Recruitment of the transcriptional machinery through GAL11P: structure and interactions of the GAL4 dimerization domain

Abstract

The GAL4 dimerization domain (GAL4-dd) is a powerful transcriptional activator when tethered to DNA in a cell bearing a mutant of the GAL11 protein, named GAL11P. GAL11P (like GAL11) is a component of the RNA-polymerase II holoenzyme. Nuclear magnetic resonance (NMR) studies of GAL4-dd revealed an elongated dimer structure with C(2) symmetry containing three helices that mediate dimerization via coiled-coil contacts. The two loops between the three coiled coils form mobile bulges causing a variation of twist angles between the helix pairs. Chemical shift perturbation analysis mapped the GAL11P-binding site to the C-terminal helix alpha3 and the loop between alpha1 and alpha2. One GAL11P monomer binds to one GAL4-dd dimer rendering the dimer asymmetric and implying an extreme negative cooperativity mechanism. Alanine-scanning mutagenesis of GAL4-dd showed that the NMR-derived GAL11P-binding face is crucial for the novel transcriptional activating function of the GAL4-dd on GAL11P interaction. The binding of GAL4 to GAL11P, although an artificial interaction, represents a unique structural motif for an activating region capable of binding to a single target to effect gene expression.

Figure 1

Functional domains of GAL4 and primary sequence of the GAL4 dimerization domain (GAL4_50–106). (A) GAL4 contains several separable functional regions, including a DNA-binding domain, a dimerization domain, and three activating regions (the activating region between 94–106 is nonfunctional in yeast; Simkovich 1996). The C-terminal region of GAL4 (851–881) recognizes the GAL80 inhibitory protein (not shown). (B) Partial amino acid sequences of the GAL4-dd and LAC9. Conserved residues are shaded in yellow. The double mutation on GAL4-dd (87 and 90) are colored red (bold). The secondary structure elements, α1, α2, and α3 are indicated by blue cylinders. (C) Temperature dependence of the unfolding of GAL4-dd. The denaturation and renaturation curves are shown in pink and blue, respectively. The CD spectra were taken in a sample 0.5 mg/mL in 50 mM phosphate buffer at pH 7.2 in an AVIV instrument, model 62DS (AVIV Associates) at 222 nm.

Figure 1

Functional domains of GAL4 and primary sequence of the GAL4 dimerization domain (GAL4_50–106). (A) GAL4 contains several separable functional regions, including a DNA-binding domain, a dimerization domain, and three activating regions (the activating region between 94–106 is nonfunctional in yeast; Simkovich 1996). The C-terminal region of GAL4 (851–881) recognizes the GAL80 inhibitory protein (not shown). (B) Partial amino acid sequences of the GAL4-dd and LAC9. Conserved residues are shaded in yellow. The double mutation on GAL4-dd (87 and 90) are colored red (bold). The secondary structure elements, α1, α2, and α3 are indicated by blue cylinders. (C) Temperature dependence of the unfolding of GAL4-dd. The denaturation and renaturation curves are shown in pink and blue, respectively. The CD spectra were taken in a sample 0.5 mg/mL in 50 mM phosphate buffer at pH 7.2 in an AVIV instrument, model 62DS (AVIV Associates) at 222 nm.

Figure 2

Three-dimensional structure of the GAL4-dd. (A) A stereoview of the ribbon diagram of a representative structure of the GAL4-dd showing residues H53 to D100. The N and C termini are labeled N and C, respectively. The two monomers are colored differently. Unassigned residues P73 to L77 within the loop connecting α1 and α2 are colored orange. This figure was generated with the program MOLMOL (Koradi et al. 1996). (B) A stereoview of the backbone atoms from residues L54 to D100 of the 17 lowest energy structures with helices α1, α2, and α3 (blue) simultaneously superimposed. The average backbone RMSD of α1, α2, and α3 to the mean structure is 3.8 Å. (C) A stereoview of the 17 lowest energy structures whereby the backbone atoms from residues H53 to D100 of each of the three helices were separately superimposed. For clarity, the loops regions are displayed for only one structure. The backbone atoms of the unassigned P73 to L77 segment are colored in orange as in A. The average backbone RMSD of α1, α2, and α3 to the mean structure is 0.45, 1.7, and 0.75 Å, respectively. (D) Ensemble of the 17 structures aligned by superimposing the backbone heavy atoms of helix α1.

Figure 3

Dimerization interface within α1 and α3 of GAL4 (50–106). (A) A stereoview of the superimposed backbone atoms within α1 for the 17 lowest energy structures. The side chains of V57, L61, and L64 forming intermonomer contacts are displayed in different colors for each monomer. (B) A stereoview of the superimposed backbone atoms within α3 for the 17 lowest energy structures. The side chains of I89, L93 forming intermonomer contacts and F97 are shown in different colors for each monomer. (C) Helical wheel representation of α1 with hydrophobic, polar, and charged residues displayed in red, green. and blue, respectively. (D) Helical wheel representation of α3. Same color code for the residues as C but glycine in black.

