Hypersensitive termination of the hypoxic response by a disordered protein switch

Abstract

The cellular response to hypoxia is critical for cell survival and is fine-tuned to allow cells to recover from hypoxic stress and adapt to heterogeneous or fluctuating oxygen levels1,2. The hypoxic response is mediated by the α subunit of the transcription factor HIF-1 (HIF-1α)3, which interacts via its intrinsically disordered C-terminal transactivation domain with the TAZ1 (CH1) domain of the general transcriptional coactivators CBP and p300 to control transcription of critical adaptive genes4–6. One such gene is CITED2, a negative feedback regulator that attenuates HIF transcriptional activity by competing for TAZ1 binding through its own disordered transactivation domain7–9. Little is known about the molecular mechanism by which CITED2 displaces the tightly bound HIF-1α from their common cellular target. The HIF-1α and CITED2 transactivation domains bind to TAZ1 through helical motifs that flank a conserved LP(Q/E)L sequence that is essential for negative feedback regulation5,6,8,9. We show that CITED2 displaces HIF-1α by forming a transient ternary complex with TAZ1 and HIF-1α and competing for a shared binding site via its LPEL motif, thus promoting a conformational change in TAZ1 that increases the rate of HIF-1α dissociation. Through allosteric enhancement of HIF-1α release, CITED2 activates a highly responsive negative feedback circuit that rapidly and efficiently attenuates the hypoxic response, even at modest CITED2 concentrations. This hypersensitive regulatory switch is entirely dependent on the unique flexibility and binding properties of these intrinsically disordered proteins and exemplifies a likely common strategy used by the cell to respond rapidly to environmental signals.

Investigations of the Underlying Mechanisms of HIF-1α and CITED2 Binding to TAZ1

“…The lowest basin corresponds to CITED2 bound state (CB state, 0.00 kT), which means that TAZ1-CITED2 is the dominant state of all. This is consistent with the NMR results of ref 15 Fig. 2 The free energy surface at experimental temperature as a function of Q inter (TAZ1-HIF-1α) and Q inter (TAZ1-CITED2), as well as the main states on the free energy surface (HB1 is not a basin on free energy surface but an area near HB state with part of CITED2 bound).…”

“…The NMR experiments of Wright et al 15 observed that TAZ1-CITED2 complex is dominant in the TAZ1:HIF-1α:CITED2 solvation with 1:1:1 molar ratio. The fluorescence anisotropy competition experiments found that CITED2 exhibits an apparent K d of 0.2±0.1 nM to TAZ1-HIF-1α complex, while HIF-1α displaces TAZ1-bound CITED2 with a much higher apparent K d (0.9±0.1 µM) 15 . These experimental results indicate that CITED2 is extremely effective in displacing HIF-1α from the TAZ1-HIF-1α complex.…”

“…The possible mechanism for displacement of HIF-1α from its complex with TAZ1 by CITED2 15 was proposed that CITED2 binds to TAZ1-HIF-1α complex through its N-terminal region, displacing the dynamical and weakly interacting α helix of HIF-1α, then competing through an intramolecular process for binding to the LP(Q/E)L site. However, it is challenging to prove such replacing mechanism experimentally.…”

“…and HIF-1α as well as between TAZ1 and CITED2 were adapted according to the experimental dissociation constant (K d about 10 nM for both TAZ1-HIF-1α and TAZ1-CITED2 15 ). In our model, both TAZ1-HIF-1α…”

Allosteric control of a bacterial stress response system by an anti‐σ factor

“…Structural disorder is often advantageous for proteins that perform multiple roles, interact with numerous partners, or rapidly transition between conformational states (Wright and Dyson, 2015). In the case of HIF-1a, protein disorder is proposed to enable efficient protein partner switching even when cellular concentrations of competing partners are similar (Berlow et al, 2017). Whether unstructured regions of NepR enable partner switching between equimolar PhyR and r EcfG remains to be determined and requires additional experimentation.…”