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. 2011 Dec 23;44(6):942-53.
doi: 10.1016/j.molcel.2011.11.008.

The acidic transcription activator Gcn4 binds the mediator subunit Gal11/Med15 using a simple protein interface forming a fuzzy complex

Peter S Brzovic  1 , Clemens C Heikaus, Leonid Kisselev, Robert Vernon, Eric Herbig, Derek Pacheco, Linda Warfield, Peter Littlefield, David Baker, Rachel E Klevit, Steven Hahn
Affiliations

The acidic transcription activator Gcn4 binds the mediator subunit Gal11/Med15 using a simple protein interface forming a fuzzy complex

Peter S Brzovic et al. Mol Cell. .

Abstract

The structural basis for binding of the acidic transcription activator Gcn4 and one activator-binding domain of the Mediator subunit Gal11/Med15 was examined by NMR. 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. 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. Gcn4 uses a similar mechanism to bind two other unrelated activator-binding domains. Functional studies in yeast show the importance of residues at the protein interface, define the minimal requirements for a functional activator, and suggest a mechanism by which activators bind to multiple unrelated targets.

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Figures

Fig 1
Fig 1. Gcn4 cAD Forms a Short α-helix Upon Binding to Gal11 (also see Fig S1)
(A) Position of the two Gcn4 ADs and three Gal11 domains (ABD-1, 2, 3) that bind Gcn4. Conserved regions of Gal11 are shown in grey. (B) 1H,15N-HSQC spectra of 0.3 mM 15N-labeled Gcn4 (101–134) in the absence (black) and presence of 0.125 (red), 0.25 (green), 0.5 (blue), 1 (yellow), 2 (magenta), or 3 equivalents (cyan) of Gal11 ABD1 (158–238). Amides with the largest chemical shift perturbations (residues 121–125) are labeled and highlighted by arrows. (C) Backbone amide chemical shift perturbations of Gcn4 upon addition of 3 equivalents of ABD1. The formula [(ΔδH)2-(ΔδN/5)2]1/2 was used to calculate the combined chemical shifts of 15N and 1HN. No 1HN-peaks were observed for residues 101 and 102. (D) Combined chemical shift perturbations of 13Cα and 13Cβ of Gcn4 (101–134) bound to ABD1 in reference to free Gcn4. The location of the cAD α-helix is indicated. (E) Probability for the formation of α-helical secondary structure elements predicted by CS-Rosetta (Shen et al., 2008) for Gcn4 (101–134) in the absence (red) and presence (black) of ABD1. NMR chemical shift assignments of 13Cα, 13Cβ, 13C’, 15N, and 1HN for free and bound Gcn4 were input and used for the generation of 100 9-residue fragments starting at each residue. The percentage of fragments showing helical secondary structure at each position is shown.
Fig 2
Fig 2. Solution Structure of Gal11-ABD1
(A) NMR ensemble of 20 low-energy Gal11-ABD1 structures. Average pairwise RMSDs for the ordered backbone atoms of residues 163–187, 191–193, 195–232 is 0.9Å. (B) Ribbon representation of the Gal11-ABD1. (C) Orientation from (A) was rotated ~90 degrees about the x-axis to highlight the residues from α1, α3, and α4 that form the ABD1 hydrophobic cleft (shown in stick representation with carbons in blue, oxygens in red, and sulfur in yellow. (D) The surface electrostatic potential of ABD1 oriented as in (B). Red, negatively polarized; blue, positive; white, non-polar.
Fig 3
Fig 3. NMR spectra of the Gal11-ABD1/Gcn4-cAD complex and effects of Gcn4 paramagnetic spin labels (also see Fig S2)
(A) Titration of 13C-labeled Gcn4-cAD with unlabeled ABD1. The portion of the 13C-HSQC shows the chemical shift perturbations of the T121 and L113 methyl groups upon binding to ABD1. The trajectories of these groups were used to assign resonances that arise from intermolecular interactions observed in NOESY spectra. (B) Portions of the 13C-edited, 13C-filtered NOESY spectrum showing crosspeaks that arise from M213 and V170. Labeled crosspeaks could be unambiguously assigned to specific Gcn4 residues. M213 and V170 are located at opposite ends of the ABD1 hydrophobic cleft (Fig 2B), yet show crosspeaks to the same Gcn4 residues, suggesting that Gcn4 binds to ABD1 in multiple orientations. (C,D) Paramagnetic spin labels were incorporated at four different positions of the cAD (104, 117, 126, 133), where positions 117 and 126 flank the nascent Gcn4-cAD helix. Observed intensity perturbations in ABD1 upon complex formation with Gcn4 spin-labeled at positions 126 and 117 are shown. Gal11 (gray ribbon), with strongly affected residues (intensity decrease > 80% relative to reference spectrum) in red and significantly affected residues (intensity decrease between 50–80%) in orange.
Fig 4
Fig 4. Models of the ABD1-cAD Complex Derived from NOE and Spin-Labeling Data
(A) Ribbon representations for the ensemble of HADDOCK generated structures for Gcn4-cAD (magenta) binding to the ABD1 (gray). Gcn4 residues 101–112 and 131–134 have been removed for clarity and L113 (cyan) marks the N-terminus. (B) Three different orientations of the Gcn4 peptide are evident in the ensemble of structures depicted in (A). (C) Positions of key Gcn4 side chains W120 (orange), L123 (green), and F124 (magenta) relative to ABD1 (gray ribbon) are shown from the ensemble in (A). ABD1 residues V170 and M213 are labeled. The different modes of binding bring W120, L123, and F124 in proximity to both residues, consistent with observations derived from the (13C-edited, 13C-filtered)-NOESY (see Fig 3B).
Fig 5
Fig 5. Effect of Mutations in the cAD-ABD1 Interface on Transcription Activation in vivo (also see Fig S4)
(A) Cells with the indicated Gcn4 mutations and Gal11 Δ418–696 were induced for 90 min with SM (sulfometuron methyl; except where noted, -SM) to induce starvation. mRNA was extracted and quantitated by RT qPCR. Error bars represent the SEM. (B) Sequence of the cAD. Residues with the largest chemical shift perturbations (Fig 1B) are red; acidic residues outside of this region are blue. The arrow indicates the position of the α-helix formed upon binding Gal11. * indicates the position of alanine substitutions at hydrophobic residues and brackets indicate the positions of acidic residues substituted with Ala. (C) Schematic of the Gal11 derivative used for mutagenesis of ABD1 where black bars represent regions deleted from Gal11. Conserved regions of Gal11 are shown by shaded boxes. (D) Cell grown as in (A) and mRNA quantitated by RT qPCR. Error bars represent the SEM.
Fig 6
Fig 6. Gcn4 Uses Similar Mechanisms for Recognition of Taf12 and Gal11 ABD3 (also see Fig S5)
(A) 1H,15N-HSQC spectra of 0.3 mM 15N-labeled Gcn4 (101–134) in the absence of (black) and presence of 0.125 (red), 0.25 (green), 0.5 (blue), 1 (yellow), 2 (magenta), or 3 equivalents (cyan) of Taf12 (29–259). (B) 1H,15N-HSQC spectra of 0.3 mM 15N-labeled Gcn4 (101–134) in the absence of (black) and presence of 0.1 (red) or 0.5 equivalents (blue) of Gal11 (496–651). In each spectrum, amides with the largest chemical shift perturbations (residues 120–125) are labeled and highlighted by arrows. A complete titration could not be performed due to the limited solubility of ABD3.
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