Figure 4

¹⁵N relaxation rates and heteronuclear NOE enhancements for GAL4-dd. (A) Relaxation rates for the nitrogen longitudinal magnetization, R_N(N_z). (B) Relaxation rate for the nitrogen transverse magnetization, R_N(N_x,y). (C) Heteronuclear NOE enhancements, XNOE = (I_sat-I_eq)/I_eq, where I_sat and I_eq represent the steady state heteronuclear NOE and equilibrium value, respectively. All relaxation parameters where measured at 400 MHz with a 1-mM ¹⁵N isotopically labeled sample at pH 7.4 and at 35°C. Relaxation rates and XNOE values for residues with unassigned amide resonances and P73 are represented as zero values in all graphs. The signal of residue F68 in the XNOE experiment was too weak to allow analysis. The helices are indicated below the residue number by blue rectangles. L61 and L64 within the α1 are indicated by arrows. The numbers above each data point of the XNOE plot indicate the number of intermonomers NOEs determined for each residue as described (Walters et al. 1997a).

Figure 5

Alanine scanning mutagenesis experiments identify GAL4 residues crucial for the activation in vivo and electrophoretic mobility shift assays identify GAL4 mutants that bind GAL11P_263–352. (A) In vivo activation assay. (Top) Design of the experiment: Two LexA-binding sites are located 50 bp upstream of the GAL1 promoter TATA box. The expression of the reporter gene (lacZ) is regulated by the ability of LexA derivatives to interact with GAL11P and recruit the transcriptional machinery to this promoter. (Bottom) Summary of the alanine scanning mutagenesis of GAL4, which identify residues that are essential for activation by GAL11P in vivo. The levels of activation of each mutant were normalized against the wild-type LexA(1–202) + Gal4(50–97) derivative. Ala 91 in the wild-type GAL4 was replaced with a leucine. (B) Control for the gel-shift experiments. GAL4 (1–97) binds to its cognate site whereas neither GST–GAL11 nor GST–GAL11P binds this oligonucleotide. As shown in lanes 11–16, GST–GAL11P but not GST–GAL11 binds the GAL4–DNA complex, causing a further decrease in the electrophoretic mobility of the olignucleotide–protein complex (supershift). GST–GAL11_263–352 was at the final concentration of 1.6, 3.2, and 4.8 μM in lanes 2–4 and 11–13, respectively and so was GST–GAL11P_263–352 in lanes 5–7 and 14–16. GAL4 (30 nM) was present in lanes 9 and 11–16. GAL4 (15 nM) was added to lane 8, and 45 nM GAL4 was added to lane 10. (C–G) Each of the alanine substituted derivatives of GAL4 (1–97) binds to the cognate DNA-binding site, and their GST–GAL11P_263–352 binding properties are approximated by the intensity of the supershift. In this experiment, 3 μM GST–GAL11P_263–352 was added to each GAL4–DNA complex. GAL4 mutants were at ∼30-nM concentrations; at this concentration, each derivative saturably binds the labeled DNA probe.

Figure 6

GAL11P binds the GAL4-dd dimer with slow exchange kinetics, and as a monomer. (A) Functional domains and partial amino acid sequence of GAL11P_261–352. The N342V mutation is labeled in red. Position 342 is surrounded by a relatively high number of hydrophobic and basic residues. (B) NMR titration of ¹⁵N-labeled GAL4-dd (50–106) with unlabeled GAL11P (261–352). (¹H,¹⁵N) HSQC spectra of 1.0 mM GAL4 (50–106) dimer alone (left), in the presence of 0.5 mM GAL11P (middle), and in the presence of 1.0 mM GAL11P (right). The sample contained 50 mM phosphate buffer at pH 6.8, 100 mM NaCl. Cross peaks are labeled in the spectra of free GAL4 (left). The signal of G95 that splits in two asymmetric peaks on GAL11P binding is marked with a box at the top of the spectra. Several other signals, primarily from helix α3 disappear in the presence of GAL11P. The assignment of these signals is highlighted in the left panel. Numerous new signals of lower intensity appear in the right panel manifesting widespread asymmetry of GAL4-dd in the complex with GAL11P.

Figure 7

GAL4 residues critical for binding GAL11P and transcriptional activation in vivo map to the hydrophobic α1–α2 loop, α3 and beyond. (A) Molecular surface representation of one representative structure of GAL4-dd. Residues experiencing large and small or no changes in their chemical shifts on addition of GAL11P are displayed in red and blue, respectively. Unassigned residues are displayed in gray. The mutations in the double mutant version of GAL4-dd, Q87R, and K90E, are labeled. (B) Residues that critically affect transcriptional activation in vivo when mutated to alanine are displayed in yellow onto the same molecular structure as in Fig. 7A. A ribbon diagram of the GAL4-dd, with the same color code, with the side chains atoms of the residues sensitive to alanine mutagenesis within α1, α2, and α3 is displayed in the right panel. This figure was generated with the program MOLMOL (Koradi et al. 1996).