The c-Myc protein induces cell cycle progression and apoptosis through dimerization with Max

The c-Myc protein (Myc) is involved in cellular transformation and mitogenesis, but is also a potent inducer of programmed cell death, or apoptosis. Whether these apparently opposite functions are mediated through common or distinct molecular mechanisms remains unclear. Myc and its partner protein, Max, dimerize and bind DNA in vitro and in vivo through basic/helix-loop-helix/leucine zipper motifs (bHLH-LZ). By using complementary leucine zipper mutants (termed MycEG and MaxEG), which dimerize efficiently with each other but not with their wild-type partners, we demonstrate that both cell cycle progression and apoptosis in nontransformed rodent fibroblasts are induced by Myc-Max dimers. MycEG or MaxEG alone are inactive, but co-expression restores ability to prevent withdrawal from the cell cycle and to induce cell death upon removal of growth factors. Thus, Myc can control two alternative cell fates through dimerization with a single partner, Max.     

A dominant-negative mutant of Max that inhibits sequence-specific DNA binding by Myc proteins

Myc proteins are basic helix-loop-helix/leucine-zipper proteins that bind to specific DNA sequences. In vivo, Myc proteins have been found associated with Max, another basic helix-loop-helix/leucine-zipper protein. However, it is not known to what extent the dimerization of Myc with Max is required for the manifestation of the Myc-induced phenotype. To investigate this, we constructed a dominant-negative mutant of Max, named dMax, that inhibits sequence-specific DNA binding of Myc proteins. Using a rat neuroblastoma model system, we show that dMax reverts N-Myc-induced changes in cellular gene expression. A control mutant of dMax that contains a proline residue in the leucine-zipper region was unable to bind to N-Myc and did not revert the N-Myc-induced changes in cellular gene expression. These data support the hypothesis that N-Myc affects neuroblastoma gene expression through the formation of a DNA-binding heterodimeric complex with Max in vivo.     

A single-headed dimer of Escherichia coli ribosomal protein L7/L12 supports protein synthesis

During protein synthesis, the two elongation factors Tu and G alternately bind to the 50S ribosomal subunit at a site of which the protein L7/L12 is an essential component. L7/L12 is present in each 50S subunit in four copies organized as two dimers. Each dimer consists of distinct domains: a single N-terminal ("tail") domain that is responsible for both dimerization and binding to the ribosome via interaction with the protein L10 and two independent globular C-terminal domains ("heads") that are required for binding of elongation factors to ribosomes. The two heads are connected by flexible hinge sequences to the N-terminal domain. Important questions concerning the mechanism by which L7/L12 interacts with elongation factors are posed by us in response to the presence of two dimers, two heads per dimer, and their dynamic, mobile properties. In an attempt to answer these questions, we constructed a single-headed dimer of L7/L12 by using recombinant DNA techniques and chemical cross-linking. This chimeric molecule was added to inactive core particles lacking wild-type L7/L12 and shown to restore activity to a level approaching that of wild-type two-headed L7/L12.     

Use of a two-hybrid system to identify mutations in Max that confer increased affinity for Myc

A yeast two-hybrid system was used to identify mutants of Max that exhibit an increased affinity for Myc. Truncated forms of the Max helix-loop-helix/leucine zipper motif (HLH/Zip) were first expressed in a two- hybrid system in which the bait protein was the HLH/Zip motif of Myc. Deletion of amino acids both amino-terminal and carboxy-terminal to the leucine zipper of Max reduced Myc/Max heterodimer formation as evidenced by a 160-fold reduction in the expression of the lacZ gene. A library of partially randomized sequences encoding this minimal leucine zipper of Max was then screened using the two-hybrid system. Mutant forms of the Max leucine zipper were identified whose affinities for Myc, as measured by beta-galactosidase activity in yeast lysates, were from 8- to 200-fold greater than the wild-type Max zipper. These Max mutants were shown to interact specifically with Myc and not with wild-type Max. Of 29 mutants analyzed, all had a unique amino acid sequence. This result illustrates the value of a genetic screen in the identification of a collection of mutant forms of the Max leucine zipper whose structures would not have been predicted based on principles of structure-based design.     

