Sequence Requirements for Coiled-Coils: Analysis with λ Repressor-GCN4 Leucine Zipper Fusions

Hu, James C.; O’Shea, Erin K.; Kim, Peter; Sauer, Robert T.

doi:10.1126/science.2147779

Cited by 377 publications

(378 citation statements)

References 28 publications

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“…It might be argued that this reflects activity requirements that are too permissive. However, when the same activity criteria were used in studies of the a and d positions (Hu et al, 1990), a much smaller fraction of the randomized population was functional, even when positions is favorable to activity with a DLS score less than or equal to 0.07 and with a -if the pair is unfavorable. X indicates that the recovery of that pair was not observed in any of the activity classes, so no conclusion could be drawn as to its effect on activity.…”

Section: Discussionmentioning

confidence: 99%

“…In this respect, the structural information content of the e and g positions is less critical than that of the hydrophobic residues that make up the core of the dimer interface. For example, although leucine is not required at any specific d position, leucines must occupy at least two of four d positions to form a stable zipper (Hu et al, 1990).…”

Section: Individual Sequences As Anecdotal Evidencementioning

confidence: 99%

“…Structural properties of the GCN4 leucine zipper have also been studied in solution by two-dimensional NMR spectroscopy (Oas et al, 1990), amide proton exchange (Goodman & Kim, 1991), and biochemically using synthetic leucine zipper peptides (O'Shea et al, 1989a). The GCN4 leucine zipper has also been amenable to genetic studies, both in the intact GCN4 protein (Struhl, 1989;Pu & Struhl, 1991) and in fusions to bacterial DNAbinding proteins (Hu et al, 1990). Figure 1 shows a cartoon representation of the GCN4 leucine zipper.…”

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confidence: 99%

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Probing the roles of residues at the e and g positions of the GCN4 leucine zipper by combinatorial mutagenesis

Newell

Tidor

et al. 1993

Protein Science

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Combinatorial mutagenesis with an alphabet limited to alanine, glutamic acid, lysine, and threonine was used to probe the role of interactions involving surface residues in stabilizing a short a-helical coiled coil. The residues at eight e and g positions in the leucine zipper of the Saccharomyces cerevisiue transcription factor GCN4 were randomized to these four residues in a h repressor-leucine zipper fusion protein, resulting in 65,536 possible residue combinations. Roughly three-fourths of these combinations allowed stable coiled-coil formation as assayed by DNA binding by the fusion protein. To understand the basis for the activity differences, functional and nonfunctional mutants were sequenced and statistical tests were applied to identify structure/function correlations. Helix-forming propensity and favorable intrasubunit and intersubunit charge-charge interactions were positively correlated with activity. These studies suggest that the identities of surface side chains at the e and g positions of coiled coils contribute modestly to stability; by comparison with previous work, however, the e and g positions are far less critical than residues at the a and d positions, which form the hydrophobic core of the dimer interface.

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Section: Discussionmentioning

confidence: 99%

Section: Individual Sequences As Anecdotal Evidencementioning

confidence: 99%

mentioning

confidence: 99%

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Probing the roles of residues at the e and g positions of the GCN4 leucine zipper by combinatorial mutagenesis

Newell

Tidor

et al. 1993

Protein Science

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“…In one of these chimeras, the N-terminal transactivating domain of p53 (amino acids 1 ± 80) has been replaced by the transactivating domain of the VP16 protein of herpes simplex virus to create VP16-p53. In another construct, the C-terminal portion of p53 (amino acids 344 ± 393) was replaced by the coiled-coil (CC) domain of the yeast GCN4 protein that mediates oligomerization (Ellenberger et al, 1992;Hu et al, 1990), yielding a protein termed p53-CC. Both chimeras activated transcription from the mdm2 promoter, as shown in Figure 2 and reported previously (Pietenpol et al, 1994), and both retained the ability to activate the PIG3 promoter with an e ciency comparable to wild type p53.…”

