NORMAL GENOMIC

• Chavali GB, Vijayalakshmi C, Salunke DM
• Analysis of sequence signature defining functional specificity and structural stability in helix-loop-helix proteins.
• Proteins. 2001; 42: 471-80
• Display abstract

• Specific functional properties of many proteins directing developmental responses via transcriptional regulation are orchestrated by their characteristic helix-loop-helix (HLH) structural motif. The entire HLH motif in all these proteins assumes a common conformation irrespective of their individual biological effects. The motif controls the affinity of HLH proteins for homo- or heterodimerization, permitting mixing and matching of regulatory factors, and thereby expanding the functional repertoire. Systematic analysis of molecular contacts at the dimer interface using the models built for the functional dimers combined with the pattern of conserved/nonconserved residues within different categories of HLH proteins helped in understanding the differential role played by different residues at the dimer interface for expressing corresponding functions. The residues associated with the self and partner interactions were identified, and the signature residues contributing toward dimeric stability and functional specificity were defined. It is evident that most of the residues involved in self interactions are common among all the HLH proteins. However, while certain residues involved in partner interactions are common among all the HLH proteins, certain others are common within a category, and still others vary widely defining specificity signature at different levels. Copyright 2001 Wiley-Liss, Inc.

• Fujii Y, Shimizu T, Toda T, Yanagida M, Hakoshima T
• Structural basis for the diversity of DNA recognition by bZIP transcription factors.
• Nat Struct Biol. 2000; 7: 889-93
• Display abstract

• The basic region leucine zipper (bZIP) proteins form one of the largest families of transcription factors in eukaryotic cells. Despite relatively high homology between the amino acid sequences of the bZIP motifs, these proteins recognize diverse DNA sequences. Here we report the 2.0 A resolution crystal structure of the bZIP motif of one such transcription factor, PAP1, a fission yeast AP-1-like transcription factor that binds DNA containing the novel consensus sequence TTACGTAA. The structure reveals how the Pap1-specific residues of the bZIP basic region recognize the target sequence and shows that the side chain of the invariant Asn in the bZIP motif adopts an alternative conformation in Pap1. This conformation, which is stabilized by a Pap1-specific residue and its associated water molecule, recognizes a different base in the target sequence from that in other bZIP subfamilies.

• Sieber M, Allemann RK
• Thermodynamics of DNA binding of MM17, a 'single chain dimer' of transcription factor MASH-1.
• Nucleic Acids Res. 2000; 28: 2122-7
• Display abstract

• MASH-1, a member of the basic helix-loop-helix (bHLH) family of transcriptional regulators, is a central factor for the regulation of the differentiation of committed neuronal precursor cells of the peripheral nervous system. We have previously produced MM17, a single chain version of this dimeric protein, by linking the C-terminal end of the first subunit to the N-terminal residue of the second subunit through a flexible peptide linker. We have now determined by isothermal titration calorimetry the thermodynamic parameters characterising the DNA binding reactions of MM17. The DNA binding specificity was relatively low and comparable to that observed for wild-type MASH bHLH. At 32 degrees C and pH 7, the concentration of MM17 at which 50% DNA binding occurred was determined as 22.8 and 152 nM for binding to MCK-S and the heterologous SP-1, respectively. Similarly to MASH bHLH the free energy of the association was only slightly temperature dependent, while both the entropy and the enthalpy change were strong functions of temperature. The free energy of DNA binding was independent of the pH for the pH range between 6 and 8. Dissection of the entropy change of the association reaction suggested that the two basic domains and the linker region between the subunits underwent a folding transition from a mainly unfolded to a predominantly ordered conformation. Therefore, like wild-type MASH bHLH, the DNA binding reaction of MM17 follows an induced fit mechanism.

