Structures of AAA protein translocase Bcs1 suggest translocation mechanism of a folded protein

Abstract

The mitochondrial membrane-bound AAA protein Bcs1 translocate substrates across the mitochondrial inner membrane without previous unfolding. One substrate of Bcs1 is the iron–sulfur protein (ISP), a subunit of the respiratory Complex III. How Bcs1 translocates ISP across the membrane is unknown. Here we report structures of mouse Bcs1 in two different conformations, representing three nucleotide states. The apo and ADP-bound structures reveal a homo-heptamer and show a large putative substrate-binding cavity accessible to the matrix space. ATP binding drives a contraction of the cavity by concerted motion of the ATPase domains, which could push substrate across the membrane. Our findings shed light on the potential mechanism of translocating folded proteins across a membrane, offer insights into the assembly process of Complex III and allow mapping of human disease-associated mutations onto the Bcs1 structure.

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Fig. 1: EM density and heptameric organization of mBcs1.
Fig. 2: Bcs1-specific domain of mBcs1.
Fig. 3: AAA domain of mBcs1.
Fig. 4: Size measurement of the putative substrate-binding cavity in apo and ATPγS-bound mBcs1 structures.
Fig. 5: Model for substrate translocation by Bcs1.

Data availability

Atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) with accession codes PDB 6U1Y (ΔNmBcs1-AMP-PNP) and PDB 6UKO (FLmBcs1-ADP). Cryo-EM structures and atomic models have been deposited in the Electron Microscopy Data Bank and PDB with accession codes EMDB 20808, PDB 6UKP (Apo mBcs1) and EMDB 20811, PDB 6UKS (ATPγS-bound mBcs1). Source data for Extended Data Fig. 1 are provided with the paper.

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Acknowledgements

This research was supported by the intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health. M.J.B. is supported by US National Institute of Environmental Health Sciences (grant no. ZIC ES103326 to M.J.B). We thank the staff members of the SER-CAT and GM/CA beamlines at the Advanced Photon Source, Argonne National Laboratory for beamline support. All DNA sequencing services was conducted at the Center for Cancer Research Genomics Core, National Cancer Institute and computation for the EM image reconstruction was carried out using the Biowulf Linux cluster (biowulf.nih.gov) at the National Institutes of Health, Bethesda. We thank G. Leiman for editorial assistance.

Author information

W.K.T., M.J.B. and D.X. initiated the project. D.X. obtained funding. W.K.T. designed and conducted all the experiments. W.K.T., L.E. and D.X. analyzed the X-ray diffraction data and determined the structure. M.J.B., T.F. and N.V. acquired the EM images. W.K.T., M.J.B. and A.L.H. processed the EM images and reconstructed the maps. W.K.T. built the model into the EM maps. W.K.T. and D.X. interpreted the data and wrote the manuscript with input from all authors.

Correspondence to Di Xia.

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Peer review information Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Biochemical characterizations of recombinant mBcs1.

a, Size exclusion chromatographic profile of mBcs1 on Superdex 200. Inset: SDS-PAGE showing the purity of mBcs1 along the purification steps. b, One representative Michaelis Menten plot out of three experiments of mBcs1 ATPase activity to ATP concentration (n=3 technical replicates). c, Nucleotide-dependent conformational change of mBcs1 in the presence of different nucleotides as observed by Blue-Native PAGE. Source data

Extended Data Fig. 2 Rotation function plots showing the presence of a proper 7-fold axis.

a, Full-length mBcs1 and b, ΔNmBcs1 crystals.

Extended Data Fig. 3 Data processing workflow for mBcs1 in ATPγS-bound form.

Particles of mBcs1 in the presence of ATPγS appear to form a mix of single and dual heptamer. Initial processing showed the two heptamers in the dual heptamer do not interact uniformly, hence reducing the resolutions. Therefore, only the class that contains single heptamers was selected for subsequent refinement. To improve resolution, the AAA-domain was masked for final refinement.

Extended Data Fig. 4 Single-particle cryo-EM analysis of mBcs1 in ATPγS-bound form.

a, Representative motion-corrected micrograph of an ATPγS-bound mBcs1 sample. b, Gold-standard Fourier shell correlation (FSC) curves of the final density map of the ATPγS-bound mBcs1. The reported resolution for this structure was based on the FSC=0.143 criterion. c, Local resolution map of ATPγS-bound mBcs1 structure at 4σ contour level. The color code is given by the bottom color strip. d, Angular distribution of particles used in the final 3D reconstruction of ATPγS-bound mBcs1. The height of the cylinder is proportional to the number of particles for that view. e, Map-to-model FSC.

Extended Data Fig. 5 Structural comparison of mBcs1 domains in different nucleotide states and example densities of bound ligands.

