Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases

What is it about?

The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1–3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel–Lindau protein (VHL)–elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors.

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Why is it important?

PHD variant-associated erythrocytosis has been near exclusively linked to PHD2. Our finding that certain clinically observed PHD2 mutations substantially alter ODD selectivity, that is, the erythrocytosis-associated P317R PHD2 variant towards CODD, and the breast cancer-associated R396T PHD2 variant towards NODD, implies that altered PHD2 selectivity may have pathological consequences. Biophysical analyses employing crystallography and NMR reveal that the molecular basis of PHD isoform and variant selectivity involves enzyme–substrate interactions involving β2/β3 loop residues (that bind the LXXLAP motif) and helices α3 and α4, regions which display sequence variations between the PHD isoforms. The results reveal higher conservation of residues correlating with maintenance of CODD over NODD selectivity during the course of animal evolution that ‘CODD-type’ hydroxylation probably evolved before that of NODD, the greater importance of induced fit in CODD compared with NODD binding and clinical/genetic evidence that loss of wt PHD2 cannot be compensated for by PHD 1/3. The identification of specific interactions determining PHD selectivity for ODDs opens the way forward to PHD isoform and ODD selective inhibitors, which may be useful for the upregulation of specific sets of HIF target genes. The identification of the features determining HIFα ODD selectivity should also be of interest with respect to validating reports of non-HIF substrates for the PHDs.

Perspectives