• Littlefield O, Nelson HC
• A new use for the 'wing' of the 'winged' helix-turn-helix motif in the HSF-DNA cocrystal.
• Nat Struct Biol. 1999; 6: 464-70
• Display abstract

• The 1.75 A crystal structure of the Kluyveromyces lactis heat shock transcription factor (HSF) DNA-binding domain (DBD) complexed with DNA reveals a protein-DNA interface with few direct major groove contacts and a number of phosphate backbone contacts that are primarily water-mediated interactions. The DBD, a 'winged' helix-turn-helix protein, displays a novel mode of binding in that the 'wing' does not contact DNA like all others of that class. Instead, the monomeric DBD, which crystallized as a symmetric dimer to a pair of nGAAn inverted repeats, uses the 'wing' to form part of the protein-protein contacts. This dimer interface is likely important for increasing the DNA-binding specificity and affinity of the trimeric form of HSF, as well as for increasing cooperativity between adjacent trimers.

• Graves BJ
• Inner workings of a transcription factor partnership.
• Science. 1998; 279: 1000-2

• Juo ZS, Chiu TK, Leiberman PM, Baikalov I, Berk AJ, Dickerson RE
• How proteins recognize the TATA box.
• J Mol Biol. 1996; 261: 239-54
• Display abstract

• The crystal structure of a complex of human TATA-binding protein with TATA-sequence DNA has been solved, complementing earlier TBP/DNA analyses from Saccharomyces cerevisiae and Arabidopsis thaliana. Special insight into TATA box specificity is provided by considering the TBP/DNA complex, not as a protein molecule with bound DNA, but as a DNA duplex with a particularly large minor groove ligand. This point of view provides explanations for: (1) why T.A base-pairs are required rather than C.G; (2) why an alternation of T and A bases is needed; (3) how TBP recognizes the upstream and downstream ends of the TATA box in order to bind properly; and (4) why the second half of the TATA box can be more variable than the first.

• Jacobson RH, Tjian R
• Transcription factor IIA: a structure with multiple functions.
• Science. 1996; 272: 827-8

• Bonven BJ, Nielsen AL, Norby PL, Pedersen FS, Jorgensen P
• E-box variants direct formation of distinct complexes with the basic helix-loop-helix protein ALF1.
• J Mol Biol. 1995; 249: 564-75
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• The murine transcription factor ALF1 belongs to the class of basic helix-loop-helix proteins specific for the NCAGNTGN-version of the E-box. Binding of homodimeric ALF1 to variants of this motif was studied by a combination of binding site selection technology and DNA modification interference analysis. The results showed that substitutions at the non-conserved positions in the E-box sequence could cause profound alterations in the patterns of specific contacts at the protein-DNA interface. Thus, both the overall extent of the binding region and the backbone phosphate contact pattern differed markedly between closely related E-boxes with similar affinities for ALF1. The identity of the base at the inner N was an important determinant of contact pattern specification. The E-box variants differed in their ability to mediate ALF1 dependent transcriptional activation in vivo. We discuss the possibility that adaptability in basic helix-loop-helix protein-DNA interactions can result in complexes with different functional properties.

• Pio F et al.
• Co-crystallization of an ETS domain (PU.1) in complex with DNA. Engineering the length of both protein and oligonucleotide.
• J Biol Chem. 1995; 270: 24258-63
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• The PU.1 transcription factor is a member of the ets gene family of regulatory proteins. These molecules play a role in normal development and also have been implicated in malignant processes such as the development of erythroid leukemia. The Ets proteins share a conserved DNA-binding domain (the ETS domain) that recognizes a purine-rich sequence with the core sequence: 5'-C/AGGAA/T-3'. This domain binds to DNA as a monomer, unlike many other DNA-binding proteins. The ETS domain of the PU.1 transcription factor has been crystallized in complex with a 16-base pair oligonucleotide that contains the recognition sequence. The crystals formed in the space group C2 with a = 89.1, b = 101.9, c = 55.6 A, and beta = 111.2 degrees and diffract to at least 2.3 A. There are two complexes in the asymmetric unit. Production of large usable crystals was dependent on the length of both protein and DNA components, the use of oligonucleotides with unpaired A and T bases at the termini, and the presence of polyethylene glycol and zinc acetate in the crystallization solutions. This is the first ETS domain to be crystallized, and the strategy used to crystallize this complex may be useful for other members of the ets family.

