Resolving Conformational Switches in the Protease Ftsh using Single-Molecule FRET

Abstract

FtsH is a homohexameric AAA+ protease embedded in the prokaryote membrane, where it recognizes, unfolds, translocates and lyses protein substrates. Previous crystal structural data of Thermotoga maritima FtsH in an ADP-bound and an apo-state showed an ATPase and a protease domain linked by a flexible unresolved hinge, facilitating a large conformational change upon ADP binding. The current model of the chemo-mechanical cycle was inferred from these two crystal structures and describes FtsH with a power stroke mechanism, whereby the energy released by ATP hydrolysis is converted into a conformational switch. To date, this mechanism could not be observed experimentally. Here, we use single-molecule Förster Resonance Energy Transfer (smFRET) to study FtsH in real time. Our assay involved the vesicle encapsulation of unlabeled and double-labeled monomers to promote self-assembled FtsH hexamers. Total Internal Reflection Fluorescence (TIRF) microscopy of the immobilized molecules allowed us to collect kinetic information of conformational changes. Data analysis based on hidden Markov modeling resolved distinct conformational states, transition coordinates and transition rates. Surprisingly, without nucleotide, an FtsH monomer adopted four distinct conformational states, undergoing highly dynamic interconversion on the second timescale. Thus, FtsH performed stochastic transitions independently of an energy source, but rather using thermal activation. Upon binding of ATP, the number of conformational states remained identical, however, presence of ATP reduced some barriers of the free energy landscape. A homolog mutation in FtsH, A359V, of the pathogenic mutation A510V in paraplegin displayed an additional further closed conformational state, which was highly populated in the presence of ATP. We propose that this highly populated state could reflect the malfunctioning of the pathogenic mutation in paraplegin.