Unique structural features govern the activity of a human mitochondrial AAA+ disaggregase, Skd3.

The AAA+ protein, Skd3 (human CLPB), solubilizes proteins in the mitochondrial intermembrane space, which is critical for human health. Skd3 variants with defective protein-disaggregase activity cause severe congenital neutropenia (SCN) and 3-methylglutaconic aciduria type 7 (MGCA7). How Skd3 disaggregates proteins remains poorly understood. Here, we report a high-resolution structure of a Skd3-substrate complex. Skd3 adopts a spiral hexameric arrangement that engages substrate via pore-loop interactions in the nucleotide-binding domain (NBD). Substrate-bound Skd3 hexamers stack head-to-head via unique, adaptable ankyrin-repeat domain (ANK)-mediated interactions to form dodecamers. Deleting the ANK linker region reduces dodecamerization and disaggregase activity. We elucidate apomorphic features of the Skd3 NBD and C-terminal domain that regulate disaggregase activity. We also define how Skd3 subunits collaborate to disaggregate proteins. Importantly, SCN-linked subunits sharply inhibit disaggregase activity, whereas MGCA7-linked subunits do not. These advances illuminate Skd3 structure and mechanism, explain SCN and MGCA7 inheritance patterns, and suggest therapeutic strategies. [Display omitted] • High-resolution structure of a PARL Skd3-substrate complex • Substrate-bound PARL Skd3 hexamers stack head-to-head to form dodecamers • Mechanisms by which PARL Skd3 subunits collaborate to disaggregate proteins • Mechanisms by which Skd3 mutations cause dominant and recessive forms of disease Cupo et al. reveal the structure and mechanism of Skd3, a protein disaggregase found in mitochondria, which is critical for human health. These advances explain the inheritance patterns and suggest therapeutic strategies for debilitating diseases caused by mutations in Skd3. [ABSTRACT FROM AUTHOR]