(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



Comprehensive structural characterization of the human AAA+ disaggregase CLPB in the apo- and substrate-bound states reveals a unique mode of action driven by oligomerization [1]

['Damu Wu', 'State Key Laboratory Of Membrane Biology', 'Peking-Tsinghua Joint Center For Life Sciences', 'School Of Life Sciences', 'Peking University', 'Beijing', 'Yan Liu', 'State Key Laboratory Of Genetic Engineering', 'Zhongshan Hospital', 'Fudan University']

Date: 2023-02

The human AAA+ ATPase CLPB (SKD3) is a protein disaggregase in the mitochondrial intermembrane space (IMS) and functions to promote the solubilization of various mitochondrial proteins. Loss-of-function CLPB mutations are associated with a few human diseases with neutropenia and neurological disorders. Unlike canonical AAA+ proteins, CLPB contains a unique ankyrin repeat domain (ANK) at its N-terminus. How CLPB functions as a disaggregase and the role of its ANK domain are currently unclear. Herein, we report a comprehensive structural characterization of human CLPB in both the apo- and substrate-bound states. CLPB assembles into homo-tetradecamers in apo-state and is remodeled into homo-dodecamers upon substrate binding. Conserved pore-loops (PLs) on the ATPase domains form a spiral staircase to grip and translocate the substrate in a step-size of 2 amino acid residues. The ANK domain is not only responsible for maintaining the higher-order assembly but also essential for the disaggregase activity. Interactome analysis suggests that the ANK domain may directly interact with a variety of mitochondrial substrates. These results reveal unique properties of CLPB as a general disaggregase in mitochondria and highlight its potential as a target for the treatment of various mitochondria-related diseases.

Funding: The work was supported by the National Natural Science Foundation of China ( https://www.nsfc.gov.cn/ ) (32230051 to N.G., 31922036 to N.L.), the National Key Research and Development Program of China ( https://service.most.gov.cn/ ) (2019YFA0508904 to N.G.), and the Qidong-SLS Innovation Fund to N.G. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: Data are available from the Protein Data bank and Electron Microscopy Data Bank.The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank with the accession codes EMD-33105, EMD-33106, EMD-33109, EMD-33110 and EMD-33104 for the CLPB, CLPBE425Q, NBDE425Q-hexamer, NBDE425Qheptamer and NBDE425Q-nanomer, respectively. The atomic model of NBDE425Qnanomer has been deposited in Protein Data Bank with accession code PDB 7XBK.The coordinate and the structure factor of CLPB-ANK have been deposited in the Protein Data Bank with accession code 7XC5. All other relevant data are within the paper and its Supporting Information files.

It is currently not clear why CLPB has such a broad role in different aspects of mitochondrial function and how the ANK and NBD domains work together to fulfill its essential disaggregase activity. Here, we present a structural and functional characterization of human CLPB. Unexpectedly, we found that CLPB assembles into a homo-tetradecamers in the absence of substrate. Upon substrate binding, the CLPB complex is converted into dodecamers consisting 2 conventional hexameric units. The NBD ring within a hexamer shares common structural features of typical AAA+ unfoldase/disaggregase, with a spiral arrangement of PLs to interact with a fully threaded substrate. The N-terminal ANK domain is essential for the higher-order organization of CLPB and contributes to the disaggregase activity by directly interacting with various mitochondrial substrates. These results provide a framework for further dissection of the role of CLPB in regulating various mitochondrial functions.

