The Crystal Structure of the Novel Calcium-binding Protein AtCBL2 from Arabidopsis thaliana

Abstract

Arabidopsis thaliana calcineurin B-like protein (AtCBL2)is a member of a recently identified family of calcineurin B-like calcium-binding proteins in A. thaliana. The crystal structure of AtCBL2 has been determined at 2.1 Å resolution. The protein forms a compact ␣-helical structure with two pairs of EF-hand motifs. The structure is similar in overall folding topology to the structures of calcineurin B and neuronal calcium sensor 1, but differs significantly in local conformation. The two calcium ions are coordinated in the first and fourth EF-hand motifs, whereas the second and third EF-hand motifs are maintained in the open form by internal hydrogen bonding without coordination of calcium ions. Both a possible site and a possible mechanism for the target binding to AtCBL2 are discussed based on the three-dimensional structure.

Tuning of a Neuronal Calcium Sensor

“…Like the wild-type protein, the Rc 2-190 mutant has been crystallized in the presence of a large excess of Ca 2ϩ , and both crystal structures contain a Ca 2ϩ ion bound to EF-hand 3. Investigation of 45 Ca 2ϩ binding to non-myristoylated Rc revealed that the truncation changed the intrinsic Ca 2ϩ affinity of EF-hand 3, whereas EF-hand 2 was unaffected (Fig. 1B).…”

“…Denatured protein (after elution from a reverse phase column) was refolded as described previously (23). 45 Ca 2ϩ Binding Assay-Binding of 45 Ca 2ϩ to recoverin was investigated as described previously (17). In summary, 43 M protein was dissolved in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl and 1 mM dithiothreitol and transferred to Centricon 10 devices (Amicon).…”

“…In summary, 43 M protein was dissolved in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl and 1 mM dithiothreitol and transferred to Centricon 10 devices (Amicon). Radioactive 45 CaCl 2 was added, the samples were centrifuged for 1 min (5000 rpm, tabletop centrifuge Biofuge pico, Heraeus) and radioactivity of the filtrate was counted (free Ca 2ϩ ). Next, non-radioactive CaCl 2 was added, and the centrifugation procedure was repeated.…”

Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis

“…Interestingly, we further found that TTS was capable of directing different soluble proteins to the vacuolar membrane [32] (Supplementary information, Figure S5A), but it failed to change the subcellular localization of proteins with multiple transmembrane domains (Supplementary information, Figure S5B). CBL2 and CBL3 proteins both bind Ca 2+ Although deduced amino acid sequence and structural analysis suggest that all CBL proteins including CBL2 and CBL3 contain putative EF-hand motifs that contribute to calcium binding [10,33], experimental evidence for the calcium-binding property of CBL2 and CBL3 is limited. To determine whether CBL2 and CBL3 are indeed capable of binding Ca 2+ , we performed a 45 Caoverlay assay using the recombinant CBL2 and CBL3 proteins expressed in Escherichia coli.…”

Crystallization and preliminary crystallographic analysis of a calcineurin B-like protein 1 (CBL1) mutant fromAmmopiptanthus mongolicus

“…To date, structures of AtCBL2 (Nagae et al, 2003) and its complex with the regulatory domain of AtCIPK14 (Akaboshi et al, 2008) and of AtCBL4 (Sá nchez- Barrena et al, 2005) and its complex with the C-terminus of AtCIPK24 (Sá nchez-Barrena et al, 2007) have been determined. These structures provide clues to understanding the relationship between calcium concentration and the calcium-binding capability of certain CBLs, i.e.…”