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Molecule of the Month


240: Hypoxia-Inducible Factors

Author: David S. Goodsell

Complex of a peptide from HIF-α (pink, with proline in red), pVHL (blue), and two elongins (green). The inset shows a close-up of the hydroxylated proline.

Oxygen is essential--without it, our cells rapidly die. Because of this, we have evolved a dedicated system that monitors the amount of oxygen and mobilizes responses when it gets low (termed hypoxia). Oxygen-starved cells send out signals that tell the body to create more red blood cells and build more blood vessels. Also, oxygen-starved cells reprogram metabolism to shift energy production towards pathways that don’t need so much oxygen, for example, by decreasing pyruvate dehydrogenase and increasing lactate dehydrogenase. The Nobel Prize for Physiology or Medicine was awarded this year to three researchers who discovered the molecular details of this central oxygen-sensing process, termed the HIF system.

High Oxygen

Hypoxia-inducible factor α (HIF-α) is the central switch that enables cells to respond to limiting oxygen. It is a protein with about 800 amino acids, with several functional elements. The structure shown here (PDB entry 1lqb) includes a small portion from its central region, which has two key proline residues (one is shown here). When oxygen is plentiful, these prolines are hydroxylated by PHD enzymes (HIF prolyl hydroxylases). Then, the hydroxyproline is recognized by a complex including pVHL (von Hippel-Lindau disease tumor suppressor) that targets HIF-α for ubiquitination and degradation by proteasomes. So, at normal oxygen levels, HIF-α is continuously degraded and cells carry on as usual.

Sensing Oxygen

PHD enzymes have the job of sensing oxygen levels. They attach oxygen atoms to two key prolines in HIF-α using a metal ion and the cosubstrate α-ketoglutarate. When oxygen is scarce, PHD catalysis is slowed and the prolines are not modified. An additional enzyme, termed FIH (factor inhibiting HIF), performs a second type of hydroxylation reaction, targeting an asparagine in HIF-α and modifying the way it interacts with the transcriptional machinery (PDB entry 1h2n, not shown).

Low Oxygen

Complex of HIF-α (pink), HIF-β (yellow), and DNA (blue).

When oxygen is low, HIF-α is not hydroxylated and not degraded by proteasomes, so it springs into action. It moves to the nucleus and associates with a companion protein, called HIF-β. Together, they bind to many sites in the genome and promote transcription of genes involved in low-oxygen metabolism and remodeling the circulatory system to improve oxygen delivery. The structure shown here (PDB entry 4zpr) includes the DNA-binding portion of the complex bound to a short piece of DNA.

Exploring the Structure

PHD2 Complexes

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Drugs that bind to the PHD enzymes are being evaluated for treatment of anemia. By blocking these enzymes, the drug tricks cells into thinking they need more oxygen, so they send signals to build more red blood cells. Initial structures of PHD2 enabled structure-based design of inhibitors of the enzyme (PDB entries 2g1m, 2g19), and a drug currently under evaluation, Vadadustat, is shown here (PDB entry 5ox6). By comparing this to the structure of PHD2 bound to a small piece of HIF-α (PDB entry 3hqr), we can see that the drug mimics binding of α-ketoglutarate to the enzyme (NOG is similar to α-ketoglutarate), and is big enough to block binding of the HIF-α proline.

Topics for Further Discussion

  1. There are many structures of FIH (factor inhibiting HIF) in the PDB archive if you would like to explore it and its interaction with substrates and inhibitors.
  2. The scissor-shaped DNA-binding domain of the HIF complex is termed “basic helix-loop-helix.” You can see other examples by searching for “bHLH” on the main PDB site.

References

  1. 5ox6 Yeh, T.L., Leissing, T.M., Abboud, M.I., Thinnes, C.C., Atasoylu, O., Holt-Martyn, J.P., Zhang, D., Tumber, A., Lippl, K., Lohans, C.T., Leung, I.K.H., Morcrette, H., Clifton, I.J., Claridge, T.D.W., Kawamura, A., Flashman, E., Lu, X., Ratcliffe, P.J., Chowdhury, R., Pugh, C.W., Schofield, C.J. 2017 Molecular and cellular mechanisms of HIF prolyl hydroxylase inhibitors in clinical trials. Chem Sci 8 7651-7668
  2. 4zpr Wu, D., Potluri, N., Lu, J., Kim, Y., Rastinejad, F. 2015 Structural integration in hypoxia-inducible factors. Nature 524 303-308
  3. Semenza, G..L 2012 Hypoxia-inducible factors in physiology and medicine. Cell 148 399-408
  4. 3hqr Chowdhury, R., McDonough, M.A., Mecinovic, J., Loenarz, C., Flashman, E., Hewitson, K.S., Domene, C., Schofield, C.J. 2009 Structural basis for binding of hypoxia-inducible factor to the oxygen-sensing prolyl hydroxylases. Structure 17 981-989
  5. 2g1m2g19 McDonough, M.A., Li, V., Flashman, E., Chowdhury, R., Mohr, C., Lienard, B.M.R., Zondlo, J., Oldham, N.J., Clifton, I.J., Lewis, J., McNeill, L.A., Kurzeja, R.J.M., Hewitson, K.S., Yang, E., Jordan, S., Syed, R.S., Schofield, C.J. 2006 Cellular oxygen sensing: crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2). Proc.Natl.Acad.Sci.USA 103 9814-9819
  6. 1h2n Elkins, J.M., Hewitson, K.S., McNeill, L.A., Seibel, J.F., Schlemminger, I., Pugh, C., Ratcliffe, P., Schofield, C.J. 2003 Structure of factor-inhibiting hypoxia-inducible factor (Hif) reveals mechanism of oxidative modification of Hif-1Alpha. J.Biol.Chem. 278 1802-1806
  7. 1lqb Hon, W.C., Wilson, M.I., Harlos, K., Claridge, T.D., Schofield, C.J., Pugh, C.W., Maxwell, P.H., Ratcliffe, P.J., Stuart, D.I., Jones, E.Y. 2002 Structural basis for the recognition of hydroxyproline in HIF-1 alpha by pVHL. Nature 417 975-978