LAUSR

Significance Intrinsically disordered proteins must frequently compete for binding to shared interaction hubs to perform their cellular functions. Here, we describe the mechanism by which two disordered proteins that regulate the transcriptional response to hypoxia compete for binding to the folded TAZ1 domain of the transcriptional coactivators CBP and p300. CITED2, a negative feedback regulator of HIF-1α, displaces HIF-1α from TAZ1 in a unidirectional, switch-like manner. Efficient competition for binding of the TAZ1 domain is highly dependent on the flexibility and multivalency of the HIF-1α and CITED2 activation domains. Differences in the strength of coupling of the CITED2 and HIF-1α binding motifs are key determinants of unidirectionality and underscore the role of multivalency in regulation of cellular processes by disordered proteins. Intrinsically disordered proteins must compete for binding to common regulatory targets to carry out their biological functions. Previously, we showed that the activation domains of two disordered proteins, the transcription factor HIF-1α and its negative regulator CITED2, function as a unidirectional, allosteric molecular switch to control transcription of critical adaptive genes under conditions of oxygen deprivation. These proteins achieve transcriptional control by competing for binding to the TAZ1 domain of the transcriptional coactivators CREB-binding protein (CBP) and p300 (CREB: cyclic-AMP response element binding protein). To characterize the mechanistic details behind this molecular switch, we used solution NMR spectroscopy and complementary biophysical methods to determine the contributions of individual binding motifs in CITED2 to the overall competition process. An N-terminal region of the CITED2 activation domain, which forms a helix when bound to TAZ1, plays a critical role in initiating competition with HIF-1α by enabling formation of a ternary complex in a process that is highly dependent on the dynamics and disorder of the competing partners. Two other conserved binding motifs in CITED2, the LPEL motif and an aromatic/hydrophobic motif that we term ϕC, function synergistically to enhance binding of CITED2 and inhibit rebinding of HIF-1α. The apparent unidirectionality of competition between HIF-1α and CITED2 is lost when one or more of these binding regions is altered by truncation or mutation of the CITED2 peptide. Our findings illustrate the complexity of molecular interactions involving disordered proteins containing multivalent interaction motifs and provide insight into the unique mechanisms by which disordered proteins compete for occupancy of common molecular targets within the cell.