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Cellular oxygen sensing: Crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2).

ABSTRACT: Cellular and physiological responses to changes in dioxygen levels in metazoans are mediated via the posttranslational oxidation of hypoxia-inducible transcription factor (HIF). Hydroxylation of conserved prolyl residues in the HIF-alpha subunit, catalyzed by HIF prolyl-hydroxylases (PHDs), signals for its proteasomal degradation. The requirement of the PHDs for dioxygen links changes in dioxygen levels with the transcriptional regulation of the gene array that enables the cellular response to chronic hypoxia; the PHDs thus act as an oxygen-sensing component of the HIF system, and their inhibition mimics the hypoxic response. We describe crystal structures of the catalytic domain of human PHD2, an important prolyl-4-hydroxylase in the human hypoxic response in normal cells, in complex with Fe(II) and an inhibitor to 1.7 A resolution. PHD2 crystallizes as a homotrimer and contains a double-stranded beta-helix core fold common to the Fe(II) and 2-oxoglutarate-dependant dioxygenase family, the residues of which are well conserved in the three human PHD enzymes (PHD 1-3). The structure provides insights into the hypoxic response, helps to rationalize a clinically observed mutation leading to familial erythrocytosis, and will aid in the design of PHD selective inhibitors for the treatment of anemia and ischemic disease.

SUBMITTER: McDonough MA

PROVIDER: S-EPMC1502536 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

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Cellular oxygen sensing: Crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2).

McDonough Michael A MA Li Vivian V Flashman Emily E Chowdhury Rasheduzzaman R Mohr Christopher C Liénard Benoît M R BM Zondlo James J Oldham Neil J NJ Clifton Ian J IJ Lewis Jeffrey J McNeill Luke A LA Kurzeja Robert J M RJ Hewitson Kirsty S KS Yang Evelyn E Jordan Steven S Syed Rashid S RS Schofield Christopher J CJ

Proceedings of the National Academy of Sciences of the United States of America 20060616 26

Cellular and physiological responses to changes in dioxygen levels in metazoans are mediated via the posttranslational oxidation of hypoxia-inducible transcription factor (HIF). Hydroxylation of conserved prolyl residues in the HIF-alpha subunit, catalyzed by HIF prolyl-hydroxylases (PHDs), signals for its proteasomal degradation. The requirement of the PHDs for dioxygen links changes in dioxygen levels with the transcriptional regulation of the gene array that enables the cellular response to c ...[more]

PMID: 16782814

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Project description:Prolyl hydroxylase domain 2 (PHD2) is deemed a primary oxygen sensor in humans, yet many details of its underlying mechanism are still not fully understood. (Fe(2+) + ?KG)PHD2 is 6-coordinate, with a 2His/1Asp facial triad occupying three coordination sites, a bidentate ?-ketoglutarate occupying two sites, and an aquo ligand in the final site. Turnover is thought to be initiated upon release of the aquo ligand, creating a site for O(2) to bind at the iron. Herein we show that steady-state turnover is faster under acidic conditions, with k(cat) exhibiting a kinetic pK(a) = 7.22. A variety of spectroscopic probes were employed to identify the active-site acid, through comparison of (Fe(2+) + ?KG)PHD2 at pH 6.50 with pH 8.50. The near-UV circular dichroism spectrum was virtually unchanged at elevated pH, indicating that the secondary structure did not change as a function of pH. UV-visible and Fe X-ray absorption spectroscopy indicated that the primary coordination sphere of Fe(2+) changed upon increasing the pH; extended X-ray absorption fine structure analysis found a short Fe-(O/N) bond length of 1.96 Å at pH 8.50, strongly suggesting that the aquo ligand was deprotonated at this pH. Solvent isotope effects were measured during steady-sate turnover over a wide pH-range, with an inverse solvent isotope effect (SIE) of k(cat) observed ((D(2)O)k(cat) = 0.91 ± 0.03) for the acid form; a similar SIE was observed for the basic form of the enzyme ((D(2)O)k(cat) = 0.9 ± 0.1), with an acid equilibrium offset of ?pK(a) = 0.67 ± 0.04. The inverse SIE indicated that aquo release from the active site Fe(2+) immediately precedes a rate-limiting step, suggesting that turnover in this enzyme may be partially limited by the rate of O(2) binding or activation, and suggesting that aquo release is relatively slow. The unusual kinetic pK(a) further suggested that PHD2 might function physiologically to sense both intracellular pO(2) as well as pH, which could provide for feedback between anaerobic metabolism and hypoxia sensing.

Dataset Information

Cellular oxygen sensing: Crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2).

Publications

Cellular oxygen sensing: Crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2).

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