• Petersen JM, Skalicky JJ, Donaldson LW, McIntosh LP, Alber T, Graves BJ
• Modulation of transcription factor Ets-1 DNA binding: DNA-induced unfolding of an alpha helix.
• Science. 1995; 269: 1866-9
• Display abstract

• Conformational changes, including local protein folding, play important roles in protein-DNA interactions. Here, studies of the transcription factor Ets-1 provided evidence that local protein unfolding also can accompany DNA binding. Circular dichroism and partial proteolysis showed that the secondary structure of the Ets-1 DNA-binding domain is unchanged in the presence of DNA. In contrast, DNA allosterically induced the unfolding of an alpha helix that lies within a flanking region involved in the negative regulation of DNA binding. These findings suggest a structural basis for the intramolecular inhibition of DNA binding and a mechanism for the cooperative partnerships that are common features of many eukaryotic transcription factors.

• Meierhan D, el-Ariss C, Neuenschwander M, Sieber M, Stackhouse JF, Allemann RK
• DNA binding specificity of the basic-helix-loop-helix protein MASH-1.
• Biochemistry. 1995; 34: 11026-36
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• Despite the high degree of sequence similarity in their basic-helix-loop-helix (BHLH) domains, MASH-1 and MyoD are involved in different biological processes. In order to define possible differences between the DNA binding specificities of these two proteins, we investigated the DNA binding properties of MASH-1 by circular dichroism spectroscopy and by electrophoretic mobility shift assays (EMSA). Upon binding to DNA, the BHLH domain of MASH-1 underwent a conformational change from a mainly unfolded to a largely alpha-helical form, and surprisingly, this change was independent of the specific DNA sequence. The same conformational transition could be induced by the addition of 20% 2,2,2-trifluoroethanol. The apparent dissociation constants (KD) of the complexes of full-length MASH-1 with various oligonucleotides were determined from half-saturation points in EMSAs. MASH-1 bound as a dimer to DNA sequences containing an E-box with high affinity KD = 1.4-4.1 x 10(-14) M2). However, the specificity of DNA binding was low. The dissociation constant for the complex between MASH-1 and the highest affinity E-box sequence (KD = 1.4 x 10(-14) M2) was only a factor of 10 smaller than for completely unrelated DNA sequences (KD = approximately 1 x 10(-13) M2). The DNA binding specificity of MASH-1 was not significantly increased by the formation of an heterodimer with the ubiquitous E12 protein. MASH-1 and MyoD displayed similar binding site preferences, suggesting that their different target gene specificities cannot be explained solely by differential DNA binding. An explanation for these findings is provided on the basis of the known crystal structure of the BHLH domain of MyoD.

• Vuister GW, Kim SJ, Orosz A, Marquardt J, Wu C, Bax A
• Solution structure of the DNA-binding domain of Drosophila heat shock transcription factor.
• Nat Struct Biol. 1994; 1: 605-14
• Display abstract

• The solution structure of the DNA-binding domain of the Drosophila heat shock transcription factor, as determined by multidimensional multinuclear NMR, resembles that of the helix-turn-helix class of DNA-binding proteins. The domain comprises a four-stranded antiparallel beta-sheet, packed against a three-helix bundle. The second helix is significantly distorted and is separated from the third helix by an extended turn which is subject to conformational averaging on an intermediate time scale. Helix 3 forms a classical amphipathic helix with polar and charged residues exposed to the solvent. Upon titration with DNA, resonance shifts in the backbone and Asn and Gln side-chain amides indicate that helix 3 acts as the recognition helix of the heat shock transcription factor.

• Suzuki M
• [DNA recognition code]
• Tanpakushitsu Kakusan Koso. 1994; 39: 1597-612

• Wolberger C
• b/HLH without the zip.
• Nat Struct Biol. 1994; 1: 413-6

• Phillips SE
• Built by association: structure and function of helix-loop-helix DNA-binding proteins.
• Structure. 1994; 2: 1-4

• Tainer JA, Cunningham RP
• Molecular recognition in DNA-binding proteins and enzymes.
• Curr Opin Biotechnol. 1993; 4: 474-83
• Display abstract

• The molecular basis for the specificity and activity of protein-DNA interactions is currently being established from the combination of results on the structure and biochemistry of DNA-binding proteins and enzymes. Data detailed in the 12 most recent studies on DNA-binding protein and enzyme structures, including the major advances in the elucidation of enzyme-mediated DNA-repair processes, have both increased understanding of DNA recognition and enhanced prospects for the design of novel DNA-binding proteins in the future.