p53 DNA binding can be modulated by factors that alter the conformational equilibrium

The p53 tumor suppressor protein is a dimer of dimers that binds its consensus DNA sequence (containing two half-sites) as a pair of clamps. We show here that after one wild-type dimer of a tetramer binds to a half-site on the DNA, the other (unbound) dimer can be in either the wild-type or the mutant conformation. An equilibrium state between these two conformations exists and can be modulated by two types of regulators. One type modifies p53 biochemically and determines the intrinsic balance of the equilibrium. The other type of regulator binds directly to one or both dimers in a p53 tetramer, trapping each dimer in one or the other conformation. In the wild-type conformation, the second dimer can bind to the second DNA half-site, resulting in drastically enhanced stability of the p53-DNA complex. Importantly, a genotypically mutant p53 can also be in equilibrium with the wild-type conformation, and when trapped in this conformation can bind DNA.     

The Saccharomyces cerevisiae GATA factors Dal80p and Deh1p can form homo- and heterodimeric complexes

GATA family proteins Gln3p, Gat1p, Dal80p, and Deh1p mediate the regulation of nitrogen catabolite repression (NCR)-sensitive gene expression in Saccharomyces cerevisiae. Thus far, Gln3p, Dal80p, and Deh1p have been shown to bind to GATA sequences in NCR-sensitive promoters, in some cases to exactly the same GATA sequences. A minimal Gln3p binding site consists of a single GATA sequence, whereas a Dal80p binding site consists of two GATA sequences in specific orientation, 15 to 35 bp apart, suggesting that Dal80p may bind to DNA as a dimer. Additionally, both Dal80p and Deh1p are predicted to contain a leucine zipper motif near their C termini. Therefore, we tested whether they could form homo- and/or heterodimers in two-hybrid assays. We show that Dal80p-Dal80p, Dal80p-Dal80pLZ (leucine zipper), Dal80pLZ-Dal80pLZ, Dal80p-Deh1pLZ, Dal80pLZ-Deh1pLZ, and Deh1pLZ-Deh1pLZ complexes can form. Dal80p-Dal80p and Dal80pLZ-Dal80pLZ complexes yield 5- to 10-fold stronger signals than the other possible dimers. If Dal80p and Deh1p bind to DNA only after dimerization, then the difference in ability to form complexes could significantly affect their affinity for binding DNA and thus the degree of regulation exerted by each of the two factors.     

Linkage between operator binding and dimer to octamer self-assembly of bacteriophage lambda cI repressor

Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage. Cooperativity has long been thought to be mediated by self-assembly of repressor dimers to form tetramers which can bind simultaneously to adjacent operators. More recently, we demonstrated that when free repressor dimers self-associate in solution, tetramer is an intermediate in a concerted assembly reaction leading to octamer as the predominant higher order species [Senear, D. F., et al. (1993) Biochemistry 32, 6179-6189]. Even as a minority component in the assembly reaction, tetramer can account for pairwise cooperativity. In a similar manner, were it able to bind all three operators simultaneously, octamer could account for three-way cooperativity. In fact, based solely on repressor self-assembly, the naive prediction is that the repressor-OR interactions should be substantially more cooperative than they are. Evidently, there are unfavorable contributions to cooperativity from processes other than repressor self-assembly. Here, we focus on coupling between repressor self-association and operator binding as one possible unfavorable contribution to cooperativity. Sedimentation equilibrium analysis was used to compare the dimer-octamer association reactions of a repressor dimer-OR1 complex and free repressor dimer. Fluorescence anisotropy was used to investigate OR1 binding to free dimers and dimers assembled as higher order species. The results of these experiments indicate a significant and salt-dependent unfavorable contribution generated by such coupling. Since the oligonucleotides used in these experiments are the size of single operator sites, this coupling is mediated by the protein, not by the DNA. This mechanism does not account for an additional, salt-independent, unfavorable contribution which we presume is transmitted via the DNA. Thus, unfavorable contributions generated by structural transitions in both macromolecules serve to moderate the effect of self-association alone. We speculate that this is a general feature of cooperative protein-DNA interactions.