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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 levels. The hypoxic response is mediated by the ?-subunit of the transcription factor HIF-1 (HIF-1?), which interacts through its intrinsically disordered C-terminal transactivation domain with the TAZ1 (also known as CH1) domain of the general transcriptional coactivators CBP and p300 to control the transcription of critical adaptive genes. One such gene encodes CITED2, a negative feedback regulator that attenuates HIF-1 transcriptional activity by competing for TAZ1 binding through its own disordered transactivation domain. Little is known about the molecular mechanism by which CITED2 displaces the tightly bound HIF-1? from their common cellular target. The HIF-1? 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 regulation. Here we show that human CITED2 displaces HIF-1? by forming a transient ternary complex with TAZ1 and HIF-1? and competing for a shared binding site through its LPEL motif, thus promoting a conformational change in TAZ1 that increases the rate of HIF-1? dissociation. Through allosteric enhancement of HIF-1? 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 probably exemplifies a common strategy used by the cell to respond rapidly to environmental signals.

SUBMITTER: Berlow RB

PROVIDER: S-EPMC5375031 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Hypersensitive termination of the hypoxic response by a disordered protein switch.

Berlow Rebecca B RB Dyson H Jane HJ Wright Peter E PE

Nature 20170308 7645

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 levels. The hypoxic response is mediated by the α-subunit of the transcription factor HIF-1 (HIF-1α), which interacts through its intrinsically disordered C-terminal transactivation domain with the TAZ1 (also known as CH1) domain of the general transcriptional coactivators CBP and p300 to control the transcription of criti ...[more]

PMID: 28273070

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Project description:Introduction: Protein conformational switches are often constructed by fusing an input domain, which recognizes a target ligand, to an output domain that establishes a biological response. Prior designs have employed binding-induced folding of the input domain to drive a conformational change in the output domain. Adding a second input domain can in principle harvest additional binding energy for performing useful work. It is not obvious, however, how to fuse two binding domains to a single output domain such that folding of both binding domains combine to effect conformational change in the output domain. Methods: Here, we converted the ribonuclease barnase (Bn) to a switchable enzyme by duplicating a C-terminal portion of its sequence and appending it to its N-terminus, thereby establishing a native fold (OFF state) and a circularly permuted fold (ON state) that competed for the shared core in a mutually exclusive fashion. Two copies of FK506 binding protein (FKBP), both made unstable by the V24A mutation and one that had been circularly permuted, were inserted into the engineered barnase at the junctions between the shared and duplicated sequences. Results: Rapamycin-induced folding of FK506 binding protein stretched and unfolded the native fold of barnase via the mutually exclusive folding effect, and rapamycin-induced folding of permuted FK506 binding protein stabilized the permuted fold of barnase by the loop-closure entropy principle. These folding events complemented each other to turn on RNase function. The cytotoxic switching mechanism was validated in yeast and human cells, and in vitro with purified protein. Discussion: Thermodynamic modeling and experimental results revealed that the dual action of loop-closure entropy and mutually exclusive folding is analogous to an engine transmission in which loop-closure entropy acts as the low gear, providing efficient switching at low ligand concentrations, and mutually exclusive folding acts as the high gear to allow the switch to reach its maximum response at high ligand concentrations.

Dataset Information

Hypersensitive termination of the hypoxic response by a disordered protein switch.

Publications

Hypersensitive termination of the hypoxic response by a disordered protein switch.

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