A Novel Approach to Protein-Protein Interaction: Complex Formation between the P53 Tumor Suppressor and the HIV Tat Proteins

Longo, Flavia; Marchetti, Marta; Castagnoli, Luisa; Battaglia, Piero A.; Gigliani, F.

doi:10.1006/bbrc.1995.1045

Cited by 62 publications

(60 citation statements)

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“…To this purpose, we have utilized an expression vector derived from pC135 to direct the synthesis of bHLHZip domains fused to a cI domain defective in operator recognition, cI* (Longo et al, 1995;Marchetti et al, 1995). We have then tested the ability of these constructions to relieve l Pr transcription repression by a compatible plasmid, derived from pACYC184 and containing an active cIOmomyc gene ( Figure 3a).…”

Section: Omomyc Forms Dimeric Complexes With Myc and Maxmentioning

confidence: 99%

“…All constructs were sequenced by the Sanger method to con®rm the mutations identity and the proper reading frame. pC135* is similar to pC135, but has three mutations that replace three residues in the l repressor DNA binding domain with the corresponding amino acids of the 434 phage repressor (Longo et al, 1995;Marchetti et al, 1995). pACYCbHLHZip plasmids were constructed by replacing the 1690 bp HindII-HindIII fragment of pACYC184 (Chang and Cohen, 1978) with PvuII-PvuII fragments (containing the Plac-cI-bHLHZ-a-peptide fusions) excised from pC135-bHLHZip plasmids.…”

Section: Molecular Modellingmentioning

confidence: 99%

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Design and properties of a Myc derivative that efficiently homodimerizes

et al. 1998

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bHLH and bHLHZip are highly conserved structural domains mediating DNA binding and speci®c proteinprotein interactions. They are present in a family of transcription factors, acting as dimers, and their selective dimerization is utilized to switch on and o cell proliferation, dierentiation or apoptosis. Myc is a bHLHZip protein involved in growth control and cancer, which operates in a network with the structurally related proteins Max, Mad and Mnt. It does not form homodimers, working as a heterodimer with Max; Max, instead, forms homodimers and heterodimers with Mad and Mnt. Myc/Max dimers activate gene transcription, while Mad/Max and Mnt/Max complexes are Myc/Max antagonists and act as repressors. Modifying the molecular recognition of dimers may provide a tool for interfering with Myc function and, in general, for directing the molecular switches operated via bHLH(Zip) proteins. By molecular modelling and mutagenesis, we analysed the contribution of single amino acids to the molecular recognition of Myc, creating bHLHZip domains with altered dimerization speci®city. We report that Myc recognition speci®city is encoded in a short region within the leucine zipper; mutation of four amino acids generates a protein, Omomyc, that homodimerizes eciently and can still heterodimerize with wild type Myc and Max. Omomyc sequestered Myc in complexes with low DNA binding eciency, preventing binding to Max and inhibiting Myc transcriptional activator function. Consistently with these results, Omomyc produced a proliferation arrest in NIH3T3 cells. These data demonstrate the feasibility of interfering with fundamental biological processes, such as proliferation, by modifying the dimerization selectivity of a bHLHZip protein; this may facilitate the design of peptides of potential pharmacological interest.

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Section: Omomyc Forms Dimeric Complexes With Myc and Maxmentioning

confidence: 99%

Section: Molecular Modellingmentioning

confidence: 99%

Design and properties of a Myc derivative that efficiently homodimerizes

et al. 1998

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“…Specificity assays were done using repressor fusions and dominant-negative mutations by a method similar to methods described by others (30)(31)(32)(33) while this work was in progress. Pairwise combinations of fusion proteins with active (cI ϩ ) and inactive (cI Ϫ ) DNA-binding domains were expressed from compatible plasmids in E. coli.…”

Section: Methodsmentioning

confidence: 99%

Buried asparagines determine the dimerization specificities of leucine zipper mutants

Zeng

Herndon

1997

Proc. Natl. Acad. Sci. U.S.A.

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Regulation of gene expression by many transcription factors is controlled by specific combinations of homo-and heterodimers through a short ␣-helical coiled-coil known as a leucine zipper. The dimer interface of a leucine zipper involves side chains of the residues at the a, d, e, and g positions of the (abcdefg) n heptad repeat. To understand the basis for the specificity of dimer formation, we characterized GCN4 leucine zipper mutants with all 16 possible permutations and combinations of isoleucines and asparagines at four a positions in the dimer interface, using a genetic test for the specificity of dimer formation by repressor-leucine zipper fusions. Heterodimers were detected by loss of repressor activity in the presence of a fusion to a dominant-negative mutant form of the DNA-binding domain of repressor. Reconstruction experiments using leucine zippers from GCN4, Jun, Fos, and C͞EBP showed that this assay distinguishes pairs that form heterodimers from those that do not. We found that the mutants have novel dimerization specificities determined by the positioning of buried asparagine residues at the a positions. The pattern of buried polar residues could also explain the dimerization specificities of some naturally occurring leucine zippers. The altered specificity mutants described here should be useful for the construction of artificial regulatory circuitry.The stoichiometry and specificity with which proteins interact is a key control point in many biological processes. For example, common dimerization domains allow transcription factors in the bZIP or bHLH-LZ families to form a variety of homo-and heterodimers with different properties. By expressing different sets of subunits under different conditions, cells can generate complex regulatory circuits from a relatively small number of genes. The correct functioning of this complex regulatory machinery depends on each of the component proteins assembling only with specific partners.Leucine zippers are an excellent model system to study how the stability and specificity of protein-protein interactions are determined. High-resolution x-ray crystallographic and NMR structures are available for several leucine zippers (1-7). As ␣-helical coiled coils, leucine zippers have simple secondary and tertiary structures. Moreover, the large number of naturally occurring leucine zipper proteins includes a wide variety of distinct and overlapping dimerization specificities. At the amino acid sequence level, leucine zippers are characterized by leucine appearing in every seventh position (d) over 4 to 5 heptad repeats (abcdefg) n . The hydrophobic core of the dimer interface is formed by residues at the a and d positions (Fig. 1); the solvent-accessible e and g positions are frequently occupied by charged amino acids (8, 9). In the crystal structures of leucine zippers, including GCN4 homodimers and Jun-Fos heterodimers, intersubunit salt bridges are seen between oppositely charged amino acids at the g (ith heptad) and eЈ (i ϩ 1th heptad of the other monomer)...

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“…Similar methods have been published for the bacterial two-hybrid systems. Variants have been developed in time for specific purposes or just to improve the system in general (Longo et al, 1995;Strauch & Georgiou, 2007). In this paragraph, three methods will be presented, two are transcriptional based and one reconstitutes the catalytic site of the reporter.…”

Section: The Bacterial Two-hybrid Techniquementioning

confidence: 99%

“…The dimerization of a leucine zipper was used as a proof of principle (Hu et al, 1990). The immunity assay has been applied in many publications to investigate protein-protein interactions beside the yeast two-hybrid technique (Blackwood & Eisenman, 1995;Di lallo et al, 1999a;Longo et al, 1995;Wolfe et al, 1999). Various methods to determine protein or protein-DNA interactions have been published that prevent or allow transcription Vidal & Legrain, 1999).…”

Section: Introductionmentioning

confidence: 99%