Metal binding affinity and structural properties of calmodulin‐like protein 14 from Arabidopsis thaliana

Abstract

In addition to the well-known Ca 21 sensor calmodulin, plants possess many calmodulinlike proteins (CMLs) that are predicted to have specific roles in the cell. Herein, we described the biochemical and biophysical characterization of recombinant Arabidopsis thaliana CML14. We applied isothermal titration calorimetry to analyze the energetics of Ca 21 and Mg 21 binding to CML14, and nuclear magnetic resonance spectroscopy, together with intrinsic and ANS-based fluorescence, to evaluate the structural effects of metal binding and metal-induced conformational changes. Furthermore, differential scanning calorimetry and limited proteolysis were used to characterize protein thermal and local stability. Our data demonstrate that CML14 binds one Ca 21 ion with micromolar affinity (K d~1 2 mM) and the presence of 10 mM Mg 21 decreases the Ca 21 affinity by~5-fold. Although binding of Ca 21 to CML14 increases protein stability, it does not result in a more hydrophobic protein surface and does not induce the large conformational rearrangement typical of Ca 21 sensors, but causes only localized structural changes in the unique functional EFhand. Our data, together with a molecular modelling prediction, provide interesting insights into the biochemical properties of Arabidopsis CML14 and may be useful to direct additional studies aimed at understanding its physiological role.

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

“…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.…”

“…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 ). Interestingly, CML14, binds a single Ca 2+ atom and shows no change in exposed hydrophobicity at all (Vallone et al, 2016 ). This structural diversity among CMLs is consistent with the hypothesis that they play distinct though possibly overlapping roles, predominantly as Ca 2+ sensors.…”

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

“…AtCaM1 and CML14 were produced as described (43,57). CML19 was expressed and purified as reported in the supplemental "Methods".…”

“…The hydrodynamic radius of apoCML36-C and Mg 2ϩ -and Ca 2ϩ -bound CML36-C was estimated by SEC using a Superose 12 column (10/300GL, GE Healthcare) as described previously (43,63). Each experiment was performed at least in triplicate, and the reported values represent means Ϯ S.E.…”

“…ITC experiments were performed on a TA Instruments Nano ITC as described (29,43). All solutions were degassed prior to the rinsing and loading of both the titration and sample cells.…”