Binding of NFκB Appears to Twist the Ankyrin Repeat Domain of IκBα

Morten Beck Trelle; Kristen M Ramsey; Taehyung C Lee; Weihua Zheng; Jorge Lamboy; Peter G Wolynes; Ashok Deniz; Elizabeth A Komives

doi:10.1016/j.bpj.2016.01.001

Binding of NFκB Appears to Twist the Ankyrin Repeat Domain of IκBα

Biophys J. 2016 Feb 23;110(4):887-95. doi: 10.1016/j.bpj.2016.01.001.

Authors

Morten Beck Trelle¹, Kristen M Ramsey¹, Taehyung C Lee², Weihua Zheng³, Jorge Lamboy¹, Peter G Wolynes³, Ashok Deniz², Elizabeth A Komives⁴

Affiliations

¹ Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California.
² Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California.
³ Department of Chemistry, Rice University, Houston, Texas.
⁴ Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California. Electronic address: ekomives@ucsd.edu.

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 IκBα, the temporally regulated inhibitor of canonical NFκB signaling. Under native conditions, most of the IκBα molecules showed stable, high FRET signals consistent with distances between the fluorophores estimated from the crystal structures of the NFκB(RelA/p50)-IκBα complex. Similar high FRET efficiencies were found when the IκBα molecules were either free or in complex with NFκB(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 IκBα molecules (0.67) than for the free IκBα molecules (0.74), apparently indicating that binding of NFκB(RelA/p50) stretches the ARD of IκBα. 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 NFκB(RelA/p50) and IκBα encompasses only the dimerization domains. We also performed mass spectrometry-detected amide hydrogen/deuterium exchange (HDXMS) experiments on IκBα as well as IκBα bound to dimerization-domain-only constructs or full-length NFκB(RelA/p50). Although we expected the stretched IκBα to have regions with increased exchange, instead the HDXMS experiments showed decreases in exchange in AR3 and AR6 that were more pronounced when the NFκB 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 IκBα ARD induced by interactions of the IκBα proline-glutamate-serine-threonine-rich sequence with positively charged residues in the RelA NTD.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Amides / chemistry
Ankyrin Repeat*
Molecular Dynamics Simulation*
NF-KappaB Inhibitor alpha / chemistry*
NF-KappaB Inhibitor alpha / metabolism*
NF-kappa B / chemistry
NF-kappa B / metabolism*
Protein Binding

Substances

Amides
NF-kappa B
NF-KappaB Inhibitor alpha

Abstract

Publication types

MeSH terms

Substances

Grants and funding