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• W2022731320 abstract "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 λ 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. 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 λ 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. helix-loop-helix basic helix-loop-helix region leucine zipper glutathione S-transferase horseradish peroxidase enzyme-linked immunosorbent assay phosphate-buffered saline 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 (1Massari M.E. Murre C. Mol. Cell. Biol. 2000; 20: 429-440Google Scholar, 2Grandori C. Cowley S.M. James L.P. Eisenman R.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 653-699Google Scholar, 3Baudino T.A. Cleveland J.L. Mol. Cell. Biol. 2001; 21: 691-702Google Scholar). 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 (1Massari M.E. Murre C. Mol. Cell. Biol. 2000; 20: 429-440Google Scholar, 2Grandori C. Cowley S.M. James L.P. Eisenman R.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 653-699Google Scholar, 3Baudino T.A. Cleveland J.L. Mol. Cell. Biol. 2001; 21: 691-702Google Scholar). 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 (2Grandori C. Cowley S.M. James L.P. Eisenman R.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 653-699Google Scholar, 3Baudino T.A. Cleveland J.L. Mol. Cell. Biol. 2001; 21: 691-702Google Scholar). Myc, one of the most frequently altered genes in human cancer, induces proliferation, growth, and apoptosis but inhibits differentiation (2Grandori C. Cowley S.M. James L.P. Eisenman R.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 653-699Google Scholar, 3Baudino T.A. Cleveland J.L. Mol. Cell. Biol. 2001; 21: 691-702Google Scholar, 4Nesbit C.E. Tersak J.M. Prochownik E.V. Oncogene. 1999; 18: 3004-3016Google Scholar, 5Nasi S. Ciarapica R. Jucker R. Rosati J. Soucek L. FEBS Lett. 2001; 490: 153-162Google Scholar). 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 (1Massari M.E. Murre C. Mol. Cell. Biol. 2000; 20: 429-440Google Scholar). 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 (1Massari M.E. Murre C. Mol. Cell. Biol. 2000; 20: 429-440Google Scholar). 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 (6Soucek L. Helmer-Citterich M. Sacco A. Jucker R. Cesareni G. Nasi S. Oncogene. 1998; 17: 2463-2472Google Scholar). Omomyc sequesters Myc in complexes unable to bind DNA, preventing transcriptional activation, enhancing repression, potentiating apoptosis (7Soucek L. Jucker R. Panacchia L. Ricordy R. Tato F. Nasi S. Cancer Res. 2002; 62: 3507-3510Google Scholar), and suppressing Myc-induced papillomatosis. 2L. Soucek, S. Nasi, and G. Evan, submitted for publication. 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 λ 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. DNA sequences encoding Max bHLHZip (Ala22 to Leu102) and repertoires of HLH and bHLHZip domains were PCR amplified and inserted into the λD4 vector DNA, between SpeI and NotI restriction sites at the 3′-end of a second copy of the D-gene (8Panni S. Dente L. Cesareni G. J. Biol. Chem. 2002; 277: 21666-21674Google Scholar). 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 BL21E. coli cells. Cells were grown at 37 °C to anA600 ∼ 0.5 and induced with 0.1 mmisopropyl-ॆ-d-thiogalactopyranoside for 3 h at 37 °C (MyoD, Id2) or at room temperature (Max, Mad, Rox). After lysis in the presence of 17 Triton X-100, fusion proteins were affinity-purified on glutathione-Sepharose beads (Amersham Biosciences) and analyzed by PAGE. A HLH domain repertoire was obtained by PCR amplification of the heb gene HLH domain sequence with two degenerate primers that containedSpeI 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 ॖm oligonucleotide primers, and 4.5 Pfupolymerase 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 Taqpolymerase (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 andNotI restriction enzymes and gel-purified. 20–30 ng of purified insert was ligated to 2 ॖg ofSpeI/NotI-digested λD4 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. 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 ॖg of purified GST fusion protein, immobilized on glutathione-Sepharose beads, and preincubated for 2 h in PBS, 37 bovine serum albumin. The beads were washed repeatedly in 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 0.57 Tween 20 and suspended in 100 ॖl 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. 