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

## TL;DR

This study explores how CITED2 disrupts the HIF-1α-p300/CBP interaction through structural changes, revealing a mechanism for turning off the hypoxic response.

## Contribution

A novel approach using site-directed allostery perturbation to dissect the role of CITED2 motifs in HIF-1α regulation.

## Key findings

- The N-terminal tail of CITED2 interacts with other motifs to displace HIF-1α unidirectionally.
- Backbone perturbations in CITED2 reveal structural adaptations in p300 that govern allostery.
- Motif-by-motif analysis helps evaluate contributions to negative cooperativity with HIF-1α.

## Abstract

The interaction between the intrinsically disordered transcription factor HIF-1α 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-1α 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-1α 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-1α. We introduce an advantageous approach for evaluating the roles of the different sequence parts with the help of motif-by-motif backbone perturbations.

The structural adaptation of p300 to the backbone-modified CITED2 sequences provides insight into the molecular mechanism that governs the allosteric regulation of HIF-1α.