Dysfunction of CLPB by mutations is associated with several human diseases, such as the 3-methylglutaconic aciduria (3-MGA) [ 10 , 13 – 16 ]. A common disorder of the 3-MGA patients is the increased urinary 3-methylglutaric acid excretion, often with varying degree of microcephaly, small birth weight, neutropenia, severe encephalopathy, intellectual disability, movement disorder, and cataracts [ 10 , 13 – 16 ]. Moreover, heterozygous missense variants of CLPB were also identified in patients with severe congenital neutropenia (SCN), and these variants were found to disrupt granulocyte differentiation of human hematopoietic progenitors [ 17 ]. Many of these disease-related mutations have been shown to impair the disaggregase activity of CLPB, such as T268M, R408G, R475Q, A591V, R650P in 3-MGA and N499K, E557K, R561G, R620C in SCN [ 8 , 17 ]. At the cellular level, CLPB is important in maintaining normal cristae structure of mitochondria [ 18 ]. CLPB interacts with HAX1, an anti-apoptotic factor of BCL-2 family, to promote cell survival [ 10 , 18 ]. Up-regulated cellular level of CLPB in acute myeloid leukemia (AML) cells was found to mediate the resistance to BCL-2 inhibitor venetoclax [ 18 ]. Recently, CLPB was also found to have negative correlation with the progression-free survival in castration-resistant prostate cancer [ 19 ].

( A ) Domain organization of H. sapiens CLPB. CLPB is composed of an MTS, a short hydrophobic stretch (S), an LH, 4 ankyrin-repeat (ANK) motifs, an NBD, and a CTD. ( B ) Representative 2D classification averages of CLPB and CLPB E425Q datasets. ( C , D ) The density maps of the double-heptamer (C) and double-hexamer (D), superimposed with the models of CLPB. The density maps are shown in the side and top (ATPase ring) views. The higher-order oligomer is mediated by ANK domains. The substrate was labeled as yellow. LH, linker helix; NBD, nucleotide-binding domain.

Recently, a new type of HSP100 family proteins, CLPB (also known as SKD3) was reported to act as a protein disaggregase in the intermembrane space (IMS) of mitochondria [ 8 – 11 ]. The N-terminus of CLPB 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 (AM), 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 to remove additional hydrophobic residues from the N-terminus [ 11 , 12 ]. The ANK domain is a unique feature of CLPB compared to other AAA+ ATPases, and the removal of ANK domain disrupts the disaggregase activity of CLPB [ 8 ].

Protein misfolding and aberrant aggregation are devastating to many fundamental functions of the cell and failures to remediate them are closed related to many human diseases [ 1 , 2 ]. To maintain a healthy proteome, cells have evolved multiple dedicated systems, one of which is the HSP100 chaperone family [ 3 – 5 ]. As a subfamily of the AAA+ ATPase, HSP100 proteins generally contain an N-terminal domain (NTD), 1 or 2 ATPase domains (or nucleotide-binding domain, NBD), and usually function in hexameric forms. Taking yeast Hsp104 as an example, the NTD is involved in substrate binding, while the NBD1 and NBD2 bind and hydrolyze ATP to facilitate power substrate unfolding and translocation [ 6 ]. 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 the ATPase domains [ 7 ]. The ATP-hydrolysis cycle-dependent conformational change of each subunit results in both inter- and intra-subunit structural remodeling, which collectively lead to the threading and unidirectional movement of the peptide within the central channel [ 7 ].

Results

Substrate binding induces the transition from double-heptamer to double-hexamer A major difference between the WT and E425Q CLPB structures is the presence of a peptide substrate in the central pore, which brings close CLPB subunits to form a more compact structure. As shown in the 2D classification results, most of the top-view classes of the WT CLPB particles are heptameric, and only a tiny top-view class, 1.7% of all top-view particles, display a hexameric feature (Figs 2B, 2C, and S5A and S2 Data). In contrast, all the top-view classes of the CLPBE425Q particles are hexameric exclusively (Figs 2B–2D and S5B). This suggests that the binding of substrate may induce a transition from tetradecamers to dodecamers. To test this hypothesis, we incubated the WT CLPB complexes with a model substrate casein [8] in the presence of excessive ATPγS, which has been shown to best promote the binding of casein to Hsp104 [7,31] and CLPB [20]. As a control, WT CLPB complexes were also treated with ATPγS alone. Cryo-EM 2D classification was employed to analyze their oligomeric states. For the CLPB-ATPγS dataset, only a small fraction (8.0% of all top-view particles) shows a hexameric ring (Fig 2B and 2F), indicating that the majority of particles retain the form of double-heptamer. In sharp contrast, in the presence of casein and ATPγS, the percentage of hexameric top-view particles has increased to 51.8% (Fig 2B and 2G). Consistently, Cupo and colleagues recently also found that a mixture of CLPB double-hexamers and double-heptamer in the present of casein and ATPγS. These results indicate that substrate binding is likely the most important factor that drives the formation of double-hexamers from double-heptamer.

