The role of protein-protein interactions in Fluc dimerization stability and function.

Abstract

Functional ion channel assembly often requires subunit oligomerization. Yet the molecular factors that define the stability of ion channel complexes in membranes remain to be delineated. Here we investigate this question by studying the dynamic dimerization reaction of the model system Fluc to map out the molecular and physical forces driving this reaction in membranes. Fluc is dual-topology homodimer, where dimerization confers the highly specific fluoride permeation pathway. Our previous analysis demonstrated that a sodium titratable mutant of Fluc participates in a two-step equilibrium dimerization reaction in membranes. First, membrane-dependent dimerization drives subunits to assemble and form a non-contact lipid-solvated stable dimer intermediate by burying a membrane defect imposed in the monomer. However, the fluoride conducting state requires the protein subunits to come closer and expel the lipids to form the fluoride permeation pathway. High resolution crystal structures of Fluc show numerous protein-protein interactions across the dimerization interface, suggesting these interactions might be involved in stabilizing the close-contact dimer state. Alternatively, these contacts may not play a major role in the overall dimer stability but may be important for conferring function. Here, we will investigate how disruption of these protein-protein interactions affects dimerization stability, channel activity or both. Site-directed mutagenesis was employed following four strategies of disruption: (1) reduction of van der Waals interactions by leucine-to-alanine substitutions; (2) removal of potential hydrogen bonds by threonine-to-valine substitutions; (3) decrease backbone flexibility by glycine-to-alanine mutations of the GxxxG dimerization motif; (4) and introduction of bulky tryptophan residues at the interface. Using single-molecule photobleaching analysis to investigate dimer stability in membranes combined with single channel bilayer recordings, these results will provide a quantitative characterization of the contribution of protein-protein interactions to Fluc dimer stability and activity.