ATPase and Protease Domain Movements in the Bacterial AAA+ Protease FtsH Are Driven by Thermal Fluctuations.

Abstract AAA+ proteases are essential players in cellular pathways of protein degradation. Elucidating their conformational behavior is key for understanding their reaction mechanism and, importantly, for elaborating our understanding of mutation-induced protease deficiencies. Here, we study the structural dynamics of the Thermotoga maritima AAA+ hexameric ring metalloprotease FtsH (Tm FtsH). Using a single-molecule Förster resonance energy transfer approach to monitor ATPase and protease inter-domain conformational changes in real time, we show that Tm FtsH—even in the absence of nucleotide—is a highly dynamic protease undergoing sequential transitions between five states on the second timescale. Addition of ATP does not influence the number of states or change the timescale of domain motions but affects the state occupancy distribution leading to an inter-domain compaction. These findings suggest that thermal energy, but not chemical energy, provides the major driving force for conformational switching, while ATP, through a state reequilibration, introduces directionality into this process. The Tm FtsH A359V mutation, a homolog of the human pathogenic A510V mutation of paraplegin (SPG7) causing hereditary spastic paraplegia, does not affect the dynamic behavior of the protease but impairs the ATP-coupled domain compaction and, thus, may account for protease malfunctioning and pathogenesis in hereditary spastic paraplegia. Graphical Abstract Unlabelled Image Highlights • ATPase and protease inter-domain conformational changes of Thermotoga maritima FtsH (Tm FtsH) are studied. • A single-molecule Förster resonance energy transfer assay is developed that allows for probing of structural changes in single Tm FtsH monomers within self-hexamerized Tm FtsH rings. • Inter-domain movements occur on the second timescale between five states, are thermally driven, and weakly coupled to ATP binding or hydrolysis. • Addition of ATP does not influence the number of states or change the timescale of domain motions, but affects the state occupancy distribution leading to an inter-domain compaction. • The Tm FtsH A359V mutation, which is homologous to the human A510V paraplegin mutation, hinders ATP-induced compaction and thus may account for protease malfunctioning in hereditary spastic paraplegia. [ABSTRACT FROM AUTHOR]