Abstract 2132 Investigation of individual EF-hands of Calmodulin for the interactions with NADPH Oxidase 5's dehydrogenase domain

NADP Oxidase 5 (Nox5) produces superoxide, which is crucial for intracellular signaling and immune responses. The activity of Nox5 is triggered by the interaction among its Ca2+ binding domain (EFD), transmembrane domain, and dehydrogenase domain (DH). Furthermore, this activity is regulated by Calmodulin (CaM). Previous studies have shown that the DH of Nox5 primarily exists as a tetramer, and binding to CaM induces the formation of a dimeric DH complex with CaM. CaM consists of four cooperative EF-hands (EFs) and removing one Ca2+ binding site significantly impacts others' Ca2+ binding and overall structure. To elucidate the role of each EF, we generated chimera proteins by combining CaM and Troponin C (TnC), both of which exhibit similar helical structures but exert opposite effects on target activation. In these chimera proteins, each EF of CaM was replaced with the corresponding EF in TnC, resulting in constructs like CaM(1TnC), where EF1 of CaM was replaced with EF1 of TnC. Our findings revealed that the degrees of helical changes and the values of heat capacity change (ΔCP) upon Ca2+ binding were similar when exchanging the EFs at the 1st and 2nd positions. However, these changes were significantly lower and less negative, respectively, when the 3rd and 4th were exchanged. This observation aligns with EF cooperativity in each half domain. These results also indicate that exchanging the EFs at the C-terminal end of CaM leads to a reduction in helical content and protein flexibility. This reduction may impair CaM's ability to induce DH dimeric complex formation. Currently, we are determining their bindings to Nox5's DH using isothermal titration calorimetry and investigating the oligomeric states induced by chimera binding through size exclusion chromatography and electrophoretic mobility measurements. Furthermore, the anticipated results using CaM/TnC chimeras will validate their application in investigating the role of EF-hands for recognition and activation of target enzymes. This work was supported by the grant from the National Science Foundation grant CHE-1608484.