(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 ¹H^N. No ¹H^N-peaks were observed for residues 101 and 102. (D) Combined chemical shift perturbations of ¹³C^α and ¹³C^β 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 ¹³C^α, ¹³C^β, ¹³C’, ¹⁵N, and ¹H^N 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.

(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.

(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.

(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).

(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.

(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.