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

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) αketoglutarate-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.

Hepatic HIF2 is a key determinant of manganese excess and polycythemia in SLC30A10 deficiency

“…FIH hydroxylates HIF1A more efficiently than HIF2A. FIH requires iron for activity but has been shown to bind Mn in vitro, though the impact of Mn binding on FIH activity has yet to be explored (36). In addition to FIH, histone deacetylases known as sirtuins are also candidates.…”

Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase

“…In addition to halogenation, these enzymes have also been shown to functionalize C-H bonds with azide (Büchler et al, 2022; Chan et al, 2022; Matthews et al, 2014; Neugebauer et al, 2019) and nitrite (Matthews et al, 2014), albeit with disappointingly low yields. A variety of methods have been used to determine substrate affinity in these and related classes of enzymes, including tryptophan fluorescence (Martin et al, 2019), SPR (Hu et al, 2015), and UV-Vis spectral shift-based methods (Chan et al, 2022; Matthews et al, 2014; Price et al, 2003; Ryle et al, 1999) to name a few. During our investigations of BesD, a non-heme iron halogenase that natively chlorinates lysine at the γ-position (Marchand et al, 2019), we characterized a positive heterotropic cooperativity that exists between the primary substrate lysine and the co-substrate chloride (Smithwick et al, 2023).…”

Dynamic Domain Links Substrate Binding and Catalysis in the Factor-Inhibiting-HIF-1

“…So long as these half-reactions are synchronized, substrate hydroxylation and O 2 sensing will be tightly coupled to O 2 activation. Cofactor and αKG binding markedly decrease the global flexibility of FIH and increase the affinity for CTAD, centrally connecting protein flexibility to the sequential reaction mechanism …”

“…Cofactor and αKG binding markedly decrease the global flexibility of FIH and increase the affinity for CTAD, centrally connecting protein flexibility to the sequential reaction mechanism. 19 Inspection of the CTAD binding contacts shows a loop at FIH 102−118 that forms extensive contacts with the CTAD peptide, forming part of the substrate binding interface (Figure 1). 15,16 Both the residue at the base of this loop, Tyr102, and Gln239 form close contacts with the FIH-Asn803 target residue in a tight registry with the Fe cofactor.…”