A transcriptional activating region with two contrasting modes of protein interaction

Ansari, Aseem Z.; Reece, Richard J.; Ptashne, Mark

doi:10.1073/pnas.95.23.13543

Cited by 54 publications

(66 citation statements)

References 62 publications

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“…In the M118 and M121 derivatives, replacement of the tryptophan, leucine, or phenylalanine residues by alanine led to an approximately twofold decrease in activity but had little or no effect on function of the M122 motif. Such behavior is similar to that seen in some natural ADs where it has been difficult to assign important residues within the AD by mutagenesis of single residues (35,53). Together, the results suggest that many ADs, including our synthetic ADs, contain redundant sequence motifs that allow alternative modes of binding to ABDs.…”

Section: A Short Ad Sequence Motif With a Preference For Surroundingsupporting

confidence: 59%

A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface

Warfield

Tuttle

Pacheco

et al. 2014

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

173

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Although many transcription activators contact the same set of coactivator complexes, the mechanism and specificity of these interactions have been unclear. For example, do intrinsically disordered transcription activation domains (ADs) use sequence-specific motifs, or do ADs of seemingly different sequence have common properties that encode activation function? We find that the central activation domain (cAD) of the yeast activator Gcn4 functions through a short, conserved sequence-specific motif. Optimizing the residues surrounding this short motif by inserting additional hydrophobic residues creates very powerful ADs that bind the Mediator subunit Gal11/Med15 with high affinity via a "fuzzy" protein interface. In contrast to Gcn4, the activity of these synthetic ADs is not strongly dependent on any one residue of the AD, and this redundancy is similar to that of some natural ADs in which few if any sequence-specific residues have been identified. The additional hydrophobic residues in the synthetic ADs likely allow multiple faces of the AD helix to interact with the Gal11 activator-binding domain, effectively forming a fuzzier interface than that of the wild-type cAD.Mediator complex | protein NMR T ranscription activators are regulators of cell identity, cell growth, and the response to environmental conditions. These highly regulated factors contain one or more activation domains (ADs) that typically bind coactivators-the complexes that contact the transcription machinery and/or have chromatin modifying activity (1-5). AD-coactivator binding initiates a cascade of events leading to productive transcription including targeted chromatin remodeling and stimulation of both RNA polymerase II preinitiation complex formation and transcription elongation (6). Many broadly acting ADs bind several coactivators, allowing them to function at a wide range of promoters with different coactivator requirements (7)(8)(9)(10)(11)(12)(13)(14). The function of most tested ADs is conserved among eukaryotes (15, 16), even though some key activator targets are not conserved. For example, the herpes virus protein VP16 strongly activates transcription in both yeast and mammalian cells, although human Med25, a critical target of VP16 in humans, is not found in yeast (17,18). Thus, the ability to adapt to different coactivator targets is seemingly a key property of ADs.Defining what constitutes a functional AD has been difficult, because there is little apparent sequence similarity among different ADs. All structurally characterized eukaryotic ADs lack a stable 3D structure and are disordered in the absence of a coactivator target (19)(20)(21)(22)(23)(24)(25). Initial studies classified ADs based on enriched residues: acidic proline-, glutamine-, or serine-rich activators (26). However, these residue types later were found to be generally enriched in intrinsically disordered proteins and were termed "disorder-promoting residues" (27, 28).Attempts to define functional sequence motifs within ADs have led to ambiguous results. For many ...

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Section: A Short Ad Sequence Motif With a Preference For Surroundingsupporting

confidence: 59%

A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface

Warfield

Tuttle

Pacheco

et al. 2014

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

173

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“…of miniScGal4 for KlGal80 as compared with AD-22 is because of cooperativity resulting from dimerization of the Gal80 binding domains via the DNA binding domain-linked dimerization motif (12). Consistently, a Gst-KlGal4-AD fusion protein had a much higher affinity for NHGal80 than AD-22 (K D ϳ 15 nM; data not shown), suggesting that in this case the dimerization via the glutathione S-transferase moiety can substitute to a certain degree for the dimerization motif of Gal4 and facilitates cooperative binding to KlGal80 dimers.…”

Section: Klgal80mentioning

confidence: 99%

“…Gal4 is bound to its target genes in induced as well as noninduced cells (4 -6). Its C-terminal activation domain overlaps with the binding site for Gal80 (7)(8)(9)(10)(11)(12)(13), which in the absence of galactose binds to Gal4 and prevents recruitment of coactivators and the formation of the preinitiation complex at the promotors (14). Gal80 inhibition is relieved by a mechanism that requires direct interaction with Gal3 (15)(16)(17)(18), which is a sensor of intracellular galactose.…”

mentioning

confidence: 99%

The Galactose Switch in Kluyveromyces lactis Depends on Nuclear Competition between Gal4 and Gal1 for Gal80 Binding