Section: Phosphorylation At Carboxyterminal Residues Does Not Affect mentioning

confidence: 99%

Tumor-derived mutations within the DNA-binding domain of p53 that phenotypically resemble the deletion of the proline-rich domain

et al. 2000

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The p53 tumor suppressor protein induces apoptosis through a mechanism that may involve the transcriptional activation of cellular genes, including the PIG3 gene. A p53 protein lacking the proline-rich region (p53D62-91) induces many p53-responsive genes but not PIG3. In parallel, this mutant induces growth arrest but not apoptosis. We show here that the replacement of the N-terminal (amino acids 1 ± 80) or C-terminal (amino acids 344 ± 393) domains of p53 with heterologous domains does not interfere with transcription from the PIG3 promoter, but these chimeras still require the proline-rich region for PIG3 activation. The p53-homolog p73b also activated the PIG3 promoter, but in contrast to p53, the proline-rich domain of p73b (residues 81 ± 113) was dispensable to induce the PIG3 promoter. Some tumor-derived p53-mutants, especially M246I, retained the ability to activate transcription of mdm2 but speci®cally failed to induce the PIG3 promoter, thus resembling p53D62-91. Further, p53D62-91 and p53M246I were defective for induction of apoptosis. Finally, p53D62-91 and p53M246I both showed reduced binding to the DNA of the PIG3 promoter and also to the DNA of the mdm2 and p21 promoters in vitro. Correspondingly, at low expression levels, p53D62-91 and p53M246I poorly activated the mdm2 promoter when compared to wild type p53. Our results suggest that the proline-rich domain of p53 a ects the ability of the central domain to bind DNA. Moreover, some tumor-derived mutations within the central DNA binding domain of p53 mimic the loss of the proline-rich domain.

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“…propensity of residues in other positions can also contribute to stability (Hu et al, 1990;O'Neil et al, 1990;Zhu et al, 1993). We became interested in the folding of coiled-coil peptides when we began to use synthetic peptides to investigate the structure and function of the N-terminal region of striated muscle tropomyosin.…”

Section: A C -M D a I K K K M Q M L K L D Y E N L L D R L E Q L E A Dmentioning

confidence: 99%

Conformational intermediates in the folding of a coiled‐coil model peptide of the N‐terminus of tropomyosin and αα‐tropomyosin

Greenfield¹,

Hitchcock‐DeGregori

1993

Protein Science

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Circular dichroism was used to study the folding of aa-tropomyosin and AcTM43, a 43-residue peptide designed t o serve as a model for the N-terminal domain of tropomyosin. The sequence of the peptide is AcMDAIKKKMQMLKLDVENLLDRLEQLEADLKALEDRYKQLEGGC. The peptide appeared to form a coiled coil at low temperatures (<25 "C) in buffers with physiological ionic strength and pH. The folding and unfolding of the peptide, however, were noncooperative. When C D spectra were examined as a function of temperature, the apparent degree of folding differed when the ellipticity was followed at 222, 208, and 280 nm. Deconvolution of the spectra suggested that at least three component curves contributed to the C D in the far UV. One component curve was similar to the CD spectrum of the coiled-coil a-helix of native aa-tropomyosin. The second curve resembled the spectrum of single-stranded short a-helical segments found in globular proteins. The third was similar to that of polypeptides in the random coil conformation. These results suggested that as the peptide folded, the a-helical content increased before most of the coiled coil was formed.When the CD spectrum of striated muscle aa-tropomyosin was examined as a function of temperature, the unfolding was also not totally cooperative. As the temperature was raised from 0 to 25 "C, there was a decrease in the coiled coil and an increase in the conventional a-helix type spectrum without formation of random coil.The major transition, occurring at 40 "C, was a cooperative transition characterized by the loss of all of the remaining coiled coil and a concomitant increase in random coil.

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Sequence Requirements for Coiled-Coils: Analysis with λ Repressor-GCN4 Leucine Zipper Fusions

Cited by 377 publications

References 28 publications

Probing the roles of residues at the e and g positions of the GCN4 leucine zipper by combinatorial mutagenesis

Probing the roles of residues at the e and g positions of the GCN4 leucine zipper by combinatorial mutagenesis

Tumor-derived mutations within the DNA-binding domain of p53 that phenotypically resemble the deletion of the proline-rich domain

Conformational intermediates in the folding of a coiled‐coil model peptide of the N‐terminus of tropomyosin and αα‐tropomyosin

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