Universal two‐point interaction of mediator KIX with 9aaTAD activation domains

Abstract

The nine-amino-acid activation domain (9aaTAD) is defined by a short amino acid pattern including two hydrophobic regions (positions p3-4 and p6-7). The KIX domain of mediator transcription CBP interacts with the 9aaTAD domains of transcription factors MLL, E2A, NF-kB, and p53. In this study, we analyzed the 9aaTADs-KIX interactions by nuclear magnetic resonance. The positions of three KIX helixes α1-α2-α3 are influenced by sterically-associated hydrophobic I611, L628, and I660 residues that are exposed to solvent. The positions of two rigid KIX helixes α1 and α2 generate conditions for structural folding in the flexible KIX-L12-G2 regions localized between them. The three KIX I611, L628, and I660 residues interact with two 9aaTAD hydrophobic residues in positions p3 and p4 and together build a hydrophobic core of five residues (5R). Numerous residues in 9aaTAD position p3 and p4 could provide this interaction. Following binding of the 9aaTAD to KIX, the hydrophobic I611, L628, and I660 residues are no longer exposed to solvent and their position changes inside the hydrophobic core together with position of KIX α1-α2-α3 helixes. The new positions of the KIX helixes α1 and α2 allow the KIX-L12-G2 enhanced formation. The second hydrophobic region of the 9aaTAD (positions p6 and p7) provides strong binding with the KIX-L12-G2 region. Similarly, multiple residues in 9aaTAD position p6 and p7 could provide this interaction. In conclusion, both 9aaTAD regions p3, p4 and p6, p7 provide co-operative and highly universal binding to mediator KIX. The hydrophobic core 5R formation allows new positions of the rigid KIX α-helixes and enables the enhanced formation of the KIX-L12-G2 region. This contributes to free energy and is the key for the KIX-9aaTAD binding. Therefore, the 9aaTAD-KIX interactions do not operate under the rigid key-and-lock mechanism what explains the 9aaTAD natural variability.

The NFkB activation domain is 14-amino-acid-long variant of the 9aaTAD

“…Recently, we showed that the KIX two-point interaction with MLL requires two distinct interacting modules within the 9aaTADs. The primary interaction is mediated by MLL residues p3-4 [2]. This interaction enables generation of the joint hydrophobic core 5R inside of the MLL-KIX complexes.…”

“…The basic 9aaTAD requirements includes the presence of two hydrophobic regions in positions p3-4 and p6-7 [2]. From structural models, the essential secondary interaction of MLL and E2A with KIX is mediated by their hydrophobic residues in positions p6-7 [1].…”

“…Our aim is to identify transactivation domains in transcription factors to study their interaction with general mediators of transcription [1,2]. The Nine amino acid Transactivation Domains (9aaTAD), which represent most of the identified activation domains, have the capability to activate transcription as small peptides (9 to 14 amino acids long) [3][4][5][6][7].…”

“…The KIX domain, of CBP mediator of transcription, interacts with the 9aaTAD domains of MLL, E2A, NFkB and p53 [1,2,[12][13][14]. The KIX helixes α1-α2-α3 sterically associate hydrophobic residues α1-I611, α2-L628, and α3-I660.…”

“…Variety of heterogeneous 9aaTADs enable formation of the hydrophobic core 5R to promote KIX enhanced folding. The KIX hydrophobic interaction, which could initiate the complex formation, thus fits to vast variations of the 9aaTADs [2]. In this study, we investigated modularity of the 9aaTAD sub-domains in phylogenetically unrelated transcription factors; i) a transcription factor p65 protein from NF-kB family [15], related to immune response and ii) a transcription factor SREBP1, a regulator of sterol metabolism, containing two distinct 9aaTAD domains rising from different splicing variants (Piskacek et al 2019).…”