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, 57 milk, 0.17 Nonidet P-40) and again for 2 h with 1 ॖg/ml GST target protein in the same buffer. After washing in PBS, 0.17 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, fromAmersham Biosciences). Multiwell plates (Nunc) were coated overnight at 4 °C with 100 ॖl of anti-GST goat serum (5 ॖg/ml in PBS), washed in PBS, 0.057 Tween, and incubated in PBS, 0.057 Tween, 57 milk for 1 h at 37 °C. 0.5 ॖg 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, 0.057 Tween, incubated for 1 h at room temperature with anti-λ 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 ॖl/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. Phage DNA inserts were PCR-amplified from 1 ॖl of phage lysate with two primers flanking theSpeI and NotI cloning sites: 5′-CACGTTCCGTTATGAGGATGT-3′ and 5′-ATGTATCAGTGCCTAGC-3′. The PCR products were purified from agarose gel using the ConcertTMRapid PCR Purification system (Invitrogen), and their sequences were determined with an ABI-3700 automated sequencer. 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. 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 (9Castagnoli L. Zucconi A. Quondam M. Rossi M. Vaccaro P. Panni S. Paoluzi S. Santonico E. Dente L. Cesareni G. Comb. Chem. High Throughput Screen. 2001; 4: 121-133Google Scholar,10O'Connell D. Becerril B. Roy-Burman A. Daws M. Marks J.D. J. Mol. Biol. 2002; 321: 49-56Google Scholar), and λ phage, reported to be generally more suitable for exposing large polypeptides (11Santini C. Brennan D. Mennuni C. Hoess R.H. Nicosia A. Cortese R. Luzzago A. J. Mol. Biol. 1998; 282: 125-135Google Scholar, 12Santi E. Capone S. Mennuni C. Lahm A. Tramontano A. Luzzago A. Nicosia A. J. Mol. Biol. 2000; 296: 497-508Google Scholar, 13Hoess R.H. Curr. Pharm. Biotechnol. 2002; 3: 23-28Google Scholar). The DNA sequence encoding Max bHLHZip was cloned into the three filamentous phage vectors pC89, pC178, and pHENΔ, to obtain N-terminal fusions to pVIII or pIII coat proteins (14Felici F. Castagnoli L. Musacchio A. Jappelli R. Cesareni G. J. Mol. Biol. 1991; 222: 301-310Google Scholar, 15Saggio I. Gloaguen I. Laufer R. Gene. 1995; 152: 35-39Google Scholar) and into the λ display vector 4 (λD4) to display fusions to the D-protein C terminus (8Panni S. Dente L. Cesareni G. J. Biol. Chem. 2002; 277: 21666-21674Google Scholar, 16Cicchini C. Ansuini H. Amicone L. Alonzi T. Nicosia A. Cortese R. Tripodi M. Luzzago A. J. Mol. Biol. 2002; 322: 697Google Scholar). We asked which vector would efficiently display Max bHLHZip and allow its binding to a natural dimerization partner, the GST fusion protein Mad (2Grandori C. Cowley S.M. James L.P. Eisenman R.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 653-699Google Scholar). We found that only the λ vector particles were able to incorporate thed-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 λ phages displaying Max by 1000-fold after three cycles of affinity purification over glutathione resin containing GST-Mad (Fig.1B). Thus, λD4 was selected for the display of domain 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 (17Ferre-D'Amare A.R. Prendergast G.C. Ziff E.B. Burley S.K. Nature. 1993; 363: 38-45Google Scholar, 18Brownlie P. Ceska T. Lamers M. Romier C. Stier G. Teo H. Suck D. Structure. 1997; 5: 509-520Google Scholar)) or that of a close relative (E47, an E-protein that shares a high degree of homology with Heb (19Ellenberger T. Fass D. Arnaud M. Harrison S.C. Genes Dev. 1994; 8: 970-980Google Scholar)). 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 (17Ferre-D'Amare A.R. Prendergast G.C. Ziff E.B. Burley S.K. Nature. 1993; 363: 38-45Google Scholar, 18Brownlie P. Ceska T. Lamers M. Romier C. Stier G. Teo H. Suck D. Structure. 1997; 5: 509-520Google Scholar, 19Ellenberger T. Fass D. Arnaud M. Harrison S.C. Genes Dev. 1994; 8: 970-980Google Scholar, 20Ferre-D'Amare A.R. Pognonec P. Roeder R.G. Burley S.K. EMBO J. 1994; 13: 180-189Google Scholar, 21Ma P.C. Rould M.A. Weintraub H. Pabo C.O. Cell. 1994; 77: 451-459Google Scholar, 22Shimizu T. Toumoto A. Ihara K. Shimizu M. Kyogoku Y. Ogawa N. Oshima Y. Hakoshima T. EMBO J. 1997; 16: 4689-4697Google Scholar, 23Parraga A. Bellsolell L. Ferre-D'Amare A.R. Burley S.K. Structure. 1998; 6: 661-672Google Scholar). 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).Figure 3Design of an HLHZip domain repertoire. A, overview of Max bHLHZip dimers complexed with DNA (17Ferre-D'Amare A.R. Prendergast G.C. Ziff E.B. Burley S.K. Nature. 1993; 363: 38-45Google Scholar). 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 shownabove these sequences. Nucleotide composition and encoded amino acids for each degenerate position are shown at thetop; the classical a-b-c-d-e-f-g heptad repeat of helical structures is indicated.View Large Image Figure ViewerDownload (PPT) 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 (6Soucek L. Helmer-Citterich M. Sacco A. Jucker R. Cesareni G. Nasi S. Oncogene. 