Linker Length and Composition within Disordered Binding Motifs modulates the Avidity and Reversibility of a...

Area of Science:

• Molecular Biology
• Biochemistry
• Cellular Biology

Background:

• Intrinsically disordered proteins control gene expression during hypoxia.
• CITED2 acts as a feedback inhibitor, terminating the hypoxic response by displacing HIF-1α from CBP/p300 coactivators.
• The unidirectional nature of this switch depends on allosteric changes and thermodynamic coupling.

Purpose of the Study:

• To investigate the role of binding cooperativity in the HIF-1α/TAZ1 interaction.
• To understand how linker mutations affect the thermodynamic coupling between HIF-1α's TAZ1-binding motifs.
• To elucidate the molecular basis for the precise tuning of the hypoxic switch.

Main Methods:

• Mutagenesis of the HIF-1α activation domain linker sequence.
• Native-gel electrophoresis to assess HIF-1α/TAZ1 binding affinity.
• Fluorescence anisotropy competition assays and Nuclear Magnetic Resonance (NMR) spectroscopy to measure CITED2 competition.

Main Results:

• Specific linker mutations enhanced HIF-1α's affinity for the TAZ1 domain.
• Mutant HIF-1α variants competed more effectively with CITED2 for TAZ1 binding than wild-type.
• Linker flexibility (glycine presence) and electrostatic interactions (arginine side chain) are critical for TAZ1 affinity and CITED2 competition.

Conclusions:

• The study provides molecular insights into the regulation of the hypoxic transcriptional response.
• Linker sequence modifications in HIF-1α can modulate its interaction with coactivators, affecting the hypoxic switch.
• Flexibility and electrostatic interactions within the HIF-1α linker are key determinants of TAZ1 binding and competitive displacement by CITED2.