posted on 2020-03-06, 16:35authored byMadita Wolter, Domenico Lentini Santo, Petr Herman, Alice Ballone, Federica Centorrino, Tomas Obsil, Christian Ottmann
Inflammatory responses mediated by
the transcription factor nuclear
factor kappa-light-chain enhancer of activated B cells (NF-κB)
play key roles in immunity, autoimmune diseases, and cancer. NF-κB
is directly regulated through protein–protein interactions,
including those with IκB and 14-3-3 proteins. These two important
regulatory proteins have been reported to interact with each other,
although little is known about this interaction. We analyzed the inhibitor
of nuclear factor kappa B α (IκBα)/14-3-3σ
interaction via a peptide/protein-based approach. Structural data
were acquired via X-ray crystallography, while binding affinities
were measured with fluorescence polarization assays and time-resolved
tryptophan fluorescence. A high-resolution crystal structure (1.13
Å) of the uncommon 14-3-3 interaction motif of IκBα
(IκBαpS63) in a complex with 14-3-3σ was evaluated.
This motif harbors a tryptophan that makes this crystal structure
the first one with such a residue visible in the electron density
at that position. We used this tryptophan to determine the binding
affinity of the unlabeled IκBα peptide to 14-3-3 via tryptophan
fluorescence decay measurements.