Tracking the structural dynamics of SERCA regulation in real time.

Abstract

Calcium (Ca2+) signaling in eukaryotic cells is integral to biological processes such as neuronal activity, transcription, and muscle contraction-relaxation. Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) proteins play a vital role in this intricate signaling network by returning Ca2+ to the sarco/endoplasmic reticulum (SR/ER), thereby resetting the signal and restoring SR/ER Ca2+ levels. SERCA activity is optimised for different tissues and developmental stages by expression of different isoforms and by regulatory factors such as regulatory peptides, Ca2+ concentration and internal regulatory domains. Mutations are known to cause Brody's (SERCA1) and Darier's (SERCA2) diseases, and dysregulation of SERCA2a is associated with heart failure. To investigate the kinetic and structural effects of SERCA regulation, we performed time-resolved x-ray solution scattering (TR-XSS) experiments on two SERCA isoforms. In our TR-XSS setup the reaction is initiated by laser-induced ATP release, and the resulting structural dynamics are then monitored in real time using synchrotron x-ray pulses. We investigated the effect of Ca2+ concentration on SERCA1a in native membranes and identified three distinct structural states that developed slower under high-Ca2+ conditions and showed structural differences compared to the low-Ca2+ condition. The SERCA2b isoform has a unique regulatory domain consisting of an 11th transmembrane helix and a luminal extension loop that increases its Ca2+ affinity and lowers the maximal turnover rate. We collected TR-XSS datasets for SERCA2b with and without this regulatory region and observed differences in the reaction-cycle kinetics that indicate a loss of control of transitions between states. In summary, we have developed a synchrotron-based x-ray technique and determined the kinetic and structural basis of regulation of P-type ATPase transport across biological membranes.