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

Abstract

Intrinsically disordered proteins that mediate the cellular transcriptional response to hypoxia play important roles in turning on and turning off oxygen stress genes. In particular, 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 enhance or reduce the thermodynamic coupling between its TAZ1-binding motifs. An efficient native-gel assay shows that certain linker mutations enhance the affinity of HIF-1α for TAZ1, and fluorescence anisotropy competition and NMR measurements show that these mutants can compete with CITED2 for TAZ1 more effectively than wild-type HIF-1α. The wide range of mutants, which include insertion, deletion, replacement and scrambling of residues in the linker, provide insights into the molecular basis for the exquisite tuning of the hypoxic switch: the TAZ1 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.

Figure 1.

Solution structure of TAZ1:HIF-1α complex (PDB ID: 1L8C). The distance between the backbone Cα residues of P805 and Q814, at either end of the αB - αC linker, is indicated as a dashed black line.

Figure 2.

Alignment of CTAD sequences from representative vertebrate HIF-1α (upper section) and invertebrate HIFα (lower two sections) species. Secondary structure elements as defined previously are shown at the top of the figure. Numbers at the top of the figure refer to the human sequence.

Figure 3.

Schematic diagram of native gel competition assay and representative gel showing competition of His₆GB1-HIF-1α CTAD fusion (Ligand 1 or L1) with tag-free WT HIF-1α CTAD (Ligand2 or L2) for binding to TAZ1. Each pair of lanes contains the His₆GB1-HIF-1α CTAD (L1) constructs as annotated in the last row below the gel. Binary (b) lanes contain TAZ1 and L1 at 1:1 molar ratio. Competition (c) lanes contain TAZ1, L1, and L2 at 1:1:1. Labels at the bottom of the figure show which L1 His₆GB1 fusion (WT HIF-1α CTAD or mutants identified in Table 1) is in competition with tag-free WT HIF-1α CTAD. Note that complexes of tag-free CTAD with TAZ1 stay at the position of the injection well. Circles around the His₆GB1-HIF-1α CTAD (L1):TAZ1 complex band denote regions used for intensity determination.

Figure 4.

Fluorescence anisotropy competition assay. Unlabeled HIF variants were titrated into a preformed complex of AlexaFluor-594 labeled WT HIF-1α peptide and unlabeled TAZ1. (a) Fits of the anisotropy competition assay for respective HIF variants. (b) TAZ1 binding affinity of HIF-1α variants plotted as a function of intervening linker length. Corresponding binding free energy (at 293K) is labeled in the plot. (c) Plot of I_c/b vs K_d from fluorescence anisotropy with logarithmic trend line.

Figure 5.

Comparison of weighted average ¹H, ¹⁵N chemical shift differences (Δδ_ave = [(Δδ_HN)²+(Δδ_N/5)²]^1/2) between free and TAZ1-bound state WT HIF-1α (black) and the variants (a) ΔSR (red), (b) (GS)₂ (magenta), and (c) ΔGS (green).

Figure 6.

Fluorescence anisotropy competition assay. A pre-formed complex of unlabeled TAZ1 with CITED2 labeled with Alexa-Fluor 594 was mixed with increasing concentrations of WT (black) or ΔSR mutant (red) HIF-1α CTAD.

Figure 7.

Regions of ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled TAZ1. A. Region showing the cross peak of V405 NH in complex with CITED2 and HIF-1α TAD ΔSR (top row) and WT (second row) in the ratios 1:1:1 (red), 1:1:2 (green) and 1:1:4 (purple). B. Region showing the cross peak of W418 NεH in complex with CITED2 and HIF-1α TAD ΔSR (third row) and WT (fourth row).

Figure 8.

Region of the NMR structure family of the complex between the CTAD of HIF-1α (blue) and TAZ1 (gray), showing the disorder in the loop between αB and αC and the well-ordered side chains of I806 and L813 at either end of the disordered loop.