Recognition of the disordered p53 transactivation domain by the transcriptional adapter zinc finger domains of CREB-binding protein

Abstract

An important component of the activity of p53 as a tumor suppressor is its interaction with the transcriptional coactivators cyclic-AMP response element-binding protein (CREB)-binding protein (CBP) and p300, which activate transcription of p53-regulated stress response genes and stabilize p53 against ubiquitin-mediated degradation. The highest affinity interactions are between the intrinsically disordered N-terminal transactivation domain (TAD) of p53 and the TAZ1 and TAZ2 domains of CBP/p300. The NMR spectra of simple binary complexes of the TAZ1 and TAZ2 domains with the p53TAD suffer from exchange broadening, but innovations in construct design and isotopic labeling have enabled us to obtain high-resolution structures using fusion proteins, uniformly labeled in the case of the TAZ2-p53TAD fusion and segmentally labeled through transintein splicing for the TAZ1-p53TAD fusion. The p53TAD is bipartite, with two interaction motifs, termed AD1 and AD2, which fold to form short amphipathic helices upon binding to TAZ1 and TAZ2 whereas intervening regions of the p53TAD remain flexible. Both the AD1 and AD2 motifs bind to hydrophobic surfaces of the TAZ domains, with AD2 making more extensive hydrophobic contacts consistent with its greater contribution to the binding affinity. Binding of AD1 and AD2 is synergistic, and structural studies performed with isolated motifs can be misleading. The present structures of the fulllength p53TAD complexes demonstrate the versatility of the interactions available to an intrinsically disordered domain containing bipartite interaction motifs and provide valuable insights into the structural basis of the affinity changes that occur upon stressrelated posttranslational modification.intrinsically disordered protein | binding motif | transcriptional coactivator | intein | segmental labeling

Recognition and targeting mechanisms by chaperones in flagellum assembly and operation

“…Fusion proteins increase the effective concentration, and thus the population, of the complex and at the same time shift the kinetics of complex formation toward the slow-exchange regime. This strategy has been used extensively to determine structures of protein complexes by NMR (38). NMR analysis and comparison of the spectra of the FliT Δα4 −FliD C fusion construct with the spectra of FliT−FliD C show that the molecular interface and the structural properties are essentially identical in the two complexes (Fig.…”

p53 Proteoforms and Intrinsic Disorder: An Illustration of the Protein Structure–Function Continuum Concept

“…The next two images in Figure 6 show the NMR structures of specifically designed fusions of the N-terminal transactivation domain (TAD) of p53 (residues 1–61 or 2–61) with the TAZ1 or TAZ2 domains of the CREB-binding protein, CBP (Figure 6B,C, respectively) [123]. Similar to the TAD-p300 TAZ2 situation, structures of the CPB TAZ1 and TAZ2 domains are shown for just one illustrative member of the corresponding TAZ1 or TAZ2 conformational ensemble; this is because the structures of these domains in the fusion constructs are well-defined and similar to the structures in their un-bound states [123].…”

“…Similar to the TAD-p300 TAZ2 situation, structures of the CPB TAZ1 and TAZ2 domains are shown for just one illustrative member of the corresponding TAZ1 or TAZ2 conformational ensemble; this is because the structures of these domains in the fusion constructs are well-defined and similar to the structures in their un-bound states [123]. Figure 6B,C show that the p53 TAD region binds to different targets using a bipartite mode [123], for which TAD evolved to have two interaction motifs, AD1 (residues 18–26) and AD2 (residues 44–54). Although these regions are mostly disordered in the unbound state, upon binding to TAZ1 or TAZ2 they are able to fold into short amphipathic α-helices.…”

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

“…Several examples, including our findings with ETV4, suggest that multivalency is one route for forming specific, higher-affinity AD-cofactor interactions. For example, two ADs from p53 form bivalent interactions with both the TAZ1 and TAZ2 domains of CBP by binding to opposite faces of these domains [21]. The p53 ADs also interact with at least two other CBP domains [24], so the tetrameric form of p53 has been predicted to form a highly multivalent interaction with full-length CBP.…”

“…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]. However, higher affinity for a particular factor, and thus specificity, can be accomplished through the use of multiple ADs [21–24]. …”