Unexpected mechanism of calcium pump

In eukaryotes, calmodulin-stimulated calcium pumps including plasma-membrane Ca2+-ATPases are key regulators in intracellular Ca2+ concentration. The precise mechanism of how these pumps are regulated remained elusive. Here, we identified a bimodular, calmodulin-mediated activation mechanism of plasma-membrane Ca2+-ATPases.

HFSP Long-Term Fellow Henning Tidow and Program Grant holder Poul Nissen and colleagues
authored on Fri, 19 October 2012

Calcium ions (Ca2+) play an important role as secondary messengers in numerous signal transduction processes. Low intracellular Ca2+ concentrations are controlled and maintained by calmodulin-stimulated calcium pumps (including the plasma-membrane Ca2+-ATPases (PMCAs)). These pumps contain a unique regulatory/autoinhibitory domain. Binding of calcium-loaded calmodulin (Ca2+-CaM) to this domain releases autoinhibition and activates the pump. However, the structural basis for the activation mechanism remained elusive.

Figure1: Crystals of the (CaM7)2/ACA8R complex from A. thaliana.

We have determined the crystal structure of a PMCA regulatory domain/calmodulin complex, which revealed an unexpected 2:1 stoichiometry with two Ca2+-CaM molecules binding to different calmodulin-binding sites displayed on a long helix. In vivo and biochemical characterization showed that both calmodulin-binding sites are involved in autoinhibition and led to mechanistic insights into a novel modular, two-step Ca2+-CaM-mediated activation mechanism of PMCAs. Under low Ca2+ concentrations the pump is inactive. If the concentration rises, binding of calmodulin to one site within the regulatory domain leads to a basal activity state, while even higher Ca2+ concentration will cause full pump activity via binding of calmodulin to both sites. Mathematical modelling indicates that the role of the second calmodulin-binding site is to maintain a stable, basal level at a threshold Ca2+ concentration, where steep activation occurs. This mechanism is present in plants and mammals despite the lack of sequence similarity and different location of their regulatory domains (N-terminus in plants, C-terminus in mammals).

Figure2: Overall structure of the A. thaliana (CaM7)2/ACA8R complex. Cartoon representation with CaM molecules in red (CaMBS1) and blue (CaMBS2) and ACA8R in brown, yellow (CaMBS1) and green (CaMBS2). Ca2+ are shown in magenta.

In conclusion, we have developed a general structural model for calmodulin-mediated regulation of PMCAs allowing a stringent and highly responsive control of intracellular calcium concentration in eukaryotes.

Reference

A bimodular mechanism of calcium control in eukaryotes. Henning Tidow, Lisbeth R. Poulsen, Antonina Andreeva, Michael Knudsen, Kim L. Hein, Carsten Wiuf, Michael G. Palmgren and Poul Nissen. Nature doi:10.1038/nature11539.