• Gibson TJ, Thompson JD, Abagyan RA
• Proposed structure for the DNA-binding domain of the helix-loop-helix family of eukaryotic gene regulatory proteins.
• Protein Eng. 1993; 6: 41-50
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• A modelled tertiary structure for the dimeric HLH domain of the E47 protein is presented. Structural information was obtained from the aligned sequences of > 40 members of the HLH family. The information was used to model each monomer as an alpha-helical hairpin, with knobs-into-holes packing of side-chains as found in antiparallel coiled-coil. The dimer forms a four-helix bundle with additional knobs-into-holes packing at the dimer interface. The size and electrostatic properties of core-forming residues are all accounted for in the model. The model does not violate any known properties of protein structure. The monomers are related by two-fold rotational symmetry, in agreement with the observed DNA-binding sites which are imperfect inverted repeats. The N-terminal basic region, in which DNA binding and base specificity reside, forms the first part of helix 1. A prediction based on the model structure is that the HLH domains do not bind to DNA in its B form but require a partially unwound conformation in order to enter the major groove.

• Schwabe JW, Fairall L, Chapman L, Finch JT, Dutnall RN, Rhodes D
• The cocrystal structures of two zinc-stabilized DNA-binding domains illustrate different ways of achieving sequence-specific DNA recognition.
• Cold Spring Harb Symp Quant Biol. 1993; 58: 141-7

• Kostrewa D, Granzin J, Stock D, Choe HW, Labahn J, Saenger W
• Crystal structure of the factor for inversion stimulation FIS at 2.0 A resolution.
• J Mol Biol. 1992; 226: 209-26
• Display abstract

• The factor for inversion stimulation (FIS) binds as a homodimeric molecule to a loose 15 nucleotide consensus sequence in DNA. It stimulates DNA-related processes, such as DNA inversion and excision, it activates transcription of tRNA and rRNA genes and it regulates its own synthesis. FIS crystallizes as a homodimer, with 2 x 98 amino acid residues in the asymmetric unit. The crystal structure was determined with multiple isomorphous replacement and refined to an R-factor of 19.2% against all the 12,719 X-ray data (no sigma-cutoff) extending to 2.0 A resolution. The two monomers are related by a non-crystallographic dyad axis. The structure of the dimer is modular, with the first 23 amino acid residues in molecule M1 and the first 24 in molecule M2 disordered and not "seen" in the electron density. The polypeptide folds into four alpha-helices, with alpha A, alpha A' (amino acid residues 26 to 40) and alpha B, alpha B' (49 to 69) forming the core of the FIS dimer, which is stabilized by hydrophobic forces. To the core are attached "classical" helix-turn-helix motifs, alpha C, alpha D (73 to 81 and 84 to 94) and alpha C', alpha D'. The connections linking the helices are structured by two beta-turns for alpha A/alpha B, and alpha C1 type extensions are observed at the C termini of helices alpha B, alpha C and alpha D. Helices alpha D and alpha D' contain 2 x 6 positive charges; they are separated by 24 A and can bind adjacent major grooves in B-type DNA if it is bent 90 degrees. The modular structure of FIS is also reflected by mutation experiments; mutations in the N-terminal part and alpha A interfere with FIS binding to invertases, and mutations in the helix-turn-helix motif interfere with DNA binding.

