Abstract
Plants commonly respond to stressors by modulating the expression of a large family of calcium binding proteins including isoforms of the ubiquitous signaling protein calmodulin (CaM). The various plant CaM isoforms are thought to differentially regulate the activity of specific target proteins to modulate cellular stress responses. The mechanism(s) behind differential target activation by the plant CaMs is unknown. In this study, we used steady-state and stopped-flow fluorescence spectroscopy to investigate the strategy by which two soybean CaMs (sCaM1 and sCaM4) have evolved to differentially regulate NAD kinase (NADK), which is activated by sCaM1 but inhibited by sCaM4. Although the isolated proteins have different cation binding properties, in the presence of Mg2+ and the CaM binding domains from proteins that are differentially regulated, the two plant CaMs respond nearly identically to rapid and slow Ca2+ transients. Our data suggest that the plant CaMs have evolved to bind certain targets with comparable affinities, respond similarly to a particular Ca2+ signature, but achieve different structural states, only one of which can activate the enzyme. Understanding the basis for differential enzyme regulation by the plant CaMs is the first step to engineering a vertebrate CaM that will selectively alter the CaM signaling network.
Highlights
Ca2+ is a universal second messenger that influences nearly every function eukaryotic cells perform (Petersen et al, 2005; Clapham, 2007; Davis et al, 2016)
It has recently been suggested that the two soybean CaMs’ different Ca2+ and Mg2+ binding affinities are the reason for their differential target activation (Gifford et al, 2013)
We demonstrate that in the presence of a physiological concentration of Mg2+ and CaM binding domains from proteins that are differentially regulated, the two plant CaMs respond nearly identically to rapid and slow Ca2+ transients
Summary
Ca2+ is a universal second messenger that influences nearly every function eukaryotic cells perform (Petersen et al, 2005; Clapham, 2007; Davis et al, 2016). All eukaryotic cells express the quintessential Ca2+ binding protein calmodulin (CaM) (Klee et al, 1980; Linse et al, 1991; Means et al, 1991) This small acidic protein binds and regulates a vast assortment of proteins that are involved in nearly every cellular function (Cheung, 1980; Davis et al, 2016). It has recently been suggested that the two soybean CaMs’ different Ca2+ and Mg2+ binding affinities are the reason for their differential target activation (Gifford et al, 2013) In this manuscript, we demonstrate that in the presence of a physiological concentration of Mg2+ and CaM binding domains from proteins that are differentially regulated, the two plant CaMs respond nearly identically to rapid and slow Ca2+ transients. Our data suggest that when bound to a target protein, the plant CaMs have evolved to respond to a particular Ca2+ signature, but achieve different structural states, only one of which can activate the enzyme
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