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 (CBP) by a unidirectional allosteric mechanism involving direct competition for shared binding sites, ternary complex formation, and TAZ1 conformational changes. To obtain insight into the mechanism by which CITED2 displaces HIF-1α from TAZ1, we used NMR 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 of HIF-1α observed 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 occupancy of TAZ1 and highlight the importance of characterizing both binding partners in molecular interactions.

Competitive binding of HIF-1α and CITED2 to the TAZ1 domain of CBP from molecular simulations

“…Finally, we also capture the rank order of rigidification between the two complexes. The differences in the order parameters between both complexes indicate that CITED2:TAZ1 forms the least fluctuating complex 23 , as we also see in the simulations (Figure 5b). The main differences between the bound states are found in helices α 1 and, more importantly, α 4 of TAZ1.…”

“…Instead, they can adjust their flexibility in relation to the interaction with ligands. This conclusion has been drawn for TAZ1 from studies of a collection of IDPs, including HIF-1α, CITED2, RelA-TA2 or TAD-STAT2 23,49 .…”

“…This result is qualitatively consistent with NMR experiments. In particular, S 2 order parameters had high values for the four amphipathic helices, indicating restricted mobility, and low values in most of the loops 23 , indicating large amplitude fluctuations. This agreement with experiment validates our coarse description of the stabilizing effects of Zn 2+ cations (see Methods).…”

“…The peculiarities in the binding of IDPs may result in interesting effects, like allosteric modulation of one partner by another 13,15 . An interesting case, involving two different IDPs, is that of the α-subunit of the hypoxia inducible transcription factor HIF-1α and its negative feedback regulator CITED2 [16][17][18][19][20][21][22][23] , a system that is highly relevant for the development of cancer therapies 24 . Both proa) Donostia International Physics Center, 20018, Donostia-San Sebastián, Spain; Electronic mail: david.desancho@ehu.eus teins are able to bind to the TAZ1 domain of the CREB binding protein (CBP).…”

“…Next, we report the predictions from the consensus simulation model on the binding of each of the IDPs independently to their shared partner. We pay attention to the influence of the binding on the dynamics of TAZ1, which has been recently investigated using NMR 22,23 . Finally, we show the emergent properties that we predict for the ternary complex and discuss the implications of our findings.…”