Protein Flexibility of the α-Ketoglutarate-Dependent Oxygenase Factor-Inhibiting HIF-1: Implications for Substrate Binding, Catalysis, and Regulation

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) alpha-ketoglutarate-dependent oxygenase that is a key hypoxia (low p(O2)) sensor in humans. As these hypoxia-sensing enzymes follow a multistep chemical mechanism consuming alpha-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 la (HIF) that is hydroxylated on the side chain of Asn803. We assessed changes in protein flexibility connected to metal and alpha KG binding, finding that (M+alpha KG) 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+alpha KG) increased the affinity of FIH for CTAD as measured by titrations monitoring intrinsic tryptophan fluorescence. The decreased protein dynamics caused by (M+alpha KG) enforces a sequentially ordered substrate binding sequence in which alpha KG binds before CTAD, suggesting that selective inhibition may require inhibitors that target the binding sites of both alpha KG and the prime substrate. A consequence of the correlation between dynamics and alpha KG binding is that all relevant ligands must be included in binding-based inhibitor screens, as shown by testing permutations of M, alpha KG, and inhibitor.