NADPH oxidase 5 (Nox5) is an essential enzyme expressed in human testis, lymph nodes and spleen cells that produces superoxide to maintain cell health and for immune system. Nox5’s superoxide-generating activity is regulated by Ca2+through the interaction between its self-contained EF-hand domain (EFD) and dehydrogenase domain (DH). Recently, it has been shown that Nox5 contains a putative Calmodulin (CaM) binding domain (CMBD) within its DH, and the association of CaM to Nox5 increased its Ca2+sensitivity. However, this mechanism is largely unclear. Here we used Nox5’s CMBD peptide and partial DH constructs, including DH1 (a.a. 665-719) and DH2 (a.a 605-719), and separately studied their interactions to obtain thermodynamic parameters and to probe the conformational change. Fluorescence anisotropy measurement indicates the binding constants (Ka) for CMBD and the DH constructs to CaM to be approximately 2.5 × 106M-1, consistent with the data obtained from isothermal titration calorimetry. Currently, we are using the truncated CaMs, N-CaM and C-CaM, for the similar characterization to better assess the role of individual half lobes in binding. Furthermore, to investigate the role of each EF-hand, we utilize the chimera proteins consisting of the exchanged EF-hands between CaM and Troponin C (TnC), including 1TnC, 2TnC, 3TnC and 4TnC to perform binding studies.

Characterization of the interactions of NADPH oxidase 5 with calmodulin

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.