biostudies-literatureLiang WGSimons FoundationNIGMS NIH HHSU.S. Department of Health & Human Services | National Institutes of Health1833https://www.ebi.ac.uk/biostudies/studies/S-EPMC8983764biostudies-literatureUnknown13(1)Presequence protease (PreP), a 117 kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other aggregation-prone peptides, such as amyloid β (Aβ). PreP structures have only been determined in a closed conformation; thus, the mechanisms of substrate binding and selectivity remain elusive. Here, we leverage advanced vitrification techniques to overcome the preferential denaturation of one of two ~55 kDa homologous domains of PreP caused by air-water interface adsorption. Thereby, we elucidate cryoEM structures of three apo-PreP open states along with Aβ- and citrate synthase presequence-bound PreP at 3.3-4.6 Å resolution. Together with integrative biophysical and pharmacological approaches, theNature communicationsStructural basis for the mechanisms of human presequence protease conformational switch and substrate recognition.PMC8983764GM 121964R35 GM143052SF349247SF 349547P41 GM103310R01 GM121964U24 GM129539P41 GM103622GM 103310Tang WJNoble AJCarragher BKoehler CMLin King JVPotter CSLee DMancl JMMo SZhao MLi SLiang WGWei HLiu CWijaya JPan MfalseStructural basis for the mechanisms of human presequence protease conformational switch and substrate recognition.Presequence protease (PreP), a 117 kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other aggregation-prone peptides, such as amyloid β (Aβ). PreP structures have only been determined in a closed conformation; thus, the mechanisms of substrate binding and selectivity remain elusive. Here, we leverage advanced vitrification techniques to overcome the preferential denaturation of one of two ~55 kDa homologous domains of PreP caused by air-water interface adsorption. Thereby, we elucidate cryoEM structures of three apo-PreP open states along with Aβ- and citrate synthase presequence-bound PreP at 3.3-4.6 Å resolution. Together with integrative biophysical and pharmacological approaches, the2022-01-01T00:00:00Z2022 Apr2026-05-31T01:09:43.332Z2025-04-03T22:51:37.724ZS-EPMC89837643538316910.1038/s41467-022-29322-4