Characterization of ERM transactivation domain binding to the ACID/PTOV domain of the Mediator subunit MED25

Abstract

The N-terminal acidic transactivation domain (TAD) of ERM/ETV5 (ERM38–68), a PEA3 group member of Ets-related transcription factors, directly interacts with the ACID/PTOV domain of the Mediator complex subunit MED25. Molecular details of this interaction were investigated using nuclear magnetic resonance (NMR) spectroscopy. The TAD is disordered in solution but has a propensity to adopt local transient secondary structure. We show that it folds upon binding to MED25 and that the resulting ERM–MED25 complex displays characteristics of a fuzzy complex. Mutational analysis further reveals that two aromatic residues in the ERM TAD (F47 and W57) are involved in the binding to MED25 and participate in the ability of ERM TAD to activate transcription. Mutation of a key residue Q451 in the VP16 H1 binding pocket of MED25 affects the binding of ERM. Furthermore, competition experiments show that ERM and VP16 H1 share a common binding interface on MED25. NMR data confirms the occupancy of this binding pocket by ERM TAD. Based on these experimental data, a structural model of a functional interaction is proposed. This study provides mechanistic insights into the Mediator–transactivator interactions.

ETV4 and AP1 Transcription Factors Form Multivalent Interactions with three Sites on the MED25 Activator-Interacting Domain

“…This value is comparable to previous measurements of the interaction between the conserved AD of ETV5 and MED25 by fluorescence polarization (580 ± 20 nM) and by isothermal calorimetry (540 ± 40 nM) (Fig. S2) [7, 32]. As the AD bound MED25 with an approximately hundred-fold weaker affinity than full-length ETV4, we surmised that additional regions within ETV4 also contribute to the interaction with MED25.…”

“…Notably, the predominantly disordered AD becomes more helical in the MED25-bound state and phenylalanine and tryptophan residues in the AD are critical for this interaction [7, 32]. Based on the robust sequence conservation between the ADs of ETV1, ETV4, and ETV5 (Fig.…”

“…S3a) and the conserved sequence in ETV5 becomes more helical when interacting with MED25 (Fig. S2) [7]. By analogy to the AD, we reasoned that the surface-exposed phenylalanines in H4 might also be important for interaction with MED25.…”

“…Acidic ADs, originally noted for an enrichment of negatively-charged and non-polar residues [5, 6], have an alternating pattern of negatively-charged/nonpolar and bulky hydrophobic/aromatic residues. Although usually disordered in isolation, ADs often become more helical when interacting with cofactors [7–10]. These sequence and structural characteristics are presumably the foundation of the ability of a single AD to interact with multiple partners as a flexible hydrophobic/aromatic interface that can be presented differently to diverse proteins [11–20].…”

“…ETS binding motifs, in association with motifs recognized by JUN and FOS transcription factors (AP1 factors), are a hallmark of RAS/MAPK-responsive gene expression [31]. Previously it was demonstrated that the N-terminal AD of ETV5 binds to the ac tivator i nteracting d omain (ACID) of Mediator subunit 25 (MED25) [7, 32]. However, ~ 10% of prostate cancers frequently harbor truncations of ETV1, ETV4, or ETV5 that lack this AD due to chromosomal rearrangements [33, 34].…”