Artificial native-like lipid bilayer systems constructed from phospholipids assembling into unilamellar liposomes allow the reconstitution of detergent-solubilized transmembrane proteins into supramolecular lipid-protein assemblies called proteoliposomes, which mimic cellular membranes. Stabilization of these complexes remains challenging because of their chemical composition, the hydrophobicity and structural instability of membrane proteins, and the lability of interactions between protein, detergent, and lipids within micelles and lipid bilayers. In this work we demonstrate that metastable lipid, protein-detergent, and protein-lipid supramolecular complexes can be successfully generated and immobilized within zeolitic-imidazole framework (ZIF) to enhance their stability against chemical and physical stressors. Upon immobilization in ZIF bio-composites, blank liposomes, and model transmembrane metal transporters in detergent micelles or embedded in proteoliposomes resist elevated temperatures, exposure to chemical denaturants, aging, and mechanical stresses. Extensive morphological and functional characterization of the assemblies upon exfoliation reveal that all these complexes encapsulated within the framework maintain their native morphology, structure, and activity, which is otherwise lost rapidly without immobilization.
Copper is essential for living cells, yet toxic at elevated concentrations. Class 1B P-type (P1B-) ATPases are present in all kingdoms of life, facilitating cellular export of transition metals including copper. P-type ATPases follow an alternating access mechanism, with inward-facing E1 and outward-facing E2 conformations. Nevertheless, no structural information on E1 states is available for P1B-ATPases, hampering mechanistic understanding. Here, we present structures that reach 2.7 Å resolution of a copper-specific P1B-ATPase in an E1 conformation, with complementing data and analyses. Our efforts reveal a domain arrangement that generates space for interaction with ion donating chaperones, and suggest a direct Cu+ transfer to the transmembrane core. A methionine serves a key role by assisting the release of the chaperone-bound ion and forming a cargo entry site together with the cysteines of the CPC signature motif. Collectively, the findings provide insights into P1B-mediated transport, likely applicable also to human P1B-members.
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