Hypersensitive termination of the hypoxic response by a disordered protein switch

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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 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 genes4,5,6. 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 domain7,8,9. 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. Here we show that human CITED2 displaces HIF-1α by forming a transient ternary complex with TAZ1 and HIF-1α and competing for a shared binding site through 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 probably exemplifies a common strategy used by the cell to respond rapidly to environmental signals.

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Figure 1: HIF-1α and CITED2 bind to a partially overlapping surface of TAZ1.
Figure 2: CITED2 is an unexpectedly efficient competitor for TAZ1.
Figure 3: HIF-1α and CITED2 bind TAZ1 through both static and dynamic interactions.
Figure 4: The conserved LP(Q/E)L motif mediates competition between HIF-1α and CITED2.

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Acknowledgements

We thank M. Martinez-Yamout for expert assistance with protein preparation and construct design, G. Kroon for NMR support, and A. Deniz for fluorimeter access. This work was supported by grant CA096865 from the National Institutes of Health (P.E.W.) and the Skaggs Institute for Chemical Biology. R.B.B. was supported by a postdoctoral fellowship from the American Cancer Society (125343-PF-13-202-01-DMC).

Author information

R.B.B. performed the experiments. R.B.B., H.J.D. and P.E.W. designed experiments, analysed data, and wrote the manuscript.

Correspondence to Peter E. Wright.

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Extended data figures and tables

Extended Data Figure 1 Representative 1H–15N HSQC spectra of 15N-TAZ1, 15N-TAZ1 bound to HIF-1α, and 15N-TAZ1 bound to CITED2.

Superimposed 1H–15N HSQC spectra are shown for 15N-TAZ1 in black, 15N-TAZ1 bound to HIF-1α (residues 776–826) in orange, and 15N-TAZ1 bound to CITED2 (residues 216–269) in blue. Selected cross peaks are labelled with residue assignments. The tryptophan indole resonances are shown as an inset in the lower left corner.

Extended Data Figure 2 Determination of binding affinities for HIF-1α and CITED2 peptides by fluorescence anisotropy and bio-layer interferometry (Octet).

a, Fluorescence anisotropy data for titration of unlabelled HIF-1α peptide into a pre-formed complex of AlexaFluor-594-labelled HIF-1α peptide and unlabelled TAZ1. b, Fluorescence anisotropy data for titration of unlabelled CITED2 peptide into a pre-formed complex of AlexaFluor-594-labelled CITED2 peptide and unlabelled TAZ1. c, Fluorescence anisotropy data for titration of unlabelled CITED2(216–242) into a pre-formed complex of AlexaFluor-594-labelled CITED2 peptide and unlabelled TAZ1. d, Fluorescence anisotropy data for titration of unlabelled TAZ1 into AlexaFluor-488-labelled HIF-1α(796–826). Data shown represent the average (circles) and standard deviation (error bars) of three independent measurements (ad). e, Representative bio-layer interferometry (Octet) data for HIF-1α(776–826) binding to GST–TAZ1. Data are shown in blue for three concentrations of HIF-1α as marked. The red lines are the result of fitting the data globally to obtain a shared Kd value for the three concentrations shown. f, Representative bio-layer interferometry (Octet) data for CITED2(216–269) binding to GST–TAZ1. Data are shown in blue for three concentrations of CITED2 as marked. The red lines are the result of fitting the data globally to obtain a shared Kd value for the three concentrations shown. g, Tabulated Kd values for the HIF-1α and CITED2 peptides included in this study. ND, not determined. The reported Kd values are the average and standard deviation of the Kd values obtained from nonlinear least squares fitting of at least three independent sets of experimental data. Source data

Extended Data Figure 3 Monitoring HIF-1α and CITED2 competition for 15N-TAZ1 binding by NMR spectroscopy.

a, Full 1H–15N HSQC spectra from NMR competition experiments with HIF-1α and CITED2 transactivation domain peptides. Superimposed spectra are shown for 15N-TAZ1 in the presence of one molar equivalent of HIF-1α (black), one molar equivalent of CITED2 (cyan), and one molar equivalent of both HIF-1α and CITED2 peptides (red, with fewer contours displayed for visibility). The tryptophan indole amide resonances are shown as an inset in the lower left corner. b, Detailed view of selected 15N-TAZ1 resonances. The spectral region highlighted in b is marked by the dotted lines on the full spectra in a. The spectra are displayed as described for a.

Extended Data Figure 4 Monitoring CITED2 competition for TAZ1 binding by stopped-flow fluorescence.

a, Representative time-resolved fluorescence data for rapid mixing of unlabelled CITED2 peptide with AlexaFluor-488-labelled HIF-1α peptide in a pre-formed complex with unlabelled TAZ1 (complex concentration = 0.25 μM). The data shown are the average of 10 shots. The concentrations of CITED2 used in each experiment are indicated in the upper left corner of each graph. The red lines are fits to a single exponential function, and the residuals from fitting are shown below the graph of the data obtained at each concentration of CITED2. b, Concentration dependence of observed rates (kobs) from time-resolved fluorescence experiments monitoring CITED2 competition for TAZ1 binding, determined from stopped-flow fluorescence measurements. The data shown represent the average (circles) and standard deviation (error bars) of three independent measurements. The solid red line is the result of fitting to a linear function. Source data

Extended Data Figure 5 Monitoring HIF-1α competition for TAZ1 binding by fluorescence.

