Molecular Dynamics of the Neuronal EF-Hand Ca2+-Sensor Caldendrin

Abstract

Caldendrin, L- and S-CaBP1 are CaM-like Ca2+-sensors with different N-termini that arise from alternative splicing of the Caldendrin/CaBP1 gene and that appear to play an important role in neuronal Ca2+-signaling. In this paper we show that Caldendrin is abundantly present in brain while the shorter splice isoforms L- and S-CaBP1 are not detectable at the protein level. Caldendrin binds both Ca2+ and Mg2+ with a global Kd in the low µM range. Interestingly, the Mg2+-binding affinity is clearly higher than in S-CaBP1, suggesting that the extended N-terminus might influence Mg2+-binding of the first EF-hand. Further evidence for intra- and intermolecular interactions of Caldendrin came from gel-filtration, surface plasmon resonance, dynamic light scattering and FRET assays. Surprisingly, Caldendrin exhibits very little change in surface hydrophobicity and secondary as well as tertiary structure upon Ca2+-binding to Mg2+-saturated protein. Complex inter- and intramolecular interactions that are regulated by Ca2+-binding, high Mg2+- and low Ca2+-binding affinity, a rigid first EF-hand domain and little conformational change upon titration with Ca2+ of Mg2+-liganted protein suggest different modes of binding to target interactions as compared to classical neuronal Ca2+-sensors.

Intermotif Communication Induces Hierarchical Ca2+ Filling of Caldendrin

“…The overexpressed protein was purified using the IMPACT kit (New England Biolabs), following the manufacturers' protocol as described earlier (more details are provided in Supplementary Information). 6 While NTW, EF1W, EF3W, EF4W and H3W mutants of CDD over-expressed well in E. coli, EF2W mutant of CDD did not over-express.…”

“…3,4 The protein has a unique bipartite structure with a basic and prolinerich N-terminus, and a C-terminus that it shares with two shorter splice isoforms, S-CaBP1 and L-CaBP1 (Supplementary Figure S1), which are much less abundant in brain. [3][4][5][6][7] The structural information of CDD is not known, however, based on the structure of S-CaBP1, the C-terminus resembles the structure of CaM with four EF-hand motifs, but in contrast to CaM, the first EFhand motif can probably bind both Mg 2+ and Ca 2+ , while the second EF-hand motif is cryptic. 6,8,9 Over the years, a larger CDD interactome has been identified, 1 but still very little is known how CDD operates as a Ca 2+ sensor that accommodates different binding partners.…”

“…[3][4][5][6][7] The structural information of CDD is not known, however, based on the structure of S-CaBP1, the C-terminus resembles the structure of CaM with four EF-hand motifs, but in contrast to CaM, the first EFhand motif can probably bind both Mg 2+ and Ca 2+ , while the second EF-hand motif is cryptic. 6,8,9 Over the years, a larger CDD interactome has been identified, 1 but still very little is known how CDD operates as a Ca 2+ sensor that accommodates different binding partners. A frequently neglected aspect in this regard is to understand whether an EF-hand protein has stable conformational states during the transition from the apo to the holo form.…”

A nuclease that mediates cell death induced by DNA damage and poly(ADP-ribose) polymerase-1

“…Near-UV CD spectra were recorded between 240–320 nm using a quartz cuvette of 0.5 cm path length with protein samples at a concentration of 2 mg/ml at room temperature. Far UV CD spectra were also recorded at room temperature between 190–260 nm using quartz sandwich cuvettes of 0.1 cm path length with protein samples at a concentration of 0.2 mg/ml (48). The proteins were suspended in PBS buffer with or without magnesium chloride (5.0 mM) and/or zinc chloride (0.2 mM).…”

Protein-Ligand Interactions and Allosteric Regulation of Activity in DREAM Protein

“…Since the stronger affinity binding sites of TFP on CaM are at the C-terminus domain, the data shows that this domain is mainly responsible for binding the DRE29 peptide (Cook, Walter & Walter 1994). caldendrin (Liang et al 2009) , (Reddy et al 2014). Therefore, to test whether the Nterminus of DREAM (residues 29-44) physically interacts with the core domain we conducted titrations of DRE29 with DREAM(Δ65).…”