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Computational analysis of prolyl hydroxylase domain-containing protein 2 (PHD2) mutations promoting polycythemia insurgence in humans.

ABSTRACT: Idiopathic erythrocytosis is a rare disease characterized by an increase in red blood cell mass due to mutations in proteins of the oxygen-sensing pathway, such as prolyl hydroxylase 2 (PHD2). Here, we present a bioinformatics investigation of the pathological effect of twelve PHD2 mutations related to polycythemia insurgence. We show that few mutations impair the PHD2 catalytic site, while most localize to non-enzymatic regions. We also found that most mutations do not overlap the substrate recognition site, suggesting a novel PHD2 binding interface. After a structural analysis of both binding partners, we suggest that this novel interface is responsible for PHD2 interaction with the LIMD1 tumor suppressor.

SUBMITTER: Minervini G

PROVIDER: S-EPMC4709589 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Computational analysis of prolyl hydroxylase domain-containing protein 2 (PHD2) mutations promoting polycythemia insurgence in humans.

Minervini Giovanni G Quaglia Federica F Tosatto Silvio C E SC

Scientific reports 20160112

Idiopathic erythrocytosis is a rare disease characterized by an increase in red blood cell mass due to mutations in proteins of the oxygen-sensing pathway, such as prolyl hydroxylase 2 (PHD2). Here, we present a bioinformatics investigation of the pathological effect of twelve PHD2 mutations related to polycythemia insurgence. We show that few mutations impair the PHD2 catalytic site, while most localize to non-enzymatic regions. We also found that most mutations do not overlap the substrate rec ...[more]

PMID: 26754054

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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

Computational analysis of prolyl hydroxylase domain-containing protein 2 (PHD2) mutations promoting polycythemia insurgence in humans.

Publications

Computational analysis of prolyl hydroxylase domain-containing protein 2 (PHD2) mutations promoting polycythemia insurgence in humans.

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