RESUMO
Calbindin-D28k is a calcium binding protein that is highly expressed in the mammalian central nervous system. It has been reported that calbindin-D28k binds to and increases the activity of inositol Monophosphatase (IMPase). This is an enzyme that is involved in the homeostasis of the Inositol trisphosphate signalling cascade by catalysing the final dephosphorylation of inositol and has been implicated in the therapeutic mechanism of lithium treatment of bipolar disorder. Previously studies have shown that calbindin-D28k can increase IMPase activity by up to 250 hundred-fold. A preliminary in silico model was proposed for the interaction. Here, we aimed at exploring the shape and properties of the calbindin-IMPase complex to gain new insights on this biologically important interaction. We created several fusion constructs of calbindin-D28k and IMPase, connected by flexible amino acid linkers of different lengths and orientations to fuse the termini of the two proteins together. The resulting fusion proteins have activities 200%-400% higher the isolated wild-type IMPase. The constructs were characterized by small angle X-ray scattering to gain information on the overall shape of the complexes and validate the previous model. The fusion proteins form a V-shaped, elongated and less compact complex as compared to the model. Our results shed new light into this protein-protein interaction.
RESUMO
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 as a 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.
