Linker Length and Composition within Disordered Binding Motifs Modulates the Avidity and Reversibility of a Multivalent Protein Interaction Switch

Intrinsically disordered proteins that mediate the cellular transcriptional response to hypoxia play important roles in regulating oxygen stress genes. The feedback inhibitor CITED2 operates a unidirectional switch that efficiently terminates the hypoxic response by displacing the C-terminal activation domain of the hypoxia-inducible factor HIF-1α from its complex with the TAZ1 domain of the transcriptional coactivators CBP and p300. Unidirectionality of the switch arises from subtle allosteric conformational changes in TAZ1 and from differences in the strength of thermodynamic coupling between the TAZ1-binding motifs in the multivalent HIF-1α and CITED2 activation domains. To investigate the role of binding cooperativity, we mutated a linker sequence in the HIF-1α activation domain to alter the thermodynamic coupling between its TAZ1-binding motifs. Linker mutations that enhance the affinity of HIF-1α for TAZ1 enable the HIF-1α activation domain to compete more effectively with bound CITED2. The wide range of mutants, which include insertion, deletion, substitution, and scrambling of residues in the linker, provide insights into the molecular basis for the exquisite tuning of the hypoxic switch. The TAZ1 binding affinity and consequent CITED2 competition enhancement depends both on the flexibility of the linker sequence (particularly the presence of glycine residues) and the unfavorable electrostatic interactions of a highly conserved arginine side chain in the center of the linker with an electropositive surface of TAZ1. The conservation of the linker length and sequence in all vertebrates suggests strong evolutionary pressure to tune HIF-1α binding affinity to be sub-optimal, to ensure unidirectionality of the hypoxic switch.