Anders

Lilie

Franke

et al. 2006

Journal of Biological Chemistry

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The Gal4 protein represents a universally functional transcription activator, which in yeast is regulated by protein-protein interaction of its transcription activation domain with the inhibitor Gal80. Gal80 inhibition is relieved via galactose-mediated Gal80-Gal1-Gal3 interaction. The Gal4-Gal80-Gal1/3 regulatory module is conserved between Saccharomyces cerevisiae and Kluyveromyces lactis. Here we demonstrate that K. lactis Gal80 (KlGal80) is a nuclear protein independent of the Gal4 activity status, whereas KlGal1 is detected throughout the entire cell, which implies that KlGal80 and KlGal1 interact in the nucleus. Consistently KlGal1 accumulates in the nucleus upon KlGAL80 overexpression. Furthermore, we show that the KlGal80-KlGal1 interaction blocks the galactokinase activity of KlGal1 and is incompatible with KlGal80-KlGal4-AD interaction. Thus, we propose that dissociation of KlGal80 from the AD forms the basis of KlGal4 activation in K. lactis. Quantitation of the dissociation constants for the KlGal80 complexes gives a much lower affinity for KlGal1 as compared with Gal4. Mathematical modeling shows that with these affinities a switch based on competition between Gal1 and Gal4 for Gal80 binding is nevertheless efficient provided two monomeric Gal1 molecules interact with dimeric Gal80. Consistent with such a mechanism, analysis of the sedimentation behavior by analytical ultracentrifugation demonstrates the formation of a heterotetrameric KlGal80-KlGal1 complex of 2:2 stoichiometry.Regulation of transcription requires coupling of gene-specific transcription activators to regulatory signals. The galactose switch, which induces the synthesis of galactose metabolic enzymes in response to this sugar in the environment, serves as a textbook model of how such coupling can be achieved in a eukaryotic cell (for reviews see Refs.

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“…1B) (12,13,(24)(25)(26). Many of the KIX-binding TADs from activators such as pKID, MLL, and c-Myb are intrinsically disordered proteins that assume a helical structure only upon binding to interaction partners such as KIX (25,27,28). Conformational changes in both the TADs and KIX occur upon binding, and NMR solution studies have underscored that each TAD•KIX complex is conformationally unique (10,(12)(13)(14)25).…”

mentioning

confidence: 99%

Dissecting allosteric effects of activator–coactivator complexes using a covalent small molecule ligand

Wang

Lodge

Fierke³

et al. 2014

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

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Allosteric binding events play a critical role in the formation and stability of transcriptional activator-coactivator complexes, perhaps in part due to the often intrinsically disordered nature of one or more of the constituent partners. The kinase-inducible domain interacting (KIX) domain of the master coactivator CREB binding protein/ p300 is a conformationally dynamic domain that complexes with transcriptional activators at two discrete binding sites in allosteric communication. The complexation of KIX with the transcriptional activation domain of mixed-lineage leukemia protein leads to an enhancement of binding by the activation domain of CREB (phosphorylated kinase-inducible domain of CREB) to the second site. A transient kinetic analysis of the ternary complex formation aided by small molecule ligands that induce positive or negative cooperative binding reveals that positive cooperativity is largely governed by stabilization of the bound complex as indicated by a decrease in k off . Thus, this suggests the increased binding affinity for the second ligand is not due to an allosteric creation of a more favorable binding interface by the first ligand. This is consistent with data from us and from others indicating that the on rates of conformationally dynamic proteins approach the limits of diffusion. In contrast, negative cooperativity is manifested by alterations in both k on and k off , suggesting stabilization of the binary complex.IDP | protein-protein interaction P rotein-protein interactions (PPIs) underscore all cellular processes, and the mechanistic dissection of PPI networks is thus a high priority (1-4). A particular challenge is defining the mechanism of PPI formation between intrinsically disordered proteins (IDPs) where allosteric changes play a substantive role (5-7). Allosteric communication between binding sites is vital for the proper function of feedback and regulatory circuits in the cell (8, 9). For example, the conformationally dynamic kinase-inducible domain interacting (KIX) domain of the master transcriptional coactivator CREB binding protein (CBP) undergoes a structural shift and stabilization upon binding to a cognate ligand such as the intrinsically disordered transcriptional activation domain (TAD) of mixed-lineage leukemia protein (MLL) (Fig. 1A) (10). The interaction of a second ligand, phosphorylated kinase-inducible domain (pKID) of CREB, is enhanced (up to twofold) by the presence of MLL and several elegant studies have documented allosteric communication between the two binding sites (11-15). However, the relatively modest affinities (micromolar) of the native complexes and the aggregation propensities and the promiscuous binding profiles of the transcriptional activation domains have hampered kinetic dissection of this and other complexes (16,17).The coactivator CBP exists across metazoans (18)(19)(20) and is a transcription hub that interacts with numerous transcriptional activators, using several discrete domains (21,22). The KIX domain within CBP is a 90-residue motif ...

show abstract

A transcriptional activating region with two contrasting modes of protein interaction

Cited by 54 publications

References 62 publications

A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface

A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface

The Galactose Switch in Kluyveromyces lactis Depends on Nuclear Competition between Gal4 and Gal1 for Gal80 Binding

Dissecting allosteric effects of activator–coactivator complexes using a covalent small molecule ligand

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