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Cryo-EM of CcsBA reveals the basis for cytochrome c biogenesis and heme transport

Abstract

Although the individual structures and respiratory functions of cytochromes are well studied, the structural basis for their assembly, including transport of heme for attachment, are unknown. We describe cryo-electron microscopy (cryo-EM) structures of CcsBA, a bifunctional heme transporter and cytochrome c (cyt c) synthase. Models built from the cryo-EM densities show that CcsBA is trapped with heme in two conformations, herein termed the closed and open states. The closed state has heme located solely at a transmembrane (TM) site, with a large periplasmic domain oriented such that access of heme to the cytochrome acceptor is denied. The open conformation contains two heme moieties, one in the TM-heme site and another in an external site (P-heme site). The presence of heme in the periplasmic site at the base of a chamber induces a large conformational shift that exposes the heme for reaction with apocytochrome c (apocyt c). Consistent with these structures, in vivo and in vitro cyt c synthase studies suggest a mechanism for transfer of the periplasmic heme to cytochrome.

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Fig. 1: CcsBA cryo-EM maps reveal two conformations.
Fig. 2: CcsBA TM helices and its heme-binding domains.
Fig. 3: The active site of CcsBA with WWD domain.
Fig. 4: CcsBA-closed and CcsBA-open structures that suggest a mechanism of periplasmic domain movement.

Data availability

Cryo-EM electron density maps of CcsBA-open and CcsBA-closed have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-24941 and EMD-24942, respectively. Coordinates for atomic models of CcsBA-open and CcsBA-closed have been deposited in the PDB under accession numbers 7S9Y and 7S9Z, respectively. Source data are provided with this paper.

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Acknowledgements

This work was funded by the National Institutes of Health (R01 GM47909 to R.G.K.). M.J.R. and J.A.J.F. are supported by the Washington University Center for Cellular Imaging (WUCCI), which is funded in part by Washington University School of Medicine, The Children’s Discovery Institute of Washington University and St. Louis Children’s Hospital (CDI-CORE-2015-505 and CDI-CORE-2019-813), the Foundation for Barnes-Jewish Hospital (3770). J.A.J.F. is also supported by a Chan Zuckerberg Initiative Imaging Scientist award (2020-225726). We thank J. Jarodsky and E. Burgie for critically reading the manuscript and providing insightful feedback.

Author information

Authors and Affiliations

Authors

Contributions

D.L.M. and M.C.S. built all constructs. D.E.T. and A.L.C. purified all protein complexes. D.L.M., M.C.S. and D.E.T. conducted both in vitro and in vivo reconstitution assays under the supervision of R.G.K. M.J.R. and J.A.J.F. prepared samples for both negative staining and vitrification and conducted TEM and cryo-EM imaging experiments. Initial analysis of negative stain data and subsequent analysis and refinement of cryo-EM densities from single-particle cryo-EM datasets were undertaken by M.J.R. under the supervision of J.A.J.F. Model building and docking of structures into cryo-EM densities was undertaken by E.P.L. under the supervision of R.G.K. Figures were made by E.P.L., D.L.M. and M.J.R. Movies were made by E.P.L. All authors contributed to the writing of the manuscript. All authors reviewed and approved the final manuscript. All aspects of the project were coordinated by R.G.K. J.A.J.F. and R.G.K. were responsible for the final editing and submission of the manuscript.

Corresponding author

Correspondence to Robert G. Kranz.

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The authors declare no competing interests.

Additional information

Peer review information Nature Chemical Biology thanks Celia Goulding, Amy Medlock and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Topological map of CcsBA and proposed method of apocytc heme attachment.

a. Schematic of H. hepaticus CcsBA topology catalyzing heme attachment to apocytc. CcsBA consists of fourteen transmembrane domains and two major periplasmic domains. Conserved features are shown: two conserved histidines in the transmembrane domain (TM-His1-H858, TM-His2-H83) and two conserved periplasmic histidines (P-His1-H897, P-His2-H761) which flank the heme-handling WWD domain. The WWD domain positions heme for attachment to the CXXCH motif in apocytochrome c to form holocytc. Heme enters through a vestibule and is liganded by the TM-His1 and TM-His2. Exact, conserved substitutions and semi-conserved substitutions are all colored in red (T-Coffee analysis (http://tcoffee.crg.cat)1) derived from comparing organisms: M. tuberculosis, B. pertussis, Synechocystis, B. theta, B. subtilis, Wolinella, and H. hepaticus. b. Chemistry of thioether formation. Modified from12. Red arrows indicate two electron transfer.

Extended Data Fig. 2 Raising the threshold for CcsBA-open and CcsBA-closed reveals two and one dense regions, respectively.

a. Front view of the cryo-EM map of CcsBA-open (red, 3.56 Å) with TM-heme (green) and P-heme (yellow). Density thresholding reveals the two densest regions that indicate the iron in heme. b. Cryo-EM map of the 4.14 Å closed conformation (blue) with TM-heme (green). Filtering the electron density reveals a single dense particle which is assigned to iron in heme. c. The heme to heme distances, the vertical, horizontal and then the edge to edge distances, were measured edge to edge using Autodock Vina.

Extended Data Fig. 3 The P-His2 loop is likely disordered in closed conformation.

a. Top view of the 3.56 Å open conformation (red) with TM-heme (green), TM13 (black), and P-His2 loop (orange). A lower resolution version of the open conformation was used to allow for comparisons with the closed conformation. b. Top view of the 4.14 Å closed conformation (blue) viewed from the same angle with TM-heme, and TM13. There is no density going into TM13 in the closed conformation, so there is no orange colored P-His2 density in the closed conformation.

Extended Data Fig. 4 Thioether attachment of CXXCH to the P-Heme.

a. CXXCH is attached to heme with the His liganding the iron in heme, and cytc Cys sulfur groups forming thioether attachments to the 2- and 4-vinyl. Modified from14. b. The 3.56 Å CcsBA open conformation (red) zoomed in to the active site, with P-Heme and PDB heme (both yellow) and WWD Domain (green). The PDB of CXXCH (teal) was docked into the map by Autodock Vina51 (Supplementary Table 3). The positions of the 2-and 4-vinyl sites of thioether attachment are labeled.

Supplementary information

Supplementary Information

Supplementary Figs. 1–6, video legends 1 and 2, Tables 1–4 and references.

Reporting Summary

41589_2021_935_MOESM3_ESM.mp4

Supplementary Video 1. Heme flux from the TM-heme site to the P-heme site is accompanied by conformational change. The movie starts with the closed conformation: one heme (green) in the TM-heme site, the TM domain of the protein (red), the periplasmic region of the protein (blue), both TM-His (red) and both P-His (yellow). A new heme comes from the bottom of the protein into the vestibule where it bumps the current TM-heme into the heme channel as the movie zooms to show this action. The movie then shifts to a top view where conformational change happens: the upper heme finishes moving into the P-His site and is liganded by the P-His

41589_2021_935_MOESM4_ESM.mp4

Supplementary Video 2. Movie of CXXCH entry, thioether attachment, release of P-heme into the chamber and conformational change. The movie starts with a side view (TM6 and TM7 side) of the open conformation of CcsBA with the TM domain (red), periplasmic domain (blue), P-His (yellow), P-heme (green) and CXXCH (dark green). The CXXCH moves into the door of the chamber. The CXXCH then moves into the chamber and replaces the P-His2 (H897), and the H of the CXXCH ligands the heme. The CXXCH attaches to the 2- and 4-vinyl. Following this attachment, the view zooms out to show the release of the P-heme and ends with conformational change back to the closed conformation.

Source data

Source Data Fig. 1

Statistics source data and gels.

Source Data Fig. 2

Statistics source data and gels.

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Mendez, D.L., Lowder, E.P., Tillman, D.E. et al. Cryo-EM of CcsBA reveals the basis for cytochrome c biogenesis and heme transport. Nat Chem Biol 18, 101–108 (2022). https://doi.org/10.1038/s41589-021-00935-y

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