Site-directed allostery perturbation to probe the negative regulation of hypoxia inducible factor-1α

Petrovicz, Vencel László and Pasztuhov, István and Martinek, Tamás and Hegedüs, Zsófia (2024) Site-directed allostery perturbation to probe the negative regulation of hypoxia inducible factor-1α. RSC CHEMICAL BIOLOGY, 5 (8). pp. 711-720. ISSN 2633-0679

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Abstract

The interaction between the intrinsically disordered transcription factor HIF-1a and the coactivator proteins p300/CBP is essential in the fast response to low oxygenation. The negative feedback regulator, CITED2, switches off the hypoxic response through a very efficient irreversible mechanism. The negative cooperativity with HIF-1a relies on the formation of a ternary intermediate that leads to allosteric structural changes in p300/CBP, in which the cooperative folding/binding of the CITED2 sequence motifs plays a key role. Understanding the contribution of a binding motif to the structural changes in relation to competition efficiency provides invaluable insights into the molecular mechanism. Our strategy is to site-directedly perturb the p300–CITED2 complex’s structure without significantly affecting binding thermodynamics. In this way, the contribution of a sequence motif to the negative cooperativity with HIF-1a would mainly depend on the induced structural changes, and to a lesser extent on binding affinity. Using biophysical assays and NMR measurements, we show here that the interplay between the N-terminal tail and the rest of the binding motifs of CITED2 is crucial for the unidirectional displacement of HIF-1a. We introduce an advantageous approach for evaluating the roles of the different sequence parts with the help of motif-by-motif backbone perturbations.

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