Understanding p300-transcription factor interactions using sequence variation and hybridization
RSC Chemical Biology(2021)
Abstract
The hypoxic response is central to cell function and plays a significant role in the growth and survival of solid tumours. HIF-1 regulates the hypoxic response by activating over 100 genes responsible for adaptation to hypoxia, making it a potential target for anticancer drug discovery. Although there is significant structural and mechanistic understanding of the interaction between HIF-1α and p300 alongside negative regulators of HIF-1α such as CITED2, there remains a need to further understand the sequence determinants of binding. In this work we use a combination of protein expression, chemical synthesis, fluorescence anisotropy and isothermal titration calorimetry for HIF-1α sequence variants and a HIF-1α- CITED hybrid sequence which we term CITIF. We show the HIF-1α sequence is highly tolerant to sequence variation through reduced enthalpic and less unfavourable entropic contributions, These data imply backbone as opposed to side chain interactions and ligand folding control the binding interaction and that sequence variations are tolerated as a result of adopting a more disordered bound interaction or “fuzzy” complex.
### Competing Interest Statement
The authors have declared no competing interest.
* ALA
: scan Alanine scan
BUDE
: Bristol University Docking Engine
C-TAD
: Carboxy-terminal transactivation domain
FA
: Fluorescence Anisotropy
GFP
: Green Fluorescent Protein
GST
: Glutathione S-transferase
HIF-1α
: Hypoxia-inducible factor 1-alpha
ITC
: Isothermal titration calorimetry
mAV
: multiple alanine variant
NMR
: Nuclear magnetic resonance
PPI
: Protein-protein interaction
sAV
: Single alanine variant
wt
: wild type
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