Calmodulin interacts with androglobin and regulates the nitrite reductase activity

Abstract

Androglobin (Adgb) was discovered as the fifth mammalian globin, but its structure and function remain elusive. In this study, the heme-binding globin domain of Adgb was expressed and its interaction with calmodulin (CaM) was investigated. The protein structure of Adgb and its complex with CaM were predicted using AlphaFold3 and HDOCK. The circularly permutated globin domain of Adgb was well folded with a heme group, which can interact with CaM via the IQ motif. In experimental studies, two mutants of CaM (G41C and G114C) were constructed and labeled with a fluorescent molecule (fluorescein-5-maleimide) in the N-lobe and C-lobe, respectively. Upon binding to Adgb, a greater fluorescence quenching effect was observed for the labeling of Cys41 in the N-lobe due to energy transfer to the heme group, which is consistent with the predicted structure of the Adgb-CaM complex. Furthermore, as shown by UV-vis kinetic studies, the binding of CaM enhanced the nitrite reductase activity of Adgb. This study reveals a regulatory role of CaM for the unique Adgb and provides valuable information for understanding the structure-function relationship.