Effects of Hydroxylation at Proline 567 in HIF-1α on the Binding to pVHL

Abstract

Hypoxia is a physiological condition commonly present in tissue tumor growth and hypoxia training. Hypoxia inducible factor-1α (HIF-1α) plays an essential role in the regulation of hypoxia in animal cells. This mechanism is mediated by the von Hippel–Lindau tumor suppressor protein (pVHL) through the hydroxylation of HIF-1α proline 564. However, previous study has shown proline 567 was hydroxylated by prolyl hydroxylase-3 (PHD-3) in vitro. In addition, recent computational work has illustrated that a new hydrogen bond network localized around HYP567 was rearranged. In this work, we performed all-atom molecular dynamics (MD) simulations to investigate the structural effect and the interaction between pVHL and HIF-1α in status of non-hydroxylation proline (noHYP), hydroxylation proline 564 (HYP564), hydroxylation proline 567 (HYP567), and hydroxylation prolines 564 and 567(2HYP). Our cumulative 5 μs MD simulations reveal that HYP564 shows a lower RMSD value than the rest of variations, while 2HYP increases the structural flexibility of loop L7 (V137-P146) and α-domain (V155-P192) in pVHL comparing with HYP564. Hydrogen bond profile analysis illustrates that both HYP564 and 2HYP variations have two more hydrogen bonds (HYP564-HIS115 and SER111-HYP564) presented in an extremely steady condition, while 2HYP slightly reduces the intensity of H-bonds than HYP564. After the binding free energy calculation, HIF-1α shows a strong binding affinity to pVHL in HYP564 system, while the 2HYP obviously decreases the binding free energy between HIF-1α peptide and pVHL. In addition, our study highlights the conformational dynamics process between pVHL and variations of HIF-1α, suggesting that the hydroxylation of proline 567 may disturb the capture of HIF-1α by pVHL. Considering other evidences, we suggest that proline 567 in HIF-1α may not be hydroxylated in humans, at least in normal condition.