• Vinson CR, Garcia KC
• Molecular model for DNA recognition by the family of basic-helix-loop-helix-zipper proteins.
• New Biol. 1992; 4: 396-403
• Display abstract

• The basic-helix-loop-helix-zipper (bHLH-Zip) motif is a conserved region of approximately 70 amino acids that mediates both sequence-specific DNA binding and protein dimerization. This motif is found in protein sequences from many eukaryotic organisms and is contained in the protein sequence of the oncogene myc and its partner max, and a shortened version of the motif (bHLH) is found in the muscle determination factor myoD and its partner E12. An evaluation of the conserved amino acids that define the motif coupled with the published mutagenic studies of this region has led to our formulation of a molecular model for the binding of this motif as a dimer to specific sequences of DNA. This model has the dimeric protein interacting with an abutted, dyad-symmetric DNA sequence. Helix 2 of each monomer is modeled as a coiled-coil extension of the C-terminal "leucine zipper." Helix 1 does not interact with helix 1 from its partner in the dimer but with the hydrophobic surface created when the helix 2 regions of the dimer interact with each other as a coiled-coil. Sequence-specific interactions are proposed between the basic region and the invariant cis elements that all bHLH-Zip proteins bind.

• Tropsha A, Bowen JP, Brown FK, Kizer JS
• Do interhelical side chain-backbone hydrogen bonds participate in formation of leucine zipper coiled coils?
• Proc Natl Acad Sci U S A. 1991; 88: 9488-92
• Display abstract

• The leucine zipper proteins are a group of transcriptional regulators that dimerize to form a DNA binding domain. It has been proposed that this dimerization results from the hydrophobic association of the alpha-helices of two leucine zipper monomers into a coiled coil. We propose a model for a coiled coil based on a periodic hydrophobic-hydrophilic amino acid motif found in the leucine zipper regions of 11 transcriptional regulatory proteins. This model predicts the symmetrical formation of secondary hydrogen bonds between the polar side chains of one helix and the peptide carbonyls of the opposite chain, supplementing the interactions between hydrophobic side chains. Physical modeling (CPK) and in vacuo molecular mechanics calculations of the stability of the GCN4 leucine zipper coiled coil configured in accordance with this model demonstrate a greater stability for this conformer than for a conformer configured according to a current hydrophobic model. Molecular dynamics simulations show similar stability of the two models in vacuo but a higher stability of the hydrophobic model in water.

• Hu YF, Luscher B, Admon A, Mermod N, Tjian R
• Transcription factor AP-4 contains multiple dimerization domains that regulate dimer specificity.
• Genes Dev. 1990; 4: 1741-52
• Display abstract

• Enhancer binding protein AP-4 is a transcription factor that activates both viral and cellular genes by binding to the symmetrical DNA sequence, CAGCTG. Here, we report the molecular cloning and characterization of human AP-4 cDNAs. The deduced amino acid sequence reveals that AP-4 is a helix-loop-helix (HLH) protein. Like other members of this family, the AP-4 HLH motif and the adjacent basic domain are necessary and sufficient to confer site-specific DNA binding. However, unlike other HLH proteins, AP-4 also contains two additional protein dimerization motifs consisting of leucine repeat elements LR1 and LR2. The analysis of various deletion and point mutants for their ability to dimerize in the presence or absence of DNA reveals several unusual features. Although the HLH basic region is sufficient for DNA recognition and binding, dimer formation between different truncated versions of AP-4 in solution requires an intact LR1 or LR2 domain. AP-4 is unable to form heterodimers with other helix-loop-helix family members such as the immunoglobulin enhancer binding factor, E12. In contrast, an AP-4 derivative, delta C222, which lacks LR1 and LR2 but retains an intact HLH, can form heterodimers with E12. Moreover, AP-4 molecules containing LR2 or LR1 are unable to form mixed dimers with carboxy-terminally truncated AP-4 molecules such as delta C222, but retain the ability to form complexes with longer versions of AP-4 that contain LR1 and/or LR2. Our findings strongly suggest that AP-4 contains multiple protein-protein interfaces that function to promote homodimer formation and restrict heterocomplexes. These findings provide a mechanism by which different members of the helix-loop-helix family of transcription factors can form functional dimers in a specific fashion with their appropriate partners to control transcriptional networks during cellular differentiation.

