Structural and functional insights into IκB-α/HIV-1 Tat interaction

Protein–protein interactions play fundamental roles in physiological and pathological biological processes. The characterization of the structural determinants of protein–protein recognition represents an important step for the development of molecular entities able to modulate these interactions. We have recently found that IκB-α (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) blocks the HIV-1 expression and replication in a NF-κB-independent manner by directly binding to the virus-encoded Tat transactivator. Here, we report the evaluation of the entity of binding of IκB-α to Tat through in vitro Surface Plasmon Resonance assay. Moreover, by designing and characterizing a set of peptides of the C-terminus region of IκB-α, we show that the peptide corresponding to the IκB-α sequence 262–287 was able to bind to Tat with high affinity (300 nM). The characterization of a number of IκB-α-based peptides also provided insights into their intrinsic folding properties. These findings have been corroborated by mutagenesis studies on the full-length IκB-α, which unveil that different IκB-α residues are involved in NF-κB or Tat recognition.

► We found that IκB-α avidly binds Tat with a nanomolar KD.
► We restricted IκB-α to 262–287 region equally effective to bind Tat.
► We analyzed conformational determinants of the recognition mechanism.
► We assessed that diverse IκB-α residues are involved in NF-κB or Tat recognition.