SOUL, a heme-binding protein-2 (HEBP-2), interacts with apoptosis-linked gene 2 protein (ALG-2) in a Ca2+-dependent manner. To investigate the properties of the interaction of SOUL with ALG-2, we generated several mutants of SOUL and ALG-2 and analyzed the recombinant proteins using pulldown assay and isothermal titration calorimetry. The interaction between SOUL and ALG-2 (delta3-23ALG-2) was an exothermic reaction, with 1:1 stoichiometry and high affinity (Kd = 32.4 nM) in the presence of Ca2+. The heat capacity change (ΔCp) of the reaction showed a large negative value (-390 cal/K·mol), which suggested the burial of a significant nonpolar surface area or disruption of a hydrogen bond network that was induced by the interaction (or both). One-point mutation of SOUL Phe100 or ALG-2 Trp57 resulted in complete loss of heat change, supporting the essential roles of these residues for the interaction. Nevertheless, a truncated mutant of SOUL1-143 that deleted the domain required for the interaction with ALG-2 Trp57 still showed 1:1 binding to ALG-2 with an endothermic reaction. These results provide a better understanding of the target recognition mechanism and conformational change of SOUL in the interaction with ALG-2.
Keywords: ALG-2; HEBP-2; ITC; SOUL; binding affinity; calcium-binding protein; heme-binding protein; thermodynamics.