Reconstitution of the P-type ATPase CopA into Nanodiscs: a platform for molecular spectroscopy

Reconstitution of membrane proteins in a native-like lipidic environment is crucial for the in vitro determination of their structural and functional properties. Nanoscale protein-bounded planar lipid bilayers, so-called Nanodiscs1, provide a versatile novel model membrane system into which membrane proteins can be incorporated in a monodisperse and active form being accessible from both sides of the membrane. They exhibit less scatter than liposomes and bicelles and are soluble in aqueous solution. We succeeded in the reconstitution of the evolutionary conserved P-type ATPase CopA from Legionella pneumophila into Nanodiscs, which is a key player in copper homeostasis throughout all kingdoms of life2. This provides us with an excellent platform for spectroscopic studies of the allosteric couplings that are associated with ATP-powered copper transport in this enzyme in fully controllable lipidic environments. Our current focus lies on the real-time observation of the allosteric coupling of the cytosolic nucleotide-binding domain to the intramembranous conserved copper-binding CPC-motif (cysteine‒prolin‒cysteine) in transmembrane helix 4. Moreover, the CopA‒Nanodisc system allows addressing specifically the influence of the lipid environment in the catalytic cycle. We are particularly interested in the role of water entry into the transmembrane region at specific catalytic intermediates. To this end, we use cysteine-reactive fluorophores as molecular probes for the physical environment of the copper-binding CPC-motif. Comparison between mutated versions of the CPC-motif enables a detailed view of structural transitions in the transmembrane part of the enzyme evoked by allosteric coupling. The biochemical platform represented by the Nanodiscs also opens new routes for the analysis of structural dynamics by time-resolved fluorescence spectroscopy. Our presented data on the copper-binding site hydration can be interpreted in the context of conformational changes proposed from crystal structures of CopA3.

References
1. T.H. Bayburt, S.G. Sligar. FEBS Lett. 2010, 584, 1721–1727.
2. J.M. Argüello, E. Eren, M. González-Guerrero. Biometals, 2007, 20, 233–248.
3. M. Andersson, D. Mattle, O. Sitsel, T. Klymchuk, A.M. Nielsen, L.B. Møller, S.H. White, P. Nissen, P. Gourdon. Nat. Struct. Mol. Biol. 2013, 21, 43–50.