Membrane mimetics control internal hydration and catalytic activity of the copper ATPase LpCopA from Legionella pneumophila

Abstract

Abstract textMembrane protein function depends critically on the surrounding lipid bilayer, which supports specific lipid-protein interactions and imposes constraints through its ensemble properties. Ion-transporting ATPases undergo large conformational changes that perturb these interactions, and the high chemical potential of water can further drive conformational transitions through changes in internal hydration. Here, we investigated the relationship between membrane environment, internal hydration, and catalytic activity in the P1B-type copper ATPase LpCopA from Legionella pneumophila. BADAN-labeled LpCopA mutants revealed distinct basal hydration states near the conserved canonical binding site (CBS) cysteines C382 and C384, with C382 residing in a more hydrated environment than C384. Using the osmolyte PEG-1500, the spectral response of BADAN indicated an average internal hydration volume of about 800 [A]3 for LpCopA in E. coli lipid-doped mixed micelles (MMs), corresponding to approximately 25 water molecules. In nanodiscs (NDs), less than half of this volume responded to osmotic pressure, consistent with compaction of the transmembrane helical bundle under membrane lateral pressure. Remarkably, ATPase activity measured in different lipid reconstitution systems scaled with the extent of internal hydration, with diisobutylene/maleic acid lipid particles (DIBMALPs) imposing the tightest transmembrane compaction and lowest basal activity. These data indicate that the packing density and internal hydration of the transmembrane domain of LpCopA are strongly modulated by the membrane environment. The energetic estimates further support a model in which hydration-dependent expansion of the transmembrane domain against membrane lateral pressure contributes to the free-energy barrier of ATP hydrolysis.