Abstract
Calbindin-D28K is a widely expressed calcium-buffering cytoplasmic protein that is involved in many physiological processes. It has been shown to interact with other proteins, suggesting a role as a calcium sensor. Many of the targets of calbindin-D28K are of therapeutic interest: for example, inositol monophosphatase, the putative target of lithium therapy in bipolar disorder. Presented here is the first crystal structure of human calbindin-D28K. There are significant deviations in the tertiary structure when compared with the NMR structure of rat calbindin-D28K (PDB entry 2g9b), despite 98% sequence identity. Small-angle X-ray scattering (SAXS) indicates that the crystal structure better predicts the properties of calbindin-D28K in solution compared with the NMR structure. Here, the first direct visualization of the calcium-binding properties of calbindin-D28K is presented. Four of the six EF-hands that make up the secondary structure of the protein contain a calcium-binding site. Two distinct conformations of the N-terminal EF-hand calcium-binding site were identified using long-wavelength calcium single-wavelength anomalous dispersion (SAD). This flexible region has previously been recognized asa protein-protein interaction interface. SAXS data collected in both the presence and absence of calcium indicate that there are no large structural differences in the globular structure of calbindin-D28K between the calcium-loaded and unloaded proteins.
Highlights
Calbindin-D28K is a major calcium-buffering cytoplasmic protein that is expressed at high levels in the central nervous system (CNS) and absorptive epithelium
Calbindin-D28K was first identified in the intestine, colon, kidney and uterus of Gallus gallus domesticus, 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)
Calcium single-wavelength anomalous dispersion (SAD) at long wavelength demonstrated that the N-terminal EF-hands are flexible and possesses two calcium-binding conformations
Summary
Calbindin-D28K is a major calcium-buffering cytoplasmic protein that is expressed at high levels in the central nervous system (CNS) and absorptive epithelium (gut and kidney; Schmidt, 2012). 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). 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); 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 cells through the regulation of the cytoplasmic calcium
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