CLPB-NBD assembles into polypeptide-engaged helical structures Due to the large inter- and intra-hexamer/heptamer flexibility, the structures of full-length CLPB complexes were not solved in atomic resolution. CLPB-NBD was thus used as a surrogate for high-resolution structural determination. Two mutant versions of CLPB (NBD and NBDE425Q) were analyzed by cryo-EM (S9A–S9F Fig), which again confirmed that E425Q mutation resulted in co-purification of an endogenous peptide in the central channel. Also similar to the full-length complexes, the top-view classes of the NBDE425Q particles are exclusively hexameric, whereas both hexameric and heptameric arrangements were observed for the NBDWT particles (S9F Fig). Subsequently, we focused on the NBDE425Q dataset for high-resolution refinement. After several rounds of 3D classification, 3 different oligomeric arrangements, hexameric, heptameric, and nonameric, were identified. The nonamer appeared to be more stable and could be resolved at an overall resolution of 3.7 Å (S10 Fig), allowing the atomic modeling of the NBD of CLPB. In the nucleotide-binding pockets of the nonamer, a total of 8 ATP molecules could be modeled (S11 Fig). The conserved functional motifs on the ATPase domain are well resolved, including the Walker A motif (351-GSSGIGKT-358), sensor-1 (464-TSN-466), and sensor-2 (588-GAR-590) (Fig 5E). The conserved residues I317, I318, and F541 form a hydrophobic pocket to stabilize the adenine ring of ATP, while T358 stabilizes the β- and γ-phosphates through a Mg2+ ion (Fig 5E). The Arginine Finger (R531) from the adjacent protomer points to the γ-phosphate of ATP. In general, these structures show that the NBD of CLPB is a typical AAA+ ATPase module, underscoring a conserved mechanism of ATP-powered substrate unfolding and translocation [32]. The central channels of all 3 states are occupied by a peptide substrate, which forms successive interactions with the helically arranged CLPB protomers (Fig 4A–4C). In the nonamer, 9 protomers form a 1.5-turn helix around the central substrate. Besides the protomer number, the 3 oligomers also show obvious structural differences. While the twist angle between neighboring subunits is roughly 60° in all 3 forms, the axial rise within each oligomer is not constant and varies in the hexamer and heptamer largely. The axial displacements between P1 and P6 are roughly 25 Å, 30 Å, and 35 Å for the hexamer, heptamer, and nonamer, respectively (Fig 4D–4F). This is expected, as more axial space is required to fit in the seventh and more subunits onto the hexamer. In the full-length complexes, the ANK domain provides a steric hindrance to end the helical extension. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. Cryo-EM characterization of the NBD helical structures of CLPB. (A–C) Density maps of the NBD hexamer (A), heptamer (B), and nonamer (C), respectively. Protomers are painted in different colors. (D–F) Distances along the central substrate between the PL residue Y400 of P1 and P6 in the hexamer (D), heptamer (E), and nonamer (F). The axial rise of P6 relatively to P1 are labeled. NBD, nucleotide-binding domain; PL, pore-loop. https://doi.org/10.1371/journal.pbio.3001987.g004 Overall, these data show that the ANK domain is essential in determining the oligomeric state of CLPB complex and also explain why our CLPB-NBD still retains ATPase activity. Although this nonameric helical structure does not exist in a physiological context, it serves as a high-resolution model to demonstrate that CLPB-NBD shares many characteristics of typical AAA+ proteins.

[END]
---
[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001987

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.