Cryo-EM Reveals Regulatory Mechanisms Governing Substrate Selection and Activation of Human LONP1

The human AAA+ protease LONP1 plays a central role in maintaining mitochondrial proteostasis. LONP1 processes a vast array of substrates, ranging from damaged or unfolded proteins to specific subunits stably integrated into respiratory complexes. Previous cryo-EM studies of LONP1 uncovered two distinct conformational states corresponding to inactive or active forms of the enzyme. While these states have shed light on the intricacies of LONP1 substrate translocation and proteolytic processing, little is known about the decision-making involved in LONP1 substrate engagement and subsequent initiation of its unfoldase activity. Here, we use cryo-EM to determine a novel ADP-bound, C3-symmetric intermediate state of LONP1 (LONP1C3) with putative substrate "fold-sensing" capabilities. Our biochemical and structural data indicate that LONP1C3 is an on-pathway intermediate and that is stabilized by interaction with folded substrates. Moreover, we identify additional symmetric and asymmetric conformational states, including a two-fold symmetric split-hexamer conformation, that we associate with the transition from LONP1C3 to LONP1ENZ. We propose that the C3-state regulates substrate selection and enables LONP1 to efficiently surveil the matrix proteome to ensure selective removal of damaged and dysfunctional proteins as well as privileged LONP1 substrates. These findings collectively provide further mechanistic insights into LONP1 substrate recruitment and engagement and inform on its diverse roles in maintaining homeostasis within the mitochondria.