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 metalloprotease FtsH (TmFtsH). Using a single-molecule FRET approach to monitor the real-time ATPase and protease inter-domain conformational changes, we show that TmFtsH—even in the absence of nucleotide—is a highly dynamic protease undergoing conformational transitions between five states on the second timescale. Addition of ATP does not influence the number of states nor 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 TmFtsH A359V mutation, a homolog of the human pathogenic A510V mutation of paraplegin causing hereditary spastic paraplegia (HSP), impairs the ATP-coupled domain compaction and, thus, may account for protease malfunctioning and pathogenesis in HSP.