Structure and mechanism of a mitochondrial AAA+ disaggregase CLBP

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 apoand 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. Introduction Protein misfolding and aberrant aggregation are devastating to many fundamental processes in the cell and failures to remediate them are closed related to many human diseases (Eisele et al., 2015; Horwich, 2002). To maintain a healthy proteome, cells have evolved multiple dedicated systems, one of which is the Hsp100/Clp chaperone family. Hsp100/Clp chaperones are AAA+ ATPases that are widely found in bacteria, protozoa, fungi and plants (Kedzierska-Mieszkowska and Zolkiewski, 2021). HSP100 proteins generally contain an Nterminal domain (NTD), one or two ATPase domains (or nucleotide-binding domain, NBD), and usually function in hexameric forms. Taking yeast Hsp104 as an example, the NTD is responsible for substrate binding and recognition (Kedzierska-Mieszkowska and Zolkiewski, 2021), while the NBD1 and NBD2 bind and hydrolyze ATP to power substrate unfolding and translocation (Erzberger and Berger, 2006; Lee et al., 2013). Similar to many other AAA+ ATPases, Hsp104 unfolds and transports substrates through its central pore by a ratchet-like motion of the highly conserved pore-loops (PLs) on it ATPase domain (Gates et al., 2017). The ATP-hydrolysis cycle dependent conformational change of each subunit results in both interand intra-subunit structural remodeling, which collectively lead to the threading and unidirectional movement of peptide within the central (Gates et al., 2017). Recently, a new type of HSP100 family proteins, CLPB (aka, HSP78 and Skd3) was reported to act as a protein disaggregase in the intermembrane space of mitochondria (Cupo and Shorter, 2020; Mroz et al., 2020; Thevarajan et al., 2020; Wortmann et al., 2015). The Nterminus of CLPB coding sequence has a mitochondrial targeting signal (MTS), followed by an ankyrin repeat (ANK) domain, and ends with a C-terminal NBD (Fig. 1a). There is a short hydrophobic stretch between the MTS and the first ankyrin motif, as well as a long linker helix (LH) between ANK domain and NBD (Fig. 1a). The MTS is cleaved by mitochondrial processing peptidase (MPP), followed by a second cleavage by presenilin-associated rhomboid-like (PARL) protease by removing another 35 residues from the N-terminus (Mroz et al., 2020; Saita et al., 2017). The ANK domain is a unique feature of CLPB compared to other AAA+ ATPases, and it is also the first protein that was reported to contain both ANK domain and AAA+ activity (Li et al., 2006). Importantly, the removal of ANK domain disrupts the disaggregase activity of CLPB (Cupo and Shorter, 2020). Dysfunction of CLPB was found to be closely related to several human diseases. A number of CLPB mutations were found in patients of 3-methylglutaconic aciduria (3-MGA) (CapoChichi et al., 2015; Kanabus et al., 2015; Kiykim et al., 2016; Saunders et al., 2015; Wortmann et al., 2015). A common feature of these patients is increased urinary 3methylglutaric acid excretion, with varying degree of microcephaly, small birth weight, neutropenia, severe encephalopathy, intellectual disability, movement disorder and cataracts (Capo-Chichi et al., 2015; Kanabus et al., 2015; Kiykim et al., 2016; Saunders et al., 2015; Wortmann et al., 2015). Moreover, heterozygous variants of CLPB were also identified in severe congenital neutropenia (SCN) that disrupts granulocyte differentiation of human hematopoietic progenitors and increase apoptosis (Warren et al., 2022). Many of these mutations have been shown to impair the CLPB disaggregase activity, such as T268M, R408G, R475Q, A591V, R650P in 3-MGA and N499K, E557K, R561G, R620C in SCN (Cupo and Shorter, 2020; Warren et al., 2022). At cellular level, CLPB is important to mitochondria-mediated apoptosis and maintenance of the mitochondrial cristae structure (Chen et al., 2019b). CLPB interacts with HAX1, the anti-apoptotic factor of BCL-2 family, to promote cell survival (Chen et al., 2019b; Wortmann et al., 2015). Upregulated cellular level of CLPB in acute myeloid leukemia (AML) patients was found to mediate the resistance to BCL2 inhibitor venetoclax (Chen et al., 2019b). Recently, CLPB was also found to have negative correlation with the progression-free survival in castration-resistant prostate cancer (Pudova et al., 2020). It is currently not clear why CLBP has such a broad role in many aspects of mitochondrial function, and how the ANK and NBD domains work together to catalyze its essential disaggregase function. Here, we present a comprehensive structural characterization of human CLPB in both the apo and substrate-bound state. Unexpectedly, we found 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. While the CLPB works in a highly conserved manner to grasp and translocate substrate using the aromatic PL on its NBD, the ANK domain is responsible for recruiting a variety of substrates, including those proteins en route to the matrix. Together, our results suggest that CLPB functions as a general disaggregase in mitochondria and highlight the possibility of targeting CLPB for therapeutic purposes.