FC0069
p65 (RelA)  -  CREB Binding Protein (CBP) TAZ1

Biological function
The interaction of the RelA subunit of NF-κB with the general co-activator protein CBP/p300 is vital for RelA-dependent gene transcription, which plays role in the immune response, cell survival, differentiation, and proliferation.

Domain organization/sequence features
RelA interacts with CBP/p300 in a bipartite manner—the RHR domain (19-306) of RelA contacts the KIX domain of CBP/p300 and the TAD of RelA (426-549) contacts the TAZ1 (also known as the CH1) domain of CBP/p300. KIX interactions require RelA- phosphorylation at a well-conserved Ser276 residue. Although both the RHR:KIX and TAD:TAZ1 interactions contribute to transcriptional activation of RelA-dependent genes, phosphorylation of Ser276 is considered critical for the RelA:CBP/p300 interaction.

Structural evidence
In the RelA–TA2:TAZ1 structure, the N- and C-terminal regions of the RelA–TA2 fragment used for structural analysis (Lys425–Thr433 and Ser482–Pro490, respectively) are dynamically disordered, with zero or negative [¹H]- ¹⁵N heteronuclear NOEs. RelA–TA2, essentially the Leu434–Val481 region, entirely wraps around TAZ1 in a predominantly extended conformation by docking itself through its well-conserved bulky hydrophobic residues into the interlinked hydrophobic grooves of TAZ1. The C-terminal region (Glu471–Asn477) of RelA–TA2 folds into one short helix α_c that is anchored to the hydrophobic pocket of TAZ1. The N-terminal region (Leu434–Leu439) of RelA–TA2 also forms a short helix α_N, which is docked into the shallow hydrophobic groove formed at the junction of α₁ and α₄ of TAZ1. The α_N helix is dynamically disordered with only about 30% of the helical population, as calculated from the magnitude of the C- alpha and carbonyl secondary chemical shifts, and the small value of the [¹H]-¹⁵N heteronuclear NOE confirms that this region is highly flexible on the nanosecond time scale.

Biochemical evidence
The RelA–TAD:CBP–TAZ1 interaction was originaly mapped to RelA(477–504) region. Only a weak interaction between this RelA fragment and TAZ1 as compared to the whole RelA–TAD [RelA (425–549)] and therefore scanned the entire TAD for TAZ1 binding using GST pull-down assays. A RelA construct spanning residues Lys425–Pro490 (in the TA2 region) emerged as the minimal fragment with maximal affinity to TAZ1. Increasing the NaCl concentration from 50 to 150 mM weakened RelA–TA2:TAZ1 binding by 4-fold, consistent with a significant electrostatic contribution to binding from the complementary charges on TAZ1 and the RelA–TA2 region. The Leu449Ala and Leu465Ala substitutions each led to a 7-fold decrease in the TAZ1 binding affinity. RelA TA2 interacts with TAZ1 via multiple ψψXXψXXψ sequence motifs. N-terminal truncation to remove residues Lys425– His440, thereby eliminating the entire α_N helix, decreases the affinity by only 1.6-fold. Deletion of residues Ser486–Pro490 from the disordered C- terminal end of RelA had no effect on TAZ1 binding. However, further truncation to remove Val481–His485 greatly impaired binding by eliminating hydrophobic contacts. RelA residues beyond Val481 do not contribute to the interaction.

Structure/Mechanism
The hydrophobic interactions are complemented by electrostatic interactions between the highly acidic RelA–TA2 region and the strongly electropositive surface of TAZ1. The region from Asp444–Asp448 in RelA–TA2 is particularly acidic, with four out of five residues being negatively charged, and passes through a deep cleft in the TAZ1 surface that is lined with basic residues. While RelA becomes more ordered upon interaction with TAZ1, only limited, highly localized secondary structure is formed. Contacts between the N-terminal part of the RelA–TA2 and TAZ1 are dynamic, contributing little to binding affinity, and are an example of what is frequently termed a ’fuzzy’ interaction. The hydrophobic contacts are complemented by electrostatic interactions involving the many acidic residues in the RelA–TA2 region. Disruption of any of the hydrophobic contacts leads to a significant decrease in binding affinity toward TAZ1.

Mechanism category
tethering

Posttranslational modification
The RelA–TA2 region also contains a well-conserved Ser467 (Ser468 in human RelA), which is a known phosphorylation site, which can be phosphorylated by GSK3b, IKKe, or IKKb and plays a critical role in transcriptional regulation of NF-κB dependent genes. A phosphomimetic mutant, RelA(Ser467Asp), binds TAZ1 with about 1.4-fold higher affinity than wild type RelA–TA2 and exhibits the same dependence on ionic strength, suggesting an enhanced but still nonspecific electrostatic interaction.

Significance
Fuzziness of the RelA TA2-TAZ1 complex exerts dynamic control of RelA-CBP/p300 interactions, which enables transcription of a series of NF-κB target genes independent of RelA(Ser276) phosphorylation.