Comparison of backbone dynamics of the p50 dimerization domain of NFκB in the homodimeric transcription factor NFκB1 and in its heterodimeric complex with RelA (p65)

Abstract

The nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) transcription factors play a critical role in human immune response. The family includes homodimers and heterodimers of five component proteins, which mediate different transcriptional responses and bind preferentially to different DNA sequences. Crystal structures of DNA complexes show that the dimers of the Rel-homology regions are structurally very similar. Differing DNA sequence preference together with structural similarity suggests that the dimers may differ in their dynamics. In this study, we present the first near-complete 15 N, 13 C α/β , and H N backbone resonance assignments of two dimers of the dimerization domain (DD) of the NFκB1 (p50) protein (residues 241-351): the homodimer of two p50 domains and a heterodimer of the p50 DD with the p65 DD. As expected, the two dimers behave very similarly, with chemical shift differences between them largely concentrated in the dimer interface and attributable to specific differences in the amino acid sequences of p50 and p65. A comparison of the picosecond-nanosecond dynamics of the homo-and heterodimers also shows that the environment of p50 is similar, with an overall slightly reduced correlation time for the homodimer compared to the heterodimer, consistent with its slightly smaller molecular weight. These results demonstrate that NMR spectroscopy can be used to explore subtle changes in structure and dynamics that have the potential to give insights into differences in specificity that can be exploited in the design of new therapeutic agents. K E Y W O R D S{ 1 H} -15 N NOE, NFκB1, p50 dimerization domain, protein dynamics, R 1 /R 2 relaxation

Domain Stability Regulated through the Dimer Interface Controls the Formation Kinetics of a Specific NF-κB Dimer

“…The chemical shift perturbation (CSP), the weighted average difference in chemical shifts of RelA, in its homo- and heterodimer forms is significant at the dimer interface (Figure E, Figure S2). Similar results were seen earlier for p50 in homo- and heterodimer forms . Further, secondary structure propensity (SSP) software predicted the secondary structure of the different DDs using the backbone chemical shifts (Figure E and Figure S3).…”

“…Similar results were seen earlier for p50 in homo-and heterodimer forms. 29 Further, secondary structure propensity (SSP) software 14 predicted the secondary structure of the different DDs using the backbone chemical shifts (Figure 1E and Figure S3). The ΔSSP values, the SSP score difference between the respective homo-and the heterodimer forms, revealed only a little change for p50 with no significant change observed for that of RelA.…”

Toll-like receptor 3 (TLR3) regulation mechanisms and roles in antiviral innate immune responses

“…NF-κB is a central transcription factor crucial to innate and adaptive immunities, cell proliferation, apoptosis, and the stress response (Cartwright et al, 2016). The p50/p65 heterodimer of NF-κB activates the expression of IFN-β and proinflammatory cytokines (Kawai and Akira, 2007;Kohl et al, 2019). The K63linked polyubiquitin chains of RIP-1 and TRAF6 can recruit the transforming growth factor-β (TGF-β)activated kinase 1 (TAK1)-TAK1-binding protein 2 (TAB2) -TAB3 complex via the ubiquitin-binding activity of TAB2/3, causing TAK1 autophosphorylation and activation (Yang and Shu, 2020).…”

Slowly Relaxing Local Structure Analysis of 15N Relaxation from the Proteins p50 and Human Neutrophil Gelatinase-Associated Lipocalin: New Insights into the Dynamic Structure of β-Barrel Proteins

“…However, DNA sequence preference and DNA-binding affinity differ. Under the premise that this could stem from differences in dynamics, Kohl et al . studied in solution the p50 protein in the two environments p50-p50 and p50-p65.…”

“…The chain segments 20–40 and 70–85, which represent the monomer interfaces in the two dimers, show differences between the corresponding average amide ( 1 H, 15 N) chemical shifts . Pertinent residues, in particular of p50-p65 (meaning here and in the forthcoming “p50 within the scope of the heterodimer p50-p65”; likewise, p50-p50 means “p50 within the scope of the homodimer p50-p50”), show line-broadening in the 15 N dimension of the 15 N– 1 H HSQC spectrum.…”