Fe$^{2+}$ -catalyzed oxidative cleavages of Ca$^{2+}$ -ATPase reveal novel features of its pumping mechanism

Cédric Montigny (1) , Christine Jaxel (2) , Alla Shainskaya (3) , Joelle Vinh (4, 5) , Valérie Labas (4, 5) , Jesper V. Møller (6) , Steven J. D. Karlish (3) , Marc Le Maire (1)

1 LPSM - Laboratoire des Protéines et Systèmes Membranaires
2 SMPSD - UMR 8221 - Systèmes membranaires, photobiologie, stress et détoxification
3 Weizmann Institute of Science [Rehovot, Israël]
4 NDC - Neurobiologie et diversité cellulaire
5 ESPCI Paris - Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris
6 Aarhus University [Aarhus]

We have analyzed the Fe$^{2+}$-catalyzed oxidative cleavages of Ca$^{2+}$-ATPase in the presence of Ca$^{2+}$, with or without the ATP analog 5'-adenylyl-$\beta \gamma$-imidodiphosphate (AMP-PNP) or in the presence of the inhibitor thapsigargin. To identify the positions of cleavages as precisely as possible, we have used previously identified proteinase K and tryptic fragments as a standard, advanced mass spectrometry techniques, as well as specific antibodies. A number of cleavages are similar to those described for Na$^+$,K$^+$-ATPase or other P-type pumps and are expected on the basis of the putative Mg2$^{2+}$ binding residues near the phosphorylated Asp$^{351}$ in E1 or E2P conformations. However, intriguing new features have also been observed. These include a Fe$^{2+}$ site near M3, which cannot be due to the presence of histidine residues as it was postulated in the case of Na$^+$,K$^+$-ATPase and H$^+$,K$^+$-ATPase. This site could represent a Ca$^{2+}$ binding zone between M1 and M3, preceding Ca$^{2+}$ occlusion within M4, 5, 6, and 8. In addition, we present evidence that, in the non-crystalline state, the N- and P-domain may approach each other, at least temporarily, in the presence of Ca$^{2+}$ (E1Ca$_2$ conformation), whereas the presence of MgATP stabilizes the N to P interaction (E1MgATP conformation).

Domaines: Sciences du Vivant [q-bio]; Chimie

Cédric Montigny, Christine Jaxel, Alla Shainskaya, Joelle Vinh, Valérie Labas, et al.. Fe$^{2+}$ -catalyzed oxidative cleavages of Ca$^{2+}$ -ATPase reveal novel features of its pumping mechanism. Journal of Biological Chemistry, 2004, 279 (42), pp.43971 - 43981. ⟨10.1074/jbc.M407142200⟩. ⟨hal-01636936⟩