Role of Backbone Dynamics in Modulating the Interactions of Disordered Ligands with the TAZ1 Domain of the CREB-Binding Protein

Abstract

The intrinsically disordered transactivation domains of HIF-1α and CITED2 compete for binding of the TAZ1 domain of the CREB-binding protein by a unidirectional allosteric mechanism involving direct competition for shared binding sites, ternary complex formation, and TAZ1 conformational changes. To gain insight into the mechanism by which CITED2 displaces HIF-1α from TAZ1, we used nuclear magnetic resonance spin relaxation methods to obtain an atomic-level description of the picosecond to nanosecond backbone dynamics that contribute to TAZ1 binding and competition. We show that HIF-1α and CITED2 adopt different dynamics in their complexes with TAZ1, with flexibility observed for HIF-1α in regions that would maintain accessibility for CITED2 to bind to TAZ1 and facilitate subsequent HIF-1α dissociation. In contrast, critical regions of CITED2 adopt a rigid structure in its complex with TAZ1, minimizing the ability of HIF-1α to compete for binding. We also find that TAZ1, previously thought to be a rigid scaffold for binding of disordered protein ligands, displays altered backbone dynamics in its various bound states. TAZ1 is more rigid in its CITED2-bound state than in its free state or in complex with HIF-1α, with increased rigidity observed not only in the CITED2 binding site but also in regions of TAZ1 that undergo conformational changes between the HIF-1α- and CITED2-bound structures. Taken together, these data suggest that backbone dynamics in TAZ1, as well as in the HIF-1α and CITED2 ligands, play a role in modulating the occupancy of TAZ1 and highlight the importance of characterizing both binding partners in molecular interactions.

Figure 1.

NMR spin relaxation data for ¹⁵N HIF-1α:TAZ1 and ¹⁵N CITED2:TAZ1 complexes. ¹⁵N R1, ¹⁵N R₂, and {¹H}-¹⁵N NOE data collected at 500 MHz (black) and 600 MHz (red) are shown for ¹⁵N HIF-1α:TAZ1 (left) and ¹⁵N CITED2:TAZ1 (right). The secondary structures formed by HIF-1α (left) and CITED2 (right) in complex with TAZ1 and the conserved LP(Q/E)L motif are shown above the graphs for reference. {¹H}-¹⁵N NOE data for CITED2 residues 264-269 (to the right of the dashed vertical line) are plotted on the right y-axis.

Figure 2.

Backbone amide order parameters (S²) for ¹⁵N HIF-1α:TAZ1 and ¹⁵N CITED2:TAZ1 complexes. S² values obtained from Model-Free analysis of the NMR spin relaxation data are shown for ¹⁵N HIF-1α:TAZ1 (top) and ¹⁵N CITED2:TAZ1 (bottom).The secondary structures formed by HIF-1α (top) and CITED2 (bottom) in complex with TAZ1 and the conserved LP(Q/E)L motif are shown above the graphs for reference.

Figure 3.

Evidence for dynamic clustering within binding motifs in ¹⁵N HIF-1α:TAZ1 and ¹⁵N CITED2:TAZ1. The spectral densities J(ω_N) (top) and J(0.87ω_H) (bottom) are plotted as a function of J(0) for ¹⁵N HIF-1α:TAZ1 (left) and ¹⁵N CITED2:TAZ1 (right) for data collected at 600 MHz. Data corresponding to discrete binding motifs of HIF-1α and CITED2 are shown in the colors indicated in the legends. Data points corresponding to the residues in the conserved LP(Q/E)L motif are labeled.

Figure 4.

Backbone amide order parameters (S²) for ¹⁵N TAZ1,¹⁵N TAZ1:HIF-1α and ¹⁵N TAZ1:CITED2 complexes. S² values obtained from Model-Free analysis of the NMR spin relaxation data are shown for ¹⁵N TAZ1 (black), ¹⁵N TAZ1:HIF-1α (red), and ¹⁵N TAZ1:CITED2 (blue). The helical regions of TAZ1 are shown above the graph for reference.

Figure 5.

Changes in ¹⁵N TAZ1 backbone amide order parameters upon HIF-1α or CITED2 binding. Differences in S² values (ΔS²) obtained from Model-Free analysis are shown for ¹⁵N TAZ1:HIF-1α compared to free ¹⁵N TAZ1 (top), ¹⁵N TAZ1:CITED2 compared to free ¹⁵N TAZ1 (middle), and ¹⁵N TAZ1:CITED2 compared to ¹⁵N TAZ1:HIF-1α (bottom). The helical regions of TAZ1 are shown above the graphs for reference.

Figure 6.

Mapping changes in ¹⁵N TAZ1 backbone amide order parameters between the ¹⁵N TAZ1:HIF-1α and ¹⁵N TAZ1:CITED2 complexes. TAZ1 is shown in grey, HIF-1α is shown in red, and CITED2 is shown in blue. Zinc atoms are shown as dark blue spheres. Residues with ΔS² greater than 0.1 are shown as orange spheres. The ΔS² values were calculated as S²_CITED2 - S²_HIF1α. The secondary structural elements of HIF-1α and CITED2, the conserved LP(Q/E)L motif, and the TAZ1 helices are labeled for reference. The disordered C-terminal tail of CITED2 (residues 260-269) is not shown.

Figure 7.

Backbone dynamics for HIF-1α and CITED2 in their binary complexes with TAZ1. The ribbon width of the HIF-1α (A) and CITED2 (B) transactivation domain peptides is scaled by 1-S²; the backbone color gradient ranges from blue (1-S² = 0.1) to red (1- S² = 0.9). TAZ1 is shown in the cartoon representation in grey. Zinc atoms are shown as dark blue spheres. The disordered C-terminal tail of CITED2 (residues 260-269) is not shown. The secondary structural elements of HIF-1α and CITED2 are labeled for reference.