Unexpected specificity within dynamic transcriptional protein-protein complexes

Abstract

A key functional event in eukaryotic gene activation is the formation of dynamic protein-protein interaction networks between transcriptional activators and transcriptional coactivators. Seemingly incongruent with the tight regulation of transcription, many biochemical and biophysical studies suggest that activators use nonspecific hydrophobic and/or electrostatic interactions to bind to coactivators, with few if any specific contacts. Here a mechanistic dissection of a set of representative dynamic activator•coactivator complexes, comprised of the ETV/PEA3 family of activators and the coactivator Med25, reveals a different molecular recognition model. The data demonstrate that small sequence variations within an activator family significantly redistribute the conformational ensemble of the complex while not affecting overall affinity, and distal residues within the activator-not often considered as contributing to binding-play a key role in mediating conformational redistribution. The ETV/PEA3•Med25 ensembles are directed by specific contacts between the disordered activator and the Med25 interface, which is facilitated by structural shifts of the coactivator binding surface. Taken together, these data highlight the critical role coactivator plasticity plays in recognition of disordered activators and indicate that molecular recognition models of disordered proteins must consider the ability of the binding partners to mediate specificity.

Keywords: ETV/PEA3; Med25; coactivator; protein–protein interactions; transcriptional activator.

Fig. 1.

Recognition models of activator function. (Left) Transcriptional activators regulate gene activity via protein–protein interactions with coactivators. (Right) Comparison of nonspecific (3, 11) and specific (this work) models of activator•coactivator recognition. Nonspecific models propose that the TADs of unique activators bind the ABDs of coactivators via nonspecific intermolecular interactions, forming complexes without fixed orientation or structure via “sequence-independent” recognition (11). Rounded boxes in the TADs represent the binding motif.

Fig. 2.

ETV/PEA3 activators differentially engage with Med25. (A) Alignment of ETV/PEA3 family activation domains. The helix denotes the residues that undergo coupled folding and binding with Med25, as determined by NMR chemical shift analysis (30). (B) Mechanism of binding of ETV/PEA3 activators to Med25, determined here for ETV1 and ETV4, and previously for ETV5 (32). The range of exchange rates between analogous steps for ETV/PEA3 TADs are shown. (C) Table of relevant binding parameters for each ETV/PEA3 TAD, including the equilibrium affinity, exchange rates between C1 and C2 (k_ex,12) and between C2 and C3 (k_ex,23), and equilibrium populations of each state. All values represent the average of three to four biological replicates, and the error is the SD. (D) Equilibrium populations of the three ETV/PEA3•Med25 conformations scaled relative to the diameter of the black circle. SDs of the values are shown as the dark gray outer circle. (E) Overlay of the Ile Cδ region of the ¹H,¹³C-HSQC of Med25 in complex with 1.1 equivalents unlabeled ETV1 (blue), ETV4 (orange), or ETV5 (gray). Peaks that have chemical shift differences between complexes are labeled. Note: I449 and I541 form a single overlapped peak for the ETV1 and ETV5 complexes. Full spectra are in the supporting information. (F) Chemical shift differences of Med25 methyl resonances between ETV1 and ETV4 (orange) and ETV1 and ETV5 (gray). (G) Chemical shift perturbations induced by binding of ETV/PEA3 activators plotted on the structure of Med25 (PDB ID 2XNF) (34). Yellow = 0.040 to 0.080 ppm, red > 0.080 ppm. Cyan circles highlight general distinctions in perturbation patterns. Several residues with chemical shift differences >0.030 ppm between the ETV1•Med25 (or ETV5•Med25) and ETV4•Med25 complexes are labeled.

Fig. 3.

Variable residues in the disordered N terminus and the helical binding region mediate differences in ETV/PEA3•Med25 conformational behavior. (A) Alignment of ETV1 and ETV4 activators with regions that were selected for mutational analysis boxed. Bolded residues are conserved between ETV1 and ETV5, but not ETV4. Regions/residues that affected the conformational ensemble are color coded to ETV1 (blue) or ETV4 (orange). Effects of the Gln/His residues in the variable motif were also tested but did not affect conformational populations and are thus omitted in B and C for clarity. Populations of conformational states in B and C are scaled relative to the diameter of the black circle. (B) Results from kinetics experiments of mutant TADs, for native (Left) and nonnative (Right) combinations of variable N termini and helical binding regions. Variants were made based on the ETV4 sequence. The data shown are the average across all of the variants tested from each group, with the error (dark gray outer circle) representing the SD. (C) Results from kinetics experiments with ETV1^∆Nt (Top) and ETV4^∆Nt (Bottom). (D) Average equilibrium K_d values of variants tested. *Conformer was undetectable in kinetics experiments (see SI Appendix, Discussion of Kinetic Data Analysis for further details).

Fig. 4.

ETV/PEA3 variable regions engage in unique interactions with the Med25 surface. Effects of conservative mutations in the (A) helical binding region and (B) N termini are plotted on the structure of Med25. Yellow = 0.015 to 0.030 ppm, red ≥ 0.030 ppm. Residues discussed in the text are labeled. Gray spheres denote residues that undergo identical perturbations in both parent and mutant complexes. Residues chosen for mutation are bolded and labeled in the alignment. (C) Chemical shift perturbations of 150 µM ETV1 (Top) and ETV4 (Bottom) TADs in the absence (blue and orange, respectively) and presence (light blue and maroon, respectively) of 280 µM unlabeled Med25. TADs were selectively ¹⁵N labeled at the positions noted. Small secondary peaks in free ETV4 spectra were observed and likely arose from isomerization of the two tandem Pro residues in the N-terminal region.

Fig. 5.

Structural shifts in the Med25 ABD accompany the conformational changes of ETV/PEA3•Med25 complexes. (A) Examples of Med25 resonances undergoing shifts toward the unbound position (black) upon ETV4^F60L (cyan) mutation. (B) Comparison of chemical shift perturbations from binding of ETV4 (Left) and ETV4^F60L (Right) demonstrate that conformational changes involve the binding site and allosteric regions of Med25. Residues shown in A are labeled on the structures. Yellow = 0.040 to 0.080 ppm, red > 0.080 ppm.

Fig. 6.

Specific recognition model emerging from this work. Activator binding domains recognize a diversity of activators via conformational plasticity and unique specific intermolecular interactions. Rounded boxes in the TADs represent the “canonical” recognition motifs. Orange dashes indicate interactions made by disordered regions outside the recognition motifs.