• Murre C et al.
• Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence.
• Cell. 1989; 58: 537-44
• Display abstract

• A DNA binding and dimerization motif, with apparent amphipathic helices (the HLH motif), has recently been identified in various proteins, including two that bind to immunoglobulin enhancers (E12 and E47). We show here that various HLH proteins can bind as apparent heterodimers to a single DNA motif and also, albeit usually more weakly, as apparent homodimers. The HLH domain can mediate heterodimer formation between either daughterless, E12, or E47 (Class A) and achaete-scute T3 or MyoD (Class B) to form proteins with high affinity for the kappa E2 site in the immunoglobulin kappa chain enhancer. The achaete-scute T3 and MyoD proteins do not form kappa E2-binding heterodimers together, and no active complex with N-myc was evident. The formation of a heterodimer between the daughterless and achaete-scute T3 products may explain the similar phenotypes of mutants at these two loci and the genetic interactions between them. A role of E12 and E47 in mammalian development, analogous to that of daughterless in Drosophila, is likely.

• Wingender E
• Compilation of transcription regulating proteins.
• Nucleic Acids Res. 1988; 16: 1879-902

• Giniger E, Ptashne M
• Transcription in yeast activated by a putative amphipathic alpha helix linked to a DNA binding unit.
• Nature. 1987; 330: 670-2
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• Gene activation by a DNA-binding regulatory protein in yeast requires the protein to have two components: one to recognize a specific DNA sequence and a second, the 'activating region', to interact with a general transcription factor or perhaps with RNA polymerase. The activating regions that have been characterized are acidic, and mutational analysis of one indicates that this acidity is important for activity. Here we report the design of an artificial protein bearing a novel 15-amino acid peptide linked to a DNA binding fragment of the yeast regulatory protein GAL4). The synthetic peptide is acidic and should it form an alpha-helix, that helix would be amphipathic, having one hydrophilic face bearing the acidic residues, and one hydrophobic face. When expressed in yeast, the artificial protein bearing this peptide efficiently activates the GAL1 gene which is ordinarily activated by GAL4. An otherwise identical protein with the novel 15 amino acids in a scrambled order, and which is thus unable to form an amphipathic structure, does not activate GAL1 transcription.

• Wharton RP, Brown EL, Ptashne M
• Substituting an alpha-helix switches the sequence-specific DNA interactions of a repressor.
• Cell. 1984; 38: 361-9
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• It has been suggested that many DNA-binding proteins use an alpha-helix for specific sequence recognition. We have used amino acid sequence homologies to identify the presumptive DNA-recognition helices in two related proteins whose structures are unknown--the repressor and cro protein of bacteriophage 434. The 434 repressor and cro protein each bind to three similar sites in the rightward phage 434 operator, OR, and they make different contacts in each binding site, as revealed by the chemical probe dimethyl sulfate. We substituted the putative recognition alpha-helix of 434 repressor with the putative recognition alpha-helix of 434 cro protein to create a hybrid protein named repressor*. The specific DNA contacts made by repressor* are like those of 434 cro protein.

• Ohlendorf DH, Anderson WF, Matthews BW
• Many gene-regulatory proteins appear to have a similar alpha-helical fold that binds DNA and evolved from a common precursor.
• J Mol Evol. 1983; 19: 109-14
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• Amino acid and DNA sequence comparisons suggest that many sequence-specific DNA-binding proteins have in common an homologous region of about 22 amino acids. This region corresponds to two consecutive alpha-helices that occur in both Cro and cI repressor proteins of bacteriophage lambda and in catabolite gene activator protein of Escherichia coli and are presumed to interact with DNA. The results obtained here suggest that this alpha-helical DNA-binding fold occurs in many proteins that regulate gene expression. It also appears that this DNA-binding unit evolved from a common evolutionary precursor.

• Sauer RT, Yocum RR, Doolittle RF, Lewis M, Pabo CO
• Homology among DNA-binding proteins suggests use of a conserved super-secondary structure.
• Nature. 1982; 298: 447-51
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• The amino acid sequences of the repressor and cro proteins of phages lambda, 434 and P22 are homologous, especially in a region in which repressor and lambda cro have a similar alpha-helix-turn-alpha-helix secondary structure. Model-building studies indicate that this structure is important in DNA binding, and we suggest it may be a common feature of many DNa-binding proteins.