Calcium and Magnesium Binding Properties of Soybean Calmodulin 1 and 4

Abstract

Calmodulin (CaM) is known to bind and regulate hundreds of different proteins in a calcium dependent manner. Mammals encode a single isoform of CaM, while plants encode up to ten different isoforms (and dozens of calmodulin-like proteins). Moreover, recent studies have demonstrated that certain soybean CaMs have the ability to differentially activate several mammalian target enzymes in a competitive manner. As a first step in understanding the functional differences among these CaMs, we have studied the Ca2+ and Mg2+ binding properties of two unique soybean CaMs, sCaM1 and sCaM4. CaM has both N-terminal and C-terminal pairs of Ca2+ binding domains (EF-hands). In order to follow cation binding and dissociation from these sCaMs we utilized various fluorescent probes (Trp, Tyr, bis-ANS and quin-2). For each sCaM, our analysis revealed that the rate of Ca2+ dissociation was similar to the regional structural change occurring at each domain, which in turn was similar to the rate of closure of each domain's hydrophobic pocket. Compared to sCaM4, sCaM1 displayed a ∼2-fold lower affinity for Ca2+ at each domain. Consistent with this finding, the N- and C-terminal domains of sCaM1 had ∼5- and 2-fold faster calcium dissociation rates than the respective domains of SCaM4. Both sCaM's N-terminal domain bound Mg2+ with a physiologically relevant affinity (≤1mM), whereas their C-terminal domains had non-physiological Mg2+ binding affinities (≥6mM). The binding of Mg2+ to the N-terminal domains of the SCaMs kept the domain in the closed state and drastically delayed the rate of Ca2+ association and opening of the hydrophobic pocket. Thus, in addition to their ability to differentially regulate enzymes, these soybean CaMs also sense and respond to Ca2+ differently.