Structural and Functional Insights into the Action Mode of A Mitochondrial AAA+ Disaggregase CLPB

Abstract

The human AAA+ ATPase CLPB (aka, HSP78 and Skd3) is a protein disaggregase and functions to promote the solubilization of proteins in the mitochondrial intermembrane space. Unlike other AAA+ protein unfoldases or disaggregases, CLPB contains an ankyrin repeat containing domain (ANK) at its N-terminus. Mutations of CLPB are closely associated with a few human diseases, such as 3-methylglutaconic aciduria (3-MGA) and severe congenital neutropenia (SCN). The mechanism of CLPB functions as a disaggregase and the role of its unique ANK domain are currently unclear. Herein, we report a comprehensive structural characterization of human CLPB in both the apo- and substrate-bound states. Our data show that CLPB assembles into homo-tetradecamers in apo-state and dodecamers in the presence of a substrate, mediated by ANK domains via a "head-to-head" organization. Conserved pore-loops (PLs) on the ATPase domains form a spiral staircase to grasp and translocate the substrate in a step-size of two amino acid residues. We also show that the ANK domain is functionally essential, and more important, responsible for direct binding to various mitocondiral proteins in the inner membrane or intermembrane space. These results suggest that CLPB functions as a general disaggregase in mitochondria and highlight its potential as a target for the treatment of various mitochondria-related diseases.

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

“…Thus, substrate binding by PARL Skd3 promotes dodecamerization. Indeed, it has been independently established that PARL Skd3 forms dodecamers ( Spaulding et al, 2022 ; Wu et al, 2022 ). Moreover, Skd3 forms higher-order structures in cells, consistent with dodecamer formation ( Thevarajan et al, 2020 ).…”

“…The ANK is an unusual feature of Skd3 that is required for disaggregase activity ( Cupo and Shorter, 2020b ). Cryo-EM of SEC-purified PARL Skd3 NBD , which lacks the ANK domain, reveals well-resolved single hexamers but no dodecamers ( Figure S3A ) ( Wu et al, 2022 ). Thus, the ANK is not required for hexamerization, but is important for stabilizing the dodecamer.…”

“…Thus, dodecamerization may promote disaggregase activity. We suggest that the ANKs play multiple roles in protein disaggregation, including dodecamerization, potentially supported by the linker, and substrate engagement mediated by ankyrin repeats 1–4 ( Wu et al, 2022 ).…”

“…One of the most prominent features of the PARL Skd3 structure is the dodecamer, which is created by two hexamers making head-to-head contacts via the ANK domain. While our paper was under review, it has been independently established that PARL Skd3 forms dodecamers ( Spaulding et al, 2022 ; Wu et al, 2022 ). Moreover, Skd3 forms higher-order structures in cells, consistent with dodecamerization ( Thevarajan et al, 2020 ).…”

“…The linker region helps to promote dodecamer formation, whereas deletion of ankyrin repeats 1 and 2 or 3 and 4 does not perturb dodecamerization. The ankyrin repeats likely also play a role in substrate engagement ( Wu et al, 2022 ) or disaggregase plasticity analogous to the Hsp104 N-terminal domain ( Sweeny et al, 2015 ).…”