A bimodular mechanism of calcium control in eukaryotes

摘要

Calcium ions (Ca~(2+)) have an important role as secondary messengers in numerous signal transduction processes, and cells invest much energy in controlling and maintaining a steep gradient between intracellular (~0.1-micromolar) and extracellular (~2-millimolar) Ca~(2+) concentrations. Calmodulin-stimulated calcium pumps, which include the plasma-membrane Ca~(2+)-ATPases (PMCAs), are key regulators of intracellular Ca~(2+)in eukaryotes. They contain a unique amino- or carboxy-terminal regulatory domain responsible for autoinhibition, and binding of calcium-loaded calmodulin to this domain releases autoinhibition and activates the pump. However, the structural basis for the activation mechanism is unknown and a key remaining question is how cal-modulin-mediated PMCA regulation can cover both basal Ca~(2+) levels in the nanomolar range as well as micromolar-range Ca~(2+) transients generated by cell stimulation. Here we present an integrated study combining the determination of the high-resolution crystal structure of a PMCA regulatory-domain/calmodulin complex with in vivo characterization and biochemical, biophysical and bioinformatics data that provide mechanistic insights into a two-step PMCA activation mechanism mediated by calcium-loaded calmodulin. The structure shows the entire PMCA regulatory domain and reveals an unexpected 2:1 stoichiometry with two calcium-loaded calmodulin molecules binding to different sites on a long helix. A multifaceted characterization of the role of both sites leads to a general structural model for calmodulin-mediated regulation of PMCAs that allows stringent, highly responsive control of intracellular calcium in eukaryotes, making it possible to maintain a stable, basal level at a threshold Ca~(2+) concentration, where steep activation occurs.