Novel HIF2A mutations disrupt cellular oxygen sensing. (A) Mutation analysis showing heterozygous mutation of HIF2A gene in tumor specimen but not adjacent normal tissue. (B) Peptide sequence alignment of prolyl hydroxylation domain of HIF-2α. (C) Electrostatic changes of single-site mutations in PHD2-HIF-α complex. Interaction between leucine (L) and tyrosine (Y) and PHD2 residues (top) and electrostatic changes resulting from L529P and Y532C substitution (bottom; red, negative; blue, positive; ±5 kT/e) (D) Peptide hydroxylation assay through matrix-assisted laser desorption ionization-time-of-flight showing absent hydroxylation in ΔHIF-2α-L529P mutant. (E) Peptide binding assay of radioactive labeled VHL and HIF-2α-derived peptides, showing reduced VHL binding in HIF-2α mutant. (F) Immunoprecipitation assay showing decreased VHL binding in HIF-2α mutant.