Structural Biology of Calcium Signaling and Transport through Biological Membranes
Calcium ions (Ca2+) act as universal messengers required to regulate diverse physiological processes including fertilization, muscle contraction, apoptosis, secretion, and synaptic plasticity. Upon external stimuli, Ca2+-permeable ion channels, which are essential components of the calcium signaling toolkit, mediate the rapid transfer of Ca2+ from the extracellular space or intracellular Ca2+ stores (mainly the endoplasmic reticulum (ER) and mitochondria) to the cytoplasm generating local and global Ca2+ signals. Tight regulation of ion channel activity is critical for proper calcium signaling and aberrant channel activity is associated with many diseases and disorders.
Our major goal is understanding the molecular mechanism and regulation of Ca2+ signaling at the endoplasmic reticulum (ER)-mitochondria contact sites, where ER and mitochondria are linked through proteinaceous tethers located at the specialized ER subdomains named as mitochondria-associated membranes (MAMs) and outer membrane of mitochondria. At these sites, inositol 1,4,5-triphosphate receptors (IP3Rs) release Ca2+ from the ER, creating local hot spots necessary for Ca2+ uptake by mitochondrial calcium uniporter (MCU) in the inner mitochondrial membrane. Sustained Ca2+ transfer to mitochondria is necessary to maintain ATP generation, whereas excessive or reduced calcium transfer leads to initiation of apoptotic cell death or autophagy, respectively. Consequently, Ca2+ signaling at ER-mitochondria interface plays an essential role in cell fate decisions and could be an invaluable target when the cell fate decision machinery is compromised, as observed in cancer (evasion of apoptosis) and neurodegenerative diseases (excessive apoptosis).
Our overall approach is structural characterization of the target proteins using X-ray crystallography and electron cryo-microscopy (cryo-EM) followed by validation of the structural information and the structure-derived hypotheses. We use a repertoire of biophysical and biochemical methods such as analytical ultracentrifugation (AUC), multiangle light scattering (MALS), Isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and crosslinking to deduce the molecular mechanism of gating, oligomeric assembly, stoichiometry, protein-protein and protein-ligand interactions.