Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Aarhus University, Gustav Wieds Vej 10c, DK-8000 Aarhus C, Denmark.
Nature. 2012 Nov 15;491(7424):468-72. doi: 10.1038/nature11539. Epub 2012 Oct 21.
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 calmodulin-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.
钙离子 (Ca(2+)) 在众多信号转导过程中作为第二信使发挥着重要作用，细胞投入大量能量来控制和维持细胞内 (∼0.1 微摩尔) 和细胞外 (∼2 毫摩尔) Ca(2+) 浓度之间的陡峭梯度。钙调蛋白刺激的钙泵，包括质膜 Ca(2+)-ATP 酶 (PMCA)，是真核生物细胞内 Ca(2+) 的关键调节剂。它们包含一个独特的氨基或羧基末端调节域，负责自动抑制，并且结合钙负载的钙调蛋白到这个域释放自动抑制并激活泵。然而，激活机制的结构基础尚不清楚，一个关键的遗留问题是钙调蛋白介导的 PMCA 调节如何覆盖基础 Ca(2+) 水平在纳米摩尔范围内以及细胞刺激产生的微摩尔范围 Ca(2+) 瞬变。在这里，我们结合高分辨率的 PMCA 调节域/钙调蛋白复合物的晶体结构测定，以及体内表征和生化、生物物理和生物信息学数据进行综合研究，为钙调蛋白介导的 PMCA 激活机制提供了机制见解。该结构显示了整个 PMCA 调节域，并揭示了一个意想不到的 2:1 比例，两个钙负载的钙调蛋白分子结合到一个长螺旋上的不同位点。对两个位点作用的多方面表征导致了钙调蛋白介导的 PMCA 调节的一般结构模型，该模型允许对真核生物细胞内钙进行严格、高度响应的控制，从而有可能在阈值 Ca(2+) 浓度下维持稳定的基础水平，此时会发生陡峭的激活。
