Binding of NF?B Appears to Twist the Ankyrin Repeat Domain of I?B?.

ABSTRACT: Total internal reflection fluorescence-based single-molecule Förster resonance energy transfer (FRET) measurements were previously carried out on the ankyrin repeat domain (ARD) of I?B?, the temporally regulated inhibitor of canonical NF?B signaling. Under native conditions, most of the I?B? molecules showed stable, high FRET signals consistent with distances between the fluorophores estimated from the crystal structures of the NF?B(RelA/p50)-I?B? complex. Similar high FRET efficiencies were found when the I?B? molecules were either free or in complex with NF?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?B? molecules (0.67) than for the free I?B? molecules (0.74), apparently indicating that binding of NF?B(RelA/p50) stretches the ARD of I?B?. 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?B(RelA/p50) and I?B? encompasses only the dimerization domains. We also performed mass spectrometry-detected amide hydrogen/deuterium exchange (HDXMS) experiments on I?B? as well as I?B? bound to dimerization-domain-only constructs or full-length NF?B(RelA/p50). Although we expected the stretched I?B? 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?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?B? ARD induced by interactions of the I?B? proline-glutamate-serine-threonine-rich sequence with positively charged residues in the RelA NTD.

SUBMITTER: Trelle MB 

PROVIDER: S-EPMC4776026 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature