Structural characterization of the conformational change in calbindin-D28k upon calcium binding using differential surface modification analyzed by mass spectrometry.

The calcium binding protein, calbindin-D28k plays a unique role in eukaryotic cells, acting as both a calcium buffer and sensor (1–4). It is found in several tissue types and serves many functions. For example, it is responsible for the selective reabsorption of calcium in the kidney and the intestine, as well as regulating the release of insulin in pancreatic islet cells (5, 6). Calbindin-D28k is very abundant in the brain, making up 0.1–1.5% of the total soluble protein. It is essential in neural functioning, altering synaptic interactions in the hippocampus, modulating calcium channel activity and neuronal firing (6–8). In addition to this, calbindin-D28k has been found to modulate the activity of proteins involved in the development of neurodegenerative disorders including Alzheimer’s and Huntington’s disease, as well as bipolar disorder (9–14). Deciphering how calbindin-D28k functions is essential for further understanding of the pathogenesis of these neurodegenerative disorders. Structurally, calbindin-D28k is made up of six EF hand domains, four of which bind calcium (2, 15). The EF hand domain is a helix-loop-helix calcium binding domain. The loop consists of twelve conserved residues responsible for coordinating calcium binding. EF hand calcium binding proteins are subdivided into two general groups, calcium buffers and calcium sensors (16–18). As stated above, calbindin-D28k is unique in that it functions as both. EF hand proteins in the sensor category are characterized by undergoing a conformational change that occurs upon calcium binding (19, 20). Mass spectrometry (MS) and nuclear magnetic resonance (NMR) experiments have shown that calbindin-D28k binds four calcium ions, in EF hands 1, 3, 4 and 5 and that the calcium ions are sequentially bound with EF hand 1 first, followed by 4 and 5 and finally EF hand 3 (15). NMR titration experiments have also demonstrated that the structure of calbindin-D28k is ordered in the apo- state (15). As calcium ions are bound, calbindin-D28k transitions into a disordered state and once fully-loaded with four calcium ions it returns to an ordered state. The effect that the conformational change has on the surface hydrophobicity of calbindin-D28k has been investigated and the studies showed that the surface hydrophobicity of rat brain calbindin-D28k is lowest in the holo- form (2). Similar experiments for human calbindin-D28k also showed a lower surface hydrophobicity for the holo- form versus the apo- form (1). Circular dichroism (CD) spectroscopy has shown that the secondary structure remains unperturbed upon calcium binding but the tertiary structure is much more sensitive to calcium binding (21). These experiments have provided some insight into the effect calcium binding has on the structure of calbindin-D28k. They do not, however, define the specific areas of the protein affected by the conformational change. The high resolution structure of the disulfide-reduced holo- rat brain calbindin-D28k structure has been solved using NMR (2), however, the structure of apo- calbindin-D28k remains to be solved. In the absence of a high resolution structure of apo- calbindin-D28k it has not been possible to compare the three dimensional structures of the apo- and holo- conformational states of calbindin-D28k. This study uses differential surface modification analyzed by MS to identify, for the first time, the specific regions of calbindin-D28k affected by the conformational changes between the apo- and holo- forms. MS analysis of differential surface modifications has been used for several purposes including mapping protein surfaces, studying protein-protein complexes and to determine ligand induced conformational changes by differentially modifying specific amino acid residue side chains (22–32). These side chain modifications reflect both the reactivity and surface accessibility of a specific residue. Side chain reactivity is affected by its surface accessibility as well as its surrounding microenvironment, including the presence of electrostatic interactions, such as the formation of a salt bridge (22–24, 33). Several studies have established that the relative reactivity data from differential surface modification experiments of both lysine and histidine residues correlate with their surface accessibility (22–24, 34). This allows the differential surface modification patterns to be used to identify regions of structural change that occur as a result of a specific perturbation, such as the binding of calcium (33, 35). Lysine acetylation and histidine modification analyzed by mass spectrometry have been used in this study to identify, for the first time, the specific regions of calbindin-D28k undergoing a conformational change upon calcium binding. We have investigated the status of the potential disulfide bond in the apo- form and have found the apo- form to be predominantly reduced. We have also modeled the conformational changes occurring as a result of disulfide bond formation based on the reduced holo- calbindin-D28k NMR structure, and have interpreted our differential reactivity results in terms of these structural changes.