Detailed characterization of the regulatory domain of a plasma-membrane Ca2+-ATPase — a calcium pump — in complex with calmodulin results in a two-step structural model that explains how calmodulin-mediated regulation of pump activation affords highly responsive control over the intracellular calcium concentration in eukaryotes.
Control of cellular calcium levels: Calcium ions act as secondary messengers in many signal transduction cascades and are involved in a broad range of biological process, including muscle contraction and cellular motility. Plasma-membrane Ca2+-ATPases are key regulators of intracellular Ca2+ in eukaryotes, coupling the hydrolysis of ATP to the cellular export of calcium ions. However, it is not clear how the binding of calcium-loaded calmodulin leads to activation of the pump. In this manuscript, structural, biochemical and physiological experiments enabled the authors to propose a model that explains how the control of intracellular Ca2+ concentration is achieved by calmodulin-regulated Ca2+-ATPases over a broad range of physiological conditions.
Calcium ions (Ca2+) have an important role as secondary messengers in numerous signal transduction processes1,2,3,4, and cells invest much energy in controlling and maintaining a steep gradient between intracellular (∼0.1-micromolar) and extracellular (∼2-millimolar) Ca2+ concentrations1. Calmodulin-stimulated calcium pumps, which include the plasma-membrane Ca2+-ATPases (PMCAs), are key regulators of intracellular Ca2+ in eukaryotes5,6,7,8. 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 calmodulin-mediated PMCA regulation can cover both basal Ca2+ levels in the nanomolar range as well as micromolar-range Ca2+ transients generated by cell stimulation7. 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 Ca2+ concentration, where steep activation occurs.