Predicted disorder-to-order transition mutations in IκBα disrupt function

Area of Science:

• Molecular Biology
• Protein Structure and Function
• Transcription Regulation

Background:

• IκBα inhibits the transcription factor NFκB by binding to its dimerization domain.
• The ankyrin repeat domain (ARD) of IκBα contains an intrinsically disordered region (AR5-6).
• Previous studies showed mutations towards consensus ankyrin repeats created a 'prefolded' mutant.

Purpose of the Study:

• To investigate if consensus mutations solely order the AR5-6 region of IκBα.
• To explore the functional consequences of altering the intrinsic disorder of IκBα's ARD.

Main Methods:

• Utilized PONDR VL-XT to predict mutations (D → O) altering intrinsic disorder.
• Assessed protein ordering using amide H/D exchange and circular dichroism (CD).
• Measured NFκB-DNA dissociation efficiency using stopped-flow fluorescence.

Main Results:

• Two mutations, E282W and P261F, were predicted to increase order in the AR5-6 region.
• E282W showed increased order via CD and amide exchange; P261F did not.
• Both D → O mutants exhibited reduced efficiency in dissociating NFκB from DNA.

Conclusions:

• Altering intrinsic disorder in IκBα's ARD, even towards a more ordered state, can impair its function.
• The intrinsically disordered AR5-6 region of IκBα is functionally important for NFκB regulation.
• Protein disorder plays a critical role in the inhibitory mechanism of IκBα on NFκB.