased affinity for E2-2, contain identical amino acid changes in the HLH domain and most likely derive from a common original clone. These mutants (designated SCL/tal mutant 12) contain a total of four amino acid changes, two in helix 1 (N204D and G205E) and two in helix 2 (K225E and K234E). Two of the amino acid changes in SCL/tal mutant 12, N204D and K225E, eliminate highly conserved residues in the HLH family, which normally interact to form an intramolecular hydrogen bond(6, 13) . SCL/tal mutant 9, with moderately increased affinity for E2-2, contains a total of two amino acid changes, one in helix 1 (N202D) and one in helix 2 (M233I). An alignment of the high affinity SCL/tal mutants with MyoD and the Id family is shown in Fig. 2B. The residues shared in common by the high affinity SCL/tal mutants and the MyoD and Id proteins are highlighted. From this alignment it is evident that the majority of amino acid changes in the high affinity SCL/tal mutants introduce residues that are similar or identical to residues at corresponding positions in MyoD and the Id proteins.
Figure 2:
A, amino acid sequences of the HLH domain
of wild type and mutant SCL/tal proteins. The SCL/tal mutants were
selected, using the yeast two-hybrid approach, from a pool of randomly
mutated HLH domains. Binding of these SCL/tal mutants to E2-2 is
quantified by measuring the activation of the lacZ reporter
plasmid as described in Fig. 1. The affinities are expressed
relative to those of seven independent yeast colonies containing wild
type SCL/tal prey. B, alignment of HLH domains from MyoD, Id
proteins, wild type SCL/tal, and mutants of SCL/tal. Relative
affinities for E2-2, as measured by the yeast two-hybrid system, are
displayed as relative lacZ units. Relative lacZ units
for each interaction were calculated from three independent
-galactosidase assays. The numbering of amino acid residues
derives from the SCL/tal HLH domain. Amino acid residues shown in bold are those shared by the high affinity SCL/tal mutants and
MyoD and Id proteins.
Figure 3: A, analysis of chimeras between wild type and mutant SCL/tal HLH domains. The indicated SCL/tal HLH domains were assayed for E2-2 binding as described in Fig. 1and Fig. 2. B, Western blot analysis of prey protein expression. Equivalent quantities of yeast from Fig. 3A were subjected to SDS-PAGE followed by Western blot with the 12CA5 monoclonal antibody as described in Fig. 1B.
Figure 4:
In vitro interaction assay.
Recombinant P-labeled E2-2 peptide was incubated with the
indicated GST fusion proteins immobilized on glutathione-Sepharose
beads. After thorough washing, beads were resuspended in SDS-PAGE
loading buffer, and eluates were analyzed by SDS-PAGE/autoradiography. A, binding of [
P]E2-2 to GST fusion
proteins at 4 °C. In lane 1, the GST fusion contains the
intact bHLH domain of SCL/tal as well as the carboxyl terminus, amino
acids 200-331. In lane 2, the bHLH domain has been
deleted from SCL/tal. In lane 3, the GST fusion contains the
minimal bHLH domain of wild type SCL/tal, amino acids 186-242. In lane 4, the GST fusion contains the minimal bHLH domain of
SCL/tal mutant 12. B, binding of
[
P]E2-2 to GST fusion proteins at 30 °C. In lanes 1, 3, and 5, the GST fusion contains
the minimal bHLH domain of wild type SCL/tal. In lanes 2, 4, and 6, the GST fusion contains the minimal bHLH
domain of SCL/tal mutant 12. Varying quantities of
[
P]E2-2, as indicated in the figure, were
combined with the immobilized GST fusion
proteins.
The Id HLH proteins are potent dominant negative inhibitors of bHLH proteins, exerting their effects through the sequestration of E proteins into inactive complexes(14) . For Id proteins to function in an effective manner, they must efficiently outcompete the binding of tissue-specific bHLH proteins, such as SCL/tal, to E proteins. The significantly increased E2-2 binding affinity of Id proteins over SCL/tal, demonstrated previously and in this study, provides a mechanism for the reportedly effective antagonism of SCL/tal function by Id(11, 17) . Our current study also indicates a slightly increased E2-2 binding affinity of MyoD over SCL/tal. Through the isolation of SCL/tal mutants that bind E2-2 with increased affinities, comparable to those of the Id and MyoD proteins, we have identified amino acids that appear to behave as affinity determinants in HLH interactions. The amino acid alignment in Fig. 2B shows that these affinity determinants are present in several HLH proteins. Notably, these amino acids are present in both helix 1 and helix 2. Introduction of any one of the amino acids alone causes a modest increase in the affinity of SCL/tal for E2-2. Coordinate introduction of these amino acids into both helix 1 and helix 2 may lead to a marked increase in the affinity of SCL/tal for E2-2. Therefore, both helix 1 and helix 2 make significant contributions to the affinity of HLH interactions.
