To study the protein-protein interactions that allow Id, a negative regulator of cell differentiation, to inhibit the DNA-binding activities of MyoD and E47, we have synthesized peptides corresponding to the helix-oop-helix domains of MyoD, E47, and Id. We show that Id preferentially inhibits the sequence-specific DNA-binding activity of MyoD, a muscle-specific protein, as compared to E47, a more ubiquitous protein. The Id helix-oop-helix domain itself forms stable tetramers, and its inhibitory activity arises from the formation of a heterotetrameric structure with MyoD. The formation of this higher order complex provides a general mechanism by which inhibitory proteins can generate sufficient interaction free energy to overcome the large DNA-binding free energy of dimeric DNA-binding proteins.Id (inhibitor of DNA binding) is a protein that negatively regulates gene expression via direct protein-protein interaction to prevent DNA binding of other helix-loop-helix (HLH)-containing proteins (1). Id belongs to a rapidly growing family of DNA-binding proteins related by amino acid sequences that are predicted to fold into a common structural domain, the HLH (for recent reviews, see refs. 2-4). This domain mediates homo-and heterooligomerization of multiple transcription factors, in a similar fashion to the leucine zipper class of DNA-binding proteins, providing a higher order program of gene regulation via interactions between differentially expressed polypeptides. In some cases the leucine zipper and HLH motifs are both present in the same protein, such as in the protooncogene-encoded protein c-Myc and its partner, Max (5).In HLH-containing transcription regulators, a lysine-and arginine-rich basic region is typically found N-terminal to the HLH region and is essential for DNA binding (6). The HLH motif provides a dimerization interface that positions two DNA-binding domains to confer high-affinity site-specific binding to DNA sequences with approximate inversional symmetry. Experimental evidence for dimerization comes from electrophoretic mobility shift studies of the binding of E47 and MyoD to DNA (6, 7). Three similar models that describe the structure of basic region-HLH (bHLH) dimers bound to DNA have been proposed (8-10). More recently the cocrystal structure of Max, a bHLH/leucine zipper protein, bound to DNA has been determined (11). Heterodimer formation is an important regulatory mechanism for HLH proteins: activation of myogenesis by MyoD requires the action of heterodimers of MyoD, a muscle-specific protein, and E47 or E12 (12), two ubiquitously expressed proteins also involved in the regulation ofimmunoglobulin gene expression (6,13).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Since the discovery of the HLH motif, a new subclass of proteins has been found that can inhibit the function of HLH DNA-binding proteins by heterooligomer fo...

Limited digestion of E. coli DNA topoisomerase I with trypsin or papain generated a DNA-binding domain of MW 14,000 corresponding to the carboxyl terminal of the enzyme. This fragment binds to single-stranded DNA agarose as tightly as the intact enzyme. It required around 400 mM NaCl for elution. A truncated topoisomerase that lacks this C-terminal domain was purified. It was eluted from the single-stranded DNA agarose column at around 150 mM NaCl. Although the truncated enzyme could relax negatively supercoiled DNA as efficiently as the intact enzyme at low ionic strength, its processivity was more sensitive to increasing salt concentration. Measurement of binding to fluorescent etheno-M13 DNA also demonstrated that the presence of the C-terminal domain confers higher affinity to DNA for the enzyme.

E47 is an immunoglobulin enhancer DNA-binding protein that contains a basic region-helix-loop-helix (blHLH) domain. This structural motif defines a class of transcription factors that are central to the developmental regulation of many tissues. Its function is to provide a dimerization interface through the formation of a parallel four-helix bundle, resulting in the juxtaposition of two basic DNA-recognition a-helices that control sequence-specific DNA-binding. In order to gain insight into the biophysical nature of b/HLH domains, we have initiated structural studies of the E47 homodimer by NMR. Sequence-specific resonance assignments have been obtained using a combination of heteronuclear double-and triple-resonance NMR experiments. The secondary structure was deduced from characteristic patterns of NOEs, '3Calp chemical shifts, and measurements of 3JHNHa scalar couplings. Except for the basic region recognition helix, the secondary structural elements of the E47 homodimer are preserved in the absence DNA when compared with the co-crystal structure of E47 bound to DNA (Ellenberger T, Fass D, Arnaud M, Harrison SC, 1994, Genes & Dev 8:970-980). As expected, the DNA-binding helix is largely unstructured, but does show evidence of nascent helix formation. The HLH region of E47 is structured, but highly dynamic as judged by the rapid exchange of backbone hydrogen atoms and the relatively weak intensities of many of the NOEs defining the dimerization helices. This dynamic nature may be relevant to the ability of E47 both to homodimerize and to heterodimerize with MyoD, Id, and Tall. Abbreviations: PMSF, phenyl methyl sulfonyl fluoride; DTT, dithiothreitol; NOESY, NOE spectroscopy; TOCSY, total correlation spectroscopy; HMQC, heteronuclear multiple quantum coherence; HSQC, heteronuclear single-quantum coherence; CT, constant time; ID, 2D, 3D, 4D, one-, two-, three-, and four-dimensional NMR; P P I , time-proportional phase incrementation; Tip, rotating frame relaxation time; T i , the arithmetic mean of zero quantum and double quantum relaxation times; 'H-'D exchange, hydrogen deuterium exchange. tion domain capable of forming dimers, tetramers, and higherorder aggregates. Dimerization results in the pairing up of each HLH motif to form a four-helix bundle. Upon dimerization, this domain brings together two N-terminal arginine-and lysine-rich DNA-binding a-helices (the basic region), which promotes specific binding to the pseudo-symmetric CANNTG DNA sites found in the E-box control regions of many developmentally regulated genes.