Abstract
Eukaryotic transcription initiation is mediated by interactions between transcriptional activators and the mediator coactivator complex. Molecular interaction of p53 transcription factor with mediator complex subunit 25 (MED25) is essential for its target gene transcription. In this study, we characterized the molecular interaction between p53 transactivation domain (p53TAD) and activator interaction domain (ACID) of MED25 using nuclear magnetic resonance (NMR) spectroscopy. The NMR chemical shift perturbation and isothermal titration calorimetry (ITC) data showed that p53TAD interacted with MED25 ACID mainly through the p53TAD2 sequence motif. Taken together with the mutagenesis data, the refined structural model of MED25 ACID/p53TAD2 peptide complex showed that an amphipathic α-helix of p53TAD2 peptide bound an elongated hydrophobic groove of MED25 ACID. Furthermore, our results revealed the highly conserved mechanism of MED25 interaction with intrinsically unfolded acidic TADs from the transcriptional activators p53, ERM (Ets-related molecule), and herpes simplex virus protein 16 (VP16).
Highlights
The tumor suppressor p53 is a transcription factor that plays essential roles in various cellular processes, including cell cycle arrest, DNA repair, and apoptosis in response to stress signals such as DNA damage, hypoxia, oncogene activation, and viral infection [1,2,3,4]
We characterized the molecular interaction between p53 transactivation domain (p53TAD) (1–73) and mediator complex subunit 25 (MED25) activator interaction domain (ACID) by nuclear magnetic resonance (NMR) spectroscopy
Based on the previously reported assignments of free p53TAD [23], the backbone resonances for MED25 ACID-bound p53TAD were assigned, and the intensity ratio was plotted against each residue of p53TAD during the NMR titration (Figure 1D)
Summary
The tumor suppressor p53 is a transcription factor that plays essential roles in various cellular processes, including cell cycle arrest, DNA repair, and apoptosis in response to stress signals such as DNA damage, hypoxia, oncogene activation, and viral infection [1,2,3,4]. P53 mediates its cellular functions through transcriptional activation of target genes encoding pro-apoptotic proteins such as B-cell lymphoma 2-associated X protein (BAX) and p53 upregulated modulator of apoptosis (PUMA) and cyclin kinase inhibitors like p21. P53 contains distinct structural and functional domains: a N-terminal transactivation domain (TAD) (residues 1–73), a central DNA-binding domain (DBD) (residues 102–325), an oligomerization domain (OD) (residues 325–355), and a C-terminal regulatory domain (CTD) (residues 356–393) (Figure 1A). P53 binds enhancer regions of target genes and assembles the transcriptional machinery such as RNA polymerase II (Pol. II) general transcription factors (GTFs), and mediator, mainly through its DBD and TAD. The mediator coactivator complex acts as a central integrator of transcription [5]. The mediator coactivator complex interacts with a variety of transcriptional activators and recruits RNA Pol II to induce target gene transcription.
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