Protein Flexibility of the α-Ketoglutarate-Dependent Oxygenase Factor-Inhibiting HIF-1: Implications for Substrate Binding, Catalysis, and Regulation
Year: 2019
Type: article
Abstract: Protein dynamics are crucial for the mechanistically ordered enzymes to bind to their substrate in the correct sequence and perform catalysis. Factor-inhibiting HIF-1 (FIH) is a nonheme Fe(II) α-ketoglutarate-dependent oxygenase that is a key hypoxia (low p O 2 ) sensor in humans. As these hypoxia-sensing enzymes follow a multistep chemical mechanism consuming α-ketoglutarate, a protein substrate that is hydroxylated, and O 2, understanding protein flexibility and the order of substrate binding may aid in the development of strategies for selective targeting. The primary substrate of FIH is the C-terminal transactivation domain (CTAD) of hypoxia-inducible factor 1α (HIF) that is hydroxylated on the side chain of Asn803. We assessed changes in protein flexibility connected to metal and αKG binding, finding that (M+αKG) binding significantly stabilized the cupin barrel core of FIH as evidenced by enhanced thermal stability and decreased protein dynamics as assessed by global amide hydrogen/deuterium exchange mass spectrometry and limited proteolysis. Confirming predictions of the consensus mechanism, (M+αKG) increased the affinity of FIH for CTAD as measured by titrations monitoring intrinsic tryptophan fluorescence. The decreased protein dynamics caused by (M+αKG) enforces a sequentially ordered substrate binding sequence in which αKG binds before CTAD, suggesting that selective inhibition may require inhibitors that target the binding sites of both αKG and the prime substrate. A consequence of the correlation between dynamics and αKG binding is that all relevant ligands must be included in binding-based inhibitor screens, as shown by testing permutations of M, αKG, and inhibitor.
Source: Biochemistry
Institution: University of Massachusetts Amherst
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2972965187