a, Representative time-resolved fluorescence data for mixing of unlabelled HIF-1α peptide with AlexaFluor-488-labelled HIF-1α peptide in a pre-formed complex with unlabelled TAZ1 (complex concentration = 0.25 μM). The data shown are the average of 3 independent measurements. The concentrations of HIF-1α peptide used in each experiment are indicated in the upper left corner of each graph. The red lines are fits to a single exponential function, and the residuals from fitting are shown below the graph of the data obtained at each concentration of HIF-1α. b, Concentration dependence of observed rates (kobs) from time-resolved fluorescence experiments monitoring HIF-1α competition for TAZ1 binding, determined by standard fluorescence intensity measurements. The data shown represent the average (circles) and standard deviation (error bars) of three independent measurements. The solid black line is the result of fitting to a linear function. Source data

Extended Data Figure 6 Binding of full-length and truncated HIF-1α and CITED2 transactivation domain peptides to 15N-TAZ1.

a, c, Representative regions from superimposed 1H–15N HSQC spectra of 15N-TAZ1 bound to HIF-1α and CITED2 peptides. In a, free 15N-TAZ1 is shown in grey, 15N-TAZ1 bound to HIF-1α(796–826) is shown in red, and 15N-TAZ1 bound to HIF-1α(776–826) is shown in black. In c, 15N-TAZ1 is free (grey), bound to CITED2(216–242) (red), and bound to CITED2(216–269) (black). b, Weighted average 1H–15N chemical shift changes (Δδav) for each 15N-TAZ1 residue upon addition of HIF-1α(776–826) (black) and HIF-1α(796–826) (red). d, Weighted average 1H–15N chemical shift changes (Δδav) for each 15N-TAZ1 residue upon addition of CITED2(216–269) (black) and CITED2(216–242) (red). Weighted average 1H–15N chemical shift changes were calculated as Δδav = ((ΔδH)2 + (ΔδN/5)2)1/2. Source data

Extended Data Figure 7 Monitoring HIF-1α and CITED2(216–242) competition for 15N-TAZ1 binding by NMR spectroscopy.

a, Full 1H–15N HSQC spectra from NMR competition experiments with HIF-1α peptide and CITED2(216–242). Superimposed spectra are shown for 15N-TAZ1 in the presence of one molar equivalent of HIF-1α peptide (black), five molar equivalents of CITED2(216–242) (green), and one molar equivalent of HIF-1α peptide plus one (gold), three (orange), and five (magenta) molar equivalents of CITED2(216–242). The tryptophan indole amide resonances are shown as an inset in the lower left corner. b, Detailed view of selected 15N-TAZ1 resonances. The spectral region highlighted in b is marked by the dotted lines on the full spectra in a. The spectra are displayed as described for a. c, Weighted average 1H–15N chemical shift changes (Δδav) for each 15N-TAZ1–HIF-1α residue upon addition of one (gold), three (orange), or five (magenta) molar equivalents of CITED2(216–242). Weighted average 1H–15N chemical shift changes were calculated as Δδav = ((ΔδH)2 + (ΔδN/5)2)1/2. Source data

Extended Data Figure 8 Monitoring CITED2 and HIF-1α(796–826) competition for 15N-TAZ1 binding by NMR spectroscopy.

a, Full 1H–15N HSQC spectra from NMR competition experiments with CITED2 peptide and HIF-1α(796–826). Superimposed spectra are shown for 15N-TAZ1 in the presence of one molar equivalent of CITED2 peptide (cyan), five molar equivalents of HIF-1α(796–826) (purple), and one molar equivalent of CITED2 peptide plus one (gold), three (orange), and five (magenta) molar equivalents of HIF-1α(796–826). The tryptophan indole amide resonances are shown as an inset in the lower left corner. b, Detailed view of selected 15N-TAZ1 resonances. The spectral region highlighted in b is marked by the dotted lines on the full spectra in a. The spectra are displayed as described for a. c, Weighted average 1H–15N chemical shift changes (Δδav) for each 15N-TAZ1–CITED2 residue upon addition of one (gold), three (orange), or five (magenta) molar equivalents of HIF-1α(796–826). Weighted average 1H–15N chemical shift changes were calculated as Δδav = ((ΔδH)2 + (ΔδN/5)2)1/2. Source data

Extended Data Figure 9 Location of spectral changes for 15N-TAZ1–HIF-1α upon titration with the CITED2(216–242) peptide.

15N-TAZ1–HIF-1α resonances that shift and/or broaden upon addition of an equimolar amount of CITED2(216–242) are mapped onto the structure of the TAZ1–HIF-1α complex as red spheres. TAZ1 (residues 340–439) is shown in grey and the HIF-1α transactivation domain is shown in orange (residues 776–826). The expected structure of the CITED2(216–242) peptide in complex with TAZ1 is shown in blue. Structural motifs of HIF-1α and CITED2 are labelled for reference.

Extended Data Figure 10 Structural differences in the TAZ1 domain of CBP upon binding HIF-1α and CITED2.

Superposition of the TAZ1 structures in complex with HIF-1α (orange) and CITED2 (blue). The structures of the bound HIF-1α and CITED2 peptides are omitted for clarity, and the TAZ1 helices are labelled for reference.

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Berlow, R., Dyson, H. & Wright, P. Hypersensitive termination of the hypoxic response by a disordered protein switch. Nature 543, 447–451 (2017) doi:10.1038/nature21705

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