Cryo-EM structures of engineered active bc1-cbb3 type CIII2CIV super-complexes and electronic communication between the complexes

Steimle, Stefan; Eeuwen, Trevor van; Öztürk, Yavuz; Kim, Hee Jong; Braitbard, Merav; Selamoglu, Nur; García, Benjamin A.; Stroopinsky, Dina; Murakami, Kenji; Daldal, Fevzi

doi:10.1038/s41467-021-21051-4

Cited by 22 publications

(23 citation statements)

References 101 publications

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“…In an S. cerevisiae CIII 2 structure where the Q P site was empty, the Rieske head domain was observed in an intermediate position (Hartley et al ., 2019; Rathore et al ., 2019). Furthermore, recent cryoEM structures of CIII 2 from Rhodobacