| Autor: |
Cardoso, Rosa, Love, Robert, Nilsson, Carol L., Bergqvist, Simon, Nowlin, Dawn, Yan, Jiangli, Liu, Kevin K.-C., Zhu, Jing, Chen, Ping, Deng, Ya-Li, Dyson, H. Jane, Greig, Michael J., Brooun, Alexei |
| Zdroj: |
Protein Science: A Publication of the Protein Society; Dec2012, Vol. 21 Issue 12, p1885-1896, 12p, 3 Color Photographs, 3 Charts, 3 Graphs |
| Abstrakt: |
The heterodimer HIF-1α (hypoxia inducible factor)/HIF-β (also known as ARNT-aryl hydrocarbon nuclear translocator) is a key mediator of cellular response to hypoxia. The interaction between these monomer units can be modified by the action of small molecules in the binding interface between their C-terminal heterodimerization (PasB) domains. Taking advantage of the presence of several cysteine residues located in the allosteric cavity of HIF-1α PasB domain, we applied a cysteine-based reactomics 'hotspot identification' strategy to locate regions of HIF-1α PasB domain critical for its interaction with ARNT. COMPOUND 5 was identified using a mass spectrometry-based primary screening strategy and was shown to react specifically with Cys255 of the HIF-1α PasB domain. Biophysical characterization of the interaction between PasB domains of HIF-1α and ARNT revealed that covalent binding of COMPOUND 5 to Cys255 reduced binding affinity between HIF-1α and ARNT PasB domains approximately 10-fold. Detailed NMR structural analysis of HIF-1α-PasB-COMPOUND 5 conjugate showed significant local conformation changes in the HIF-1α associated with key residues involved in the HIF-1α/ARNT PasB domain interaction as revealed by the crystal structure of the HIF-1α/ARNT PasB heterodimer. Our screening strategy could be applied to other targets to identify pockets surrounding reactive cysteines suitable for development of small molecule modulators of protein function. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
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