1998; 17: 2463-2472Google Scholar, 24Amati B. Brooks M.W. Levy N. Littlewood T.D. Evan G.I. Land H. Cell. 1993; 72: 233-245Google Scholar, 25Muhle-Goll C. Gibson T. Schuck P. Schubert D. Nalis D. Nilges M. Pastore A. Biochemistry. 1994; 33: 11296-11306Google Scholar, 26Lavigne P. Crump M.P. Gagne S.M. Hodges R.S. Kay C.M. Sykes B.D. J. Mol. Biol. 1998; 281: 165-181Google Scholar). We introduced variations at 13 amino acids occupying the a, d, e, andg 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 (17Ferre-D'Amare A.R. Prendergast G.C. Ziff E.B. Burley S.K. Nature. 1993; 363: 38-45Google Scholar, 20Ferre-D'Amare A.R. Pognonec P. Roeder R.G. Burley S.K. EMBO J. 1994; 13: 180-189Google Scholar, 25Muhle-Goll C. Gibson T. Schuck P. Schubert D. Nalis D. Nilges M. Pastore A. Biochemistry. 1994; 33: 11296-11306Google Scholar, 27Hu Y.F. Luscher B. Admon A. Mermod N. Tjian R. Genes Dev. 1990; 4: 1741-1752Google Scholar). 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 α-helices package in a coiled-coil only near the carboxyl terminus of the dimer (19Ellenberger T. Fass D. Arnaud M. Harrison S.C. Genes Dev. 1994; 8: 970-980Google Scholar). In this case, also residues at b, c, and fpositions significantly contribute to the four-helix bundle. Moreover, loop residues, such as Gln22 and Thr23in the E-proteins, are involved in intermolecular bonds (19Ellenberger T. Fass D. Arnaud M. Harrison S.C. Genes Dev. 1994; 8: 970-980Google Scholar). 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 (28Voronova A. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4722-4726Google Scholar). Degenerate DNA sequences encoding the designed HLH and bHLHZip domain repertoires were synthesized by PCR and cloned in the display vector λD4 as fusions to the D capsid protein C terminus (8Panni S. Dente L. Cesareni G. J. Biol. Chem. 2002; 277: 21666-21674Google Scholar). Followingin 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 ∼807 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). 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 107 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 and5B). 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.Figure 5Sequence and binding affinity of selected bHLHZip domains. A, ribbon representation of Max Zip region (17Ferre-D'Amare A.R. Prendergast G.C. Ziff E.B. Burley S.K. Nature. 1993; 363: 38-45Google Scholar) depicting the residues that were mutated in the repertoire. Residues, in the same color code as described in Fig. 3legend, 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 λ-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 indicatedabove the Max sequence.View Large Image Figure ViewerDownload (PPT)Table IAmino acid frequency profile of affinity-selected HLH domainsView Large Image Figure ViewerDownload (PPT) Open table in a new tab Table IIZip region amino acid frequency profile of affinity-selected bHLHZip domainsView Large Image Figure ViewerDownload (PPT) Open table in a new tab 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 (1Massari M.E. Murre C. Mol. Cell. Biol. 2000; 20: 429-440Google Scholar, 28Voronova A. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4722-4726Google Scholar). 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 (19Ellenberger T. Fass D. Arnaud M. Harrison S.C. Genes Dev. 1994; 8: 970-980Google Scholar)). 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 537). All possible amino acids were found at position 9, where glycine occurred with a 657 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 (30Chavali G.B. Vijayalakshmi C. Salunke D.M. Proteins. 2001; 42: 471-480Google Scholar). The positive correlation between a Gly9 residue and dimerization strength can be explained by structural similarity to the E47 dimer (19Ellenberger T. Fass D. Arnaud M. Harrison S.C. Genes Dev. 1994; 8: 970-980Google Scholar), 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′ (19Ellenberger T. Fass D. Arnaud M. Harrison S.C. Genes Dev. 1994; 8: 970-980Google Scholar). 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 (22Shimizu T. Toumoto A. Ihara K. Shimizu M. Kyogoku Y. Ogawa N. Oshima Y. Hakoshi" @default.
• W2022731320 created "2016-06-24" @default.
• W2022731320 creator A5002796232 @default.
• W2022731320 creator A5029338279 @default.
• W2022731320 creator A5031926342 @default.
• W2022731320 creator A5066844987 @default.
• W2022731320 date "2003-04-01" @default.
• W2022731320 modified "2023-10-03" @default.
• W2022731320 title "Molecular Recognition in Helix-Loop-Helix and Helix-Loop-Helix-Leucine Zipper Domains" @default.
• W2022731320 cites W1485413013 @default.
• W2022731320 cites W1622668755 @default.
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