One of the amino acid changes in the high affinity SCL/tal mutants, G205E, introduces an acidic residue into helix 1 at a position where many other HLH proteins contain an acidic residue: MyoD and Id2 (Fig. 2B), as well as E12, E47, E2-2, HEB, Daughterless, Twist, myogenin, and MRF4. The crystallographic model of dimerization in Fig. 5A suggests that this amino acid is not close enough to E2-2 to form a bond (8.3 Å from Arg-199 of E2-2). However, previous biochemical studies clearly indicate that an acidic residue at this specific position strongly contributes to dimerization, possibly by forming an electrostatic bond with a basic residue in helix 2 of the partner protein(19) . A similar effect might arise from the N202D mutation in helix 1 of SCL/tal mutant 9: Id1, Id3, E12, E47, E2-2, HEB, Daughterless, and Myc all possess an acidic residue at this position.
Figure 5:
A,
ribbon model of the helix-loop-helix dimerization domains of SCL/tal
and E2-2. The amino acid numbering scheme for SCL/tal is also applied
to the bHLH domain of E2-2. Residues of interest are rendered as
ball-and-stick figures. Within the wild type SCL/tal bHLH domain (red), the epsilon amino group of Lys 225 in helix 2 forms an
intramolecular hydrogen bond with Asn-204 in helix 1. B,
ribbon model for the dimerization of SCL/tal mutant 12 with E2-2. As a
result of the Lys-225 Glu mutation in SCL/tal mutant 12, a novel
intermolecular salt bridge is formed between Glu-225 in SCL/tal mutant
12 and the guanido group of Arg-199 in E2-2. This interaction could
contribute to the increased dimerization affinity of SCL/tal mutant 12.
Glutamic acid residues 205 and 234 in SCL/tal mutant 12 are exposed to
the solvent. C, closeup of the triad of residues: SCL/tal 204
and 225 and E2-2 199. Carbon atoms for wild type SCL/tal are depicted
in black, and carbon atoms for SCL/tal mutant 12 are depicted
in white. Bonds are indicated by broken lines, with
bond distances in angstroms listed. Drawings were made with the
programs MOLSCRIPT and RASTER3D, described in (29) and (30) , respectively.
The M233I mutation in helix 2 of SCL/mutant 9 increases
the hydrophobicity at this position. Methionine has a Kyte-Doolittle
index (K) value of 1.9, and isoleucine has a K
value of 4.5. Notably, most HLH proteins,
including the Id, myogenic, E protein, and achaete-scute families,
possess a highly hydrophobic residue at this position, either
isoleucine (K
value of 4.5) or valine (K
value of 4.2). Structural data from x-ray
crystallography indicate that the residue at this position contributes
to a hydrophobic core at the dimerization interface(6) .
Correspondingly, we have shown that by simply changing methionine 233
to isoleucine one can reproducibly increase the affinity of SCL/tal for
E2-2 by 3-fold.
In the SCL/tal mutant (mutant 12) with highest affinity for E2-2, asparagine 204 and lysine 225 are replaced by acidic residues, aspartic acid and glutamic acid, respectively. These changes represent non-conservative substitutions at the two most highly conserved residues in the entire HLH family(13) . To analyze the structural consequences of these mutations, computational modeling was performed using the crystallographic coordinates of the E47 homodimer (see ``Materials and Methods''). The model shows that N204 and K225 normally interact to form an intramolecular hydrogen bond, approximating helix 1 and helix 2 of SCL/tal (Fig. 5A). Our experimental data indicate that despite stringent evolutionary conservation, these residues are not required for HLH dimerization. Fig. 5(B and C) shows a model predicting the effects of the combined N204D and K225E mutations in SCL/tal. By introducing acidic amino acids at positions 204 and 225 in SCL/tal, the intramolecular hydrogen bond between helix 1 and helix 2 is disrupted. However, the glutamic acid residue at position 225 of SCL/tal is optimally oriented to form an electrostatic bond with the guanido group of arginine 199 in helix 1 of the partner E protein. The net result is that an intramolecular hydrogen bond is lost and an intermolecular ionic bond is gained. A functional corollary is that monomeric SCL/tal, destabilized by the loss of an intramolecular bond, becomes much less energetically favorable than the heterodimeric form which is stabilized by introduction of an additional intermolecular ionic bond. From this model, one might predict that heterodimers of E2-2 with SCL/tal mutant 12 would show increased thermal stability, as compared with heterodimers of E2-2 with wild type SCL/tal (see data in Fig. 4). In order to confirm our hypothetical model, it will be necessary to perform direct crystallographic analyses on complexes of E2-2 with wild type and mutant versions of SCL/tal.
Our results show that the affinity of SCL/tal for E2-2 has not been maximized by natural evolution. Excessive affinity for an E protein partner may represent an undesirable property from an evolutionary standpoint. This excessive affinity might alter the functional characteristics, e.g. DNA binding, of SCL/tal-E protein complexes. Alternatively, the excessive affinity may diminish the reversibility of HLH complex formation, essentially locking cells into undesirable developmental pathways. In many systems of cellular differentiation, rapid dynamic alterations in the arrays of HLH complexes are required at various developmental stages(3, 12) . Usage of high affinity dimerization mutants of HLH proteins in these systems may provide insight into the function of normally transient, rapidly reversible HLH complexes.