The ATPγS-bound mBcs1 structure is shown in colored ribbons (subunit A in cyan; subunit B in green) and subunits of the apo mBcs1 structure are shown in gray ribbons. a, Superposition of the Bcs1-specific domain of subunit A in the apo state (cyan) with the ATPγS-bound structure of the same domain (gray). The distance change in residue I49 of subunits B is indicated. b, Superposition of the structure of the AAA domain of subunit A in the apo state (cyan) with of the same structure in the ATPγS-bound state (gray)The distance change in residue R218 of subunits B is indicated. c, Conformational difference in a single mBcs1 subunit in the two nucleotide states. The distance change in residue K145 from the tip of the helix H2 is indicated. d, EM density map of ATPγS binding site (5.0σ level, gray mesh). mBcs1 is represented in blue cartoon. Walker-A K236 and ATPγS are shown as stick models with carbon, oxygen, nitrogen, phosphorous and sulphur atoms in blue, red, dark blue, orange and yellow, respectively. e, Difference Fourier map showing the bound AMP-PNP in the nucleotide-binding site (3.0σ level, gray mesh) in crystal structure of ΔNmBcs1. The structure of ΔNmBcs1 is represented by gray cartoon. Green sphere is Mg2+. Walker-A K236 and ATPγS are shown as stick models with carbon, oxygen, nitrogen and phosphorous atoms in gray, red, dark blue and orange, respectively. f, Difference Fourier map showing the bound ADP in the nucleotide-binding site (3.0σ level, cyan mesh) in crystal structure of full-length mBcs1. Residues E282, D283 and ADP are shown as stick models with carbon, oxygen, nitrogen and phosphorous atoms in gray, red, dark blue and magenta, respectively.

Extended Data Fig. 6 Data processing workflow for mBcs1 in apo form.

Additional details are provided in the Methods section.

Extended Data Fig. 7 Single-particle cryo-EM analysis of mBcs1 in apo form.

a, Representative motion-corrected micrograph of apo-mBcs1 sample. b, Gold-standard Fourier shell correlation (FSC) curves of the final density map for the apo mBcs1 structure. The reported resolution for this structure was based on the FSC=0.143 criterion. c, Local resolution map for the apo mBcs1 structure at 2.5σ level. The color code is given by the bottom color strip. d, Angular distribution of particles used in the final 3D reconstruction of apo mBcs1. The height of the cylinder is proportional to the number of particles for that view. e, Map-to-model FSC.

Extended Data Fig. 8 Superposition of structural domains of mBcs1.

a, Superposition of the AAA domain from apo (gray) and ATPγS-bound (magenta) structures. b, Superposition of the Bcs1-specific domain from apo (gray) and ATPγS-bound (magenta) structures. c, Interface between two neighboring AAA domains of mBcs1 undergoes a sliding movement upon ATP binding. The structure in the vicinity of ATP binding site for the apo structure (both subunit A and B) is shown as cartoon in gray. The Arg-finger residue R343 of subunit A of the apo mBcs1 is shown as a stick model. The subunit A of the ATPγS-bound mBcs1 is shown in cyan and its neighboring subunit B is shown in green. The residue R343 of subunit A of the ATPγS-bound structure is shown as a stick model. The two structures are superimposed based on subunit B. The distances from CA atoms of R343 of apo or ATPγS-bound Bcs1 to the γ-phosphate of ATP are given. d, Interface between two neighboring AAA domains of bacterial NtrC1 in ADP- or ADP•BeF3-bound forms. Distances from CA atoms of Arg-finger residue R299 to bound ADP•BeF3 are given. e, Interface between two neighboring AAA domains of mammalian AAA protein p97 D1 domain in ADP- or ATPγS-bound forms. Distances from CA atoms of Arg-finger residue R299 to γ-phosphate of ATPγS are given.

Extended Data Fig. 9 Electrostatic surface potential of substrate and the putative substrate-binding cavity of mBcs1 in different states.

a, Electrostatic potential surface for the ISP subunit. b, Electrostatic potential surface of the apo mBcs1. The front portion of the surface was cut away to reveal the interior surface potential of the putative substrate-binding cavity. c, Electrostatic potential surface of the mBcs1 bound with ATPγS. The front portion of the surface was cut away to reveal the interior surface potential of the putative substrate-binding cavity.

Extended Data Fig. 10 Pathogenic mutations of BCS1L.

a, Pathogenic mutations of BCS1L and their locations in the Bcs1 structure. bd, Mapping of the mutations on ATPγS-bound and apo mBcs1 in two orthogonal orientations: top view (top) and side view (bottom). TM, Bcs1-specific and AAA regions are in black, blue and magenta ribbons, respectively. Mutations are showed in spheres. Mutations found in (b), Björnstad syndrome; (c) GRACILE syndrome and (d) Complex III deficiency.

Supplementary information

Reporting Summary

Supplementary Video 1

Morphing of conformational transition from apo to ATPγS-bound state of mBcs1 in a concerted motion. Side view of mBcs1

Supplementary Video 2

Morphing of conformational transition from apo to ATPγS-bound state of mBcs1 in a concerted motion. View from the matrix side

Source data

Source Data Extended Data Fig. 1

Statistical source data

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Tang, W.K., Borgnia, M.J., Hsu, A.L. et al. Structures of AAA protein translocase Bcs1 suggest translocation mechanism of a folded protein. Nat Struct Mol Biol 27, 202–209 (2020). https://doi.org/10.1038/s41594-020-0373-0

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