Date Published: October 01, 2018
Publisher: International Union of Crystallography
Author(s): James W. Noble, Rehab Almalki, S. Mark Roe, Armin Wagner, Ramona Duman, John R. Atack.
The X-ray structure of human calbindin-D28K, a calcium-buffering protein that is highly expressed in the central nervous system, is reported.
Calbindin-D28K is a major calcium-buffering cytoplasmic protein that is expressed at particularly high levels in the central nervous system (CNS) and absorptive epithelium (gut and kidney; Schmidt, 2012 ▸). Calcium signalling is tightly regulated and is involved in a myriad of physiological processes, and consequently calcium deregulation is a key factor in the pathogenesis of many diseases, such as Alzheimer’s disease (Bojarski et al., 2008 ▸; Kook et al., 2014 ▸). Calbindin-D28K was first identified in the intestine, colon, kidney and uterus of Gallus gallus domesticus (chicken; Wasserman et al., 1969 ▸), where it is involved in the transcellular movement of calcium across the absorptive epithelium, as found in the distal convoluted tubules of the kidney (Lambers et al., 2006 ▸). Calbindin-D28K is also highly expressed in the CNS, where it contributes up to 1.5% of the total soluble protein (Christakos et al., 1989 ▸; Berggård, Szczepankiewicz et al., 2002 ▸). In chicken kidney cells the expression of calbindin-D28K is vitamin D dependent, and this is true for other absorptive cells (Clemens et al., 1989 ▸); however, it is not the case in the CNS (Arnold & Heintz, 1997 ▸). Calbindin-D28K has been reported to regulate the depolarization-stimulated release of insulin from pancreatic β cells through the regulation of the cytoplasmic calcium concentration (Sooy et al., 1999 ▸). It is well documented in the literature that calbindin-D28K has neuroprotective properties in the CNS (Yenari et al., 2001 ▸; Yuan et al., 2013 ▸; Sun et al., 2011 ▸), and it has recently been demonstrated that its depletion in an Alzheimer’s disease mouse model accelerates neuronal loss, apoptosis and mitochondrial dysfunction (Kook et al., 2014 ▸).
Calbindin-D28K is a widely expressed calcium-binding protein that possesses a multiplicity of physiological functions. Its expression is particularly high in the central nervous system and absorptive epithelial tissues, where it buffers and facilitates the movement of calcium (Clemens et al., 1989 ▸; Schmidt, 2012 ▸; Wasserman et al., 1969 ▸). Recent work has hinted at a possible protective role of calbindin-D28K in inhibiting apoptosis and necrosis, and in slowing the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (Yuan et al., 2013 ▸; Yenari et al., 2001 ▸; Sun et al., 2011 ▸; Kook et al., 2014 ▸; Bellido et al., 2000 ▸). Not only does calbindin-D28K act as a buffer for calcium ions, but it has also been shown to interact with multiple protein targets to modulate their function or catalytic activity (Shamir et al., 2005 ▸; Berggård, Szczepankiewicz et al., 2002 ▸; Lutz et al., 2003 ▸). Here, we report the first X-ray structure of calbindin-D28K, allowing the first detailed high-resolution analysis of its calcium-binding properties. The X-ray structure of human calbindin-D28K also displays significant structural differences when compared with the previously published NMR structure of the rat calbindin-D28K molecule. The human and rat isoforms have a sequence identity of 98%. Gln44, Asp225, Thr232 and Cys257 in human calbindin-D28K are changed to Leu44, Glu225, Ser232 and Ser257 in the rat isoform. These residues are solvent-exposed and do not seem to explain the structural difference observed between the two structures. Human calbindin-D28K consists of six EF-hand motifs arranged in three pairs that maintain a globular structure stabilized by hydrophobic interactions (Kojetin et al., 2006 ▸). Four EF-hands bind calcium at a concentration of ∼1 mM in the crystallization buffer, a concentration that is significantly higher than the usually nanomolar physiological (cytosolic) concentration. This would imply that the structure presented here represents a calcium-saturated form of the protein with respect to physiological conditions (Berridge, 1997 ▸). There are differences in the calcium-binding residues between the individual calcium-binding loops; despite these differences in primary structure, macroscopic studies have indicated that calbindin-D28K binds calcium in a nonsequential, parallel manner (Berggård, Miron et al., 2002 ▸). However, other spectroscopic studies have indicated the opposite, that calcium binding is not simultaneous (Venters et al., 2003 ▸), and the differences in the calcium-binding mechanisms observed in the crystal structure support this. EF3 is the only calcium-binding loop that does not use water in the pentagonal bipyramidal coordination of the calcium ion; Glu119 at position 9 instead fulfils this role. Interestingly, both EF1 and EF4 also have glutamate at position 9 of the loop, but these residues are not involved in calcium coordination. Compared with the existing NMR model of calbindin-D28K, the X-ray structure is less compact, with the intramolecular distances increased by several ångströms across the molecule. This less condensed model was validated by SAXS, as the scattering curve of the protein in solution was better predicted by the crystal structure. This is surprising as it is often assumed that crystal structures would be more compact than solution NMR structures owing to crystal-packing restraints. The high overall r.m.s.d. value of 3.2 Å also reflects significant structural differences between the X-ray structure and the NMR model. There are substantial differences in side-chain conformations both across the molecule and within the calcium-binding loops. Calcium binding at the N-terminal EF-hand (EF1) motif (where the electron density is more disordered) appears to be more flexible that in the other calcium-binding loops. The calcium SAD experiment indicated that there are two distinct calcium positions in the N-terminal EF-hand, with the calcium ions bound 4.7 Å apart in the crystal form reported by Zhang et al. (2008 ▸) (crystallization condition 1). Although the calcium SAD data did yield a phasing solution, the dual calcium-binding geometry precluded full structure refinement. Consequently, new crystal-growth conditions potentially favouring a single conformation were explored, yielding crystal condition 2 in which residues are visible at the N-terminus. Nevertheless, the N-terminal flexibility still manifests as structural disorder, with higher temperature-factor values in this region. It was not possible to build the two conformations into the electron density, and only one calcium-binding site is visible in the final structure. The flexibility of this region could be important in facilitating interactions with other proteins. Site-directed mutagenesis studies have previously revealed that inositol monophosphatase (IMPase) binds to aspartate residues 24 and 26 in this flexible calcium-binding loop (EF1) of calbindin-D28K (Levi et al., 2013 ▸). Here, we demonstrate that Asp24 and Asp26 are involved in calcium binding. It has previously been demonstrated that the potentiation of IMPase catalytic activity by calbindin-D28K occurs irrespective of calcium being present (Berggård, Szczepankiewicz et al., 2002 ▸). The peptide-binding fragment of Ran-binding protein M has also been shown to induce large chemical shifts in the NMR spectrum of calbindin-D28K in the flexible N-terminal region (Lutz et al., 2003 ▸). Docking simulations predict the binding of IMPase at the grooved structure between the N- and C-terminal EF-hand bundles of calbindin-D28K, where the X-ray structure differs the most from the NMR model.
We present here the first X-ray structure of calbindin-D28K at near-atomic resolution. Elucidation of the calcium coordination geometry in the EF-hand loops is consistent with previous reports that the protein has four calcium-binding sites. Calcium SAD at long wavelength demonstrated that the N-terminal EF-hands are particularly flexible and possesses two calcium-binding conformations. Residues that have previously been shown to be involved in protein–protein interactions are demonstrated to also coordinate calcium, potentially bestowing a calcium-sensor function on calbindin-D28K. Calbindin-D28K maintains the same globular shape in both the calcium-loaded and unloaded forms. There is also no significant increase in the flexibility of the calbindin-D28K protein in the unloaded form. Intriguingly, PDB entry 6fie is a better model of calbindin-D28K in solution, as the X-ray scattering curve produced by the protein is better predicted by the crystal structure than by the previously published NMR model (PDB entry 2g9b). We surmise that the high-resolution X-ray structure of calbindin-D28K presented here should be used as the model of choice for future experimentation and in silico modelling.
The following reference is cited in the Supporting information for this article: Thorn & Sheldrick (2011 ▸).