Fig. 5.

Point mutations in the HIFα PAS-B central β-sheet disrupt the binding of ARNT PAS-B. (a) Superimposed ¹⁵N/¹H HSQC spectra of 250 μM ¹⁵N labeled HIF-2α PAS-B (Left) or triple mutant (Q322E/M338E/Y342T) (Right). Spectra in the presence of 900 μM unlabeled ARNT PAS-B are shown with red contours; those without ARNT are shown in black contours. Similar data for HIF-1α PAS-B are provided in Supporting Methods. (b) PAS-B domain interaction is important to form a biologically active HIF/ARNT complex. A construct expressing a luciferase reporter under the control of an HRE promoter was transfected into Ka-13 (columns 1–5) or CHO (column 6) cells along with various HIFα constructs. Values represent the average luciferase activity of three samples, with bars indicating standard error. Luciferase expression was induced by cotransfection of HIF-1α (column 2) or HIF-2α (column 4), particularly under hypoxic conditions. Cotransfection of trHIF-1α (column 3) or trHIF-2α (column 5), full-length HIFα proteins containing the three PAS-B mutations, shows a significant drop in luciferase activity compared with wild-type HIFα.