Arabidopsis calmodulin-like protein CML36 is a calcium (Ca2+) sensor that interacts with the plasma membrane Ca2+-ATPase isoform ACA8 and stimulates its activity

Abstract

Edited by Joseph Jez Calmodulin-like (CML) proteins are major EF-hand-containing, calcium (Ca 2؉)-binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca 2؉ sensors and identification of novel target proteins of CMLs deserve special attention. To this end, we recombinantly produced and biochemically characterized CML36 from Arabidopsis thaliana. We analyzed Ca 2؉ and Mg 2؉ binding to the individual EFhands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high-affinity Ca 2؉ /Mg 2؉ mixed binding sites and two lowaffinity Ca 2؉-specific sites. Binding of Ca 2؉ induced an increase in the ␣-helical content and a conformational change that lead to the exposure of hydrophobic regions responsible for target protein recognition. Cation binding, either Ca 2؉ or Mg 2؉ , stabilized the secondary and tertiary structures of CML36, guiding a large structural transition from a molten globule apo-state to a compact holoconformation. Importantly, through in vitro binding and activity assays, we showed that CML36 interacts directly with the regulative N terminus of the Arabidopsis plasma membrane Ca 2؉-ATPase isoform 8 (ACA8) and that this interaction stimulates ACA8 activity. Gene expression analysis revealed that CML36 and ACA8 are co-expressed mainly in inflorescences. Collectively, our results support a role for CML36 as a Ca 2؉ sensor that binds to and modulates ACA8, uncovering a possible involvement of the CML protein family in the modulation of plant-autoinhibited Ca 2؉ pumps. Calcium (Ca 2ϩ) is a crucial second messenger in plants, where it couples the perception of endogenous and environmental signals to plant responses (1-4). Ca 2ϩ signals are transmitted by stimulus-specific cytosolic Ca 2ϩ elevations that result from the concerted action of both Ca 2ϩ influx (channels) and Ca 2ϩ efflux (pumps and carriers) systems, which temporally shape and spatially define Ca 2ϩ dynamics, commonly This study was supported in part by Grant FUR2014 from the University of Verona (to A. A. and P. D.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains supplemental Figs.

Arabidopsis Calmodulin-Like Proteins, CML15 and CML16 Possess Biochemical Properties Distinct from Calmodulin and Show Non-overlapping Tissue Expression Patterns

“…Interestingly, the conserved CaM, AtCaM1, shows similar behavior when the two N-terminal EF-hands are rendered incapable of binding Ca 2+ (Astegno et al, 2016 ). Conversely, the changes in secondary structure of CML16 suggested by CD analysis are similar to previously reported data for CML37 (Scholz et al, 2014 ), CML39 (Bender et al, 2013 ) and CML36 (Astegno et al, 2017 ) where Ca 2+ -binding increases helical content. It is not yet clear how these subtle structural distinctions contribute to the functions of different CMLs but it underscores the biochemical complexity of this large family of Ca 2+ sensors.…”

“…As might be expected for a large family of Ca 2+ sensors, the Ca 2+ affinities among CMLs tested to date vary but generally fall within what would be considered a physiological range (nM–μM). The Ca 2+ dissociation values for CML15 and CML16 are comparable to those observed for CML42 (Dobney et al, 2009 ), CML43 (Bender et al, 2014 ), CML14 (Vallone et al, 2016 ), and CML36 (Astegno et al, 2017 ). The presence of both high- and low-affinity Ca 2+ sites in CMLs appears to be an emerging pattern (Bender et al, 2014 ; Astegno et al, 2017 ) but how these features relate to in vivo function remains unclear.…”

“…The Ca 2+ dissociation values for CML15 and CML16 are comparable to those observed for CML42 (Dobney et al, 2009 ), CML43 (Bender et al, 2014 ), CML14 (Vallone et al, 2016 ), and CML36 (Astegno et al, 2017 ). The presence of both high- and low-affinity Ca 2+ sites in CMLs appears to be an emerging pattern (Bender et al, 2014 ; Astegno et al, 2017 ) but how these features relate to in vivo function remains unclear. A recent study on a tobacco CML, rgsCaM, revealed a low affinity for Ca 2+ in vitro , calling into question whether rgsCaM could function as a Ca 2+ sensor in vivo (Makiyama et al, 2016 ).…”

“…The levels of exposed hydrophobicity, both in apo- and holo-forms of CMLs vary among family members. For example, CML36 resembles CMl15 and CML16 in that only a modest increase in exposed hydrophobicity is observed upon Ca 2+ binding (Astegno et al, 2017 ). In contrast, CML37, CML42, CML43, behave more like CaM, displaying low intrinsic hydrophobic exposure in the apo form and much greater relative increases in response to Ca 2+ (Dobney et al, 2009 ; Bender et al, 2014 ; Scholz et al, 2014 ).…”

Advances and current challenges in calcium signaling

“…For example, Loss of CML42 was associated with aberrant trichomes, whereas CML41 was reported to regulate plasmodesmal closure during plant immune responses. Moreover, CML24 and CML9 were found to be involved in abscisic acid (ABA) responses and ionic stress, whereas CML36 was reported to regulate the activity of the Ca 2+ ATPase ACA8 (Delk et al, 2005;Magnan et al, 2008;Dobney et al, 2009;Astegno et al, 2017;Xu et al, 2017).…”