FC0061
GCN4  -  Med15

Biological function
Yeast Gcn4 contains tandem acidic ADs that act in conjunction with the coactivators Mediator, SAGA, and SWI/SNF and directly regulates >70 genes involved in diverse processes such as the response to metabolic stress and autophagy.

Domain organization/sequence features
The two Gcn4 ADs (residues 1–100 and 101–134) are unrelated in sequence apart from their acidic character.

Structural evidence
Gal11 activator-binding domain 1 has a four-helix fold with a small shallow hydrophobic cleft at its center. In the bound complex, eight residues of Gcn4 adopt a helical conformation allowing three Gcn4 aromatic/aliphatic residues to insert into the Gal11 cleft. Placement of spin-labels near the cAD termini (residues 104 and 133) indicate that regions outside the cAD helix do not make close contact with activator binding domain of Gal11 (ABD1, 158-238). The protein-protein interface is dynamic and surprisingly simple, involving only hydrophobic interactions. This allows Gcn4 to bind Gal11 in multiple conformations and orientations, an example of a ’fuzzy complex’ where the Gcn4- Gal11 interface cannot be described by a single conformation.

Biochemical evidence
Binding affinities were measured using isothermal titration calorimetry. Wild-type cAD binds ABD1 with a K_D of 10.1 μM. As expected from NMR analysis, Ala substitutions at Gcn4 120 or 124 decreased binding affinity for ABD1 by 5.6- and 6.5-fold, respectively. Alanine substitutions were made at Gcn4-cAD W120 and F124, residues making direct contact with Gal11-ABD1. Gal11 W196 positioned in the center of the activator-binding cleft, and Gal11 residues M213 and T200, located at the edge of the binding cleft. Wild- type cAD binds ABD1 with a K_D of 10.1 μM. As expected from NMR analysis, Ala substitutions at Gcn4 120 or 124 decreased binding affinity for ABD1 by 5.6- and 6.5-fold, respectively.

Structure/Mechanism
The multiple weak Gcn4- Gal11 interactions additively contribute to overall transcription activation and illustrate an important principal of Gal11 recruitment by Gcn4; Gcn4 binds Gal11 not by a single high affinity, high specificity interaction but rather by multiple low affinity interactions. Three Gcn4 residues, W120, L123, and F124, interact with hydrophobic residues in a shallow ABD1 cleft. Although the cAD and ABD1 have opposite electrostatic surface potentials, structural and mutational analysis demonstrate that there are no specific polar or ionic interactions at the interface. This likely contributes to the second major difference from previous activator-coactivator structures: the cAD helix binds to ABD1 in multiple orientations. This binding is in fast exchange on the NMR time scale, suggesting that Gcn4 can rapidly sample multiple Gal11 activator-binding domains as a mechanism to recruit Mediator to the enhancer/promoter region.

Mechanism category
Tethering

Significance
Fuzziness of the complex explains why the Gal11 activator-binding domains act additively to increase activated transcription and why multimerization of transcription factor DNA binding sites often greatly stimulates transcription.