Binding of NF kappa B Appears to Twist the Ankyrin Repeat Domain of I kappa B alpha

MB Trelle and KM Ramsey and TC Lee and WH Zheng and J Lamboy and PG Wolynes and A Deniz and EA Komives, BIOPHYSICAL JOURNAL, 110, 887-895 (2016).

DOI: 10.1016/j.bpj.2016.01.001

Total internal reflection fluorescence-based single-molecule Forster resonance energy transfer (FRET) measurements were previously carried out on the ankyrin repeat domain (ARD) of I kappa B alpha, the temporally regulated inhibitor of canonical NF kappa B signaling. Under native conditions, most of the I kappa B alpha molecules showed stable, high FRET signals consistent with distances between the fluorophores estimated from the crystal structures of the NF kappa B(RelA/p50)-I kappa B alpha complex. Similar high FRET efficiencies were found when the I kappa B alpha molecules were either free or in complex with NF kappa B(RelA/p50), and were interpreted as being consistent with the crystallographically observed ARD structure. An exception to this was observed when the donor and acceptor fluorophores were attached in AR3 (residue 166) and AR6 (residue 262). Surprisingly, the FRET efficiency was lower for the bound I kappa B alpha molecules (0.67) than for the free I kappa B alpha molecules (0.74), apparently indicating that binding of NF kappa B(RelA/p50) stretches the ARD of I kappa B alpha. Here, we conducted confocal-based single-molecule FRET studies to investigate this phenomenon in greater detail. The results not only recapitulated the apparent stretching of the ARD but also showed that the effect was more pronounced when the N-terminal domains (NTDs) of both RelA and p50 were present, even though the interface between NF kappa B(RelA/p50) and I kappa B alpha encompasses only the dimerization domains. We also performed mass spectrometry-detected amide hydrogen/deuterium exchange (HDXMS) experiments on I kappa B alpha as well as I kappa B alpha bound to dimerization-domainonly constructs or full-length NF kappa B(RelA/p50). Although we expected the stretched I kappa B alpha to have regions with increased exchange, instead the HDXMS experiments showed decreases in exchange in AR3 and AR6 that were more pronounced when the NF kappa B NTDs were present. Simulations of the interaction recapitulated the increased distance between residues 166 and 262, and also provide a plausible mechanism for a twisting of the I kappa B alpha ARD induced by interactions of the I kappa B alpha proline-glutamate-serine-threonine-rich sequence with positively charged residues in the RelA NTD.

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