Phosphoinositide binding to the DHR-1 domain. A, the head group of PtdIns(3,4,5)P3 with phosphate positions numbered. B, thermodynamic parameters for phosphoinositide and head group binding to wild-type DHR-1 (Kd, dissociation constant; ΔH, enthalpy change; TΔS, temperature (K) × entropy change; ΔG (the free energy change) = ΔH-TΔS = −RT ln Kd); N is the apparent stoichiometry. No binding was observed for PtdIns(3,5)P2, PtdIns(3,4)P2, or PtdIns(3)P1. * indicates the experiment was performed in 145 mm NaCl. All other experiments were carried out in low salt (see “Experimental Procedures”). Representative ITC profiles are provided in supplemental Fig. S4. C, effect of point mutations on PtdIns(3,4,5)P3 binding, defined as Kd(wt)/Kd(mutant). Error estimates are from fitting of ITC titration curves. At right are melting temperatures, Tm, for each mutant, in °C. *, the Tm value given for K524A is actually for the triple mutant, K446A/K524A/K555A. D, mutation sites mapped onto the DHR-1 structure. E and F, ITC competition titrations (negative values of energy indicate an exothermic reaction; positive values, endothermic). E, DHR-1 equilibrated with a 1.5 m excess of PtdIns(4,5)P2 titrated with PtdIns(3,4,5)P3. F, the converse experiment: DHR-1/PtdIns(3,4,5)P3 titrated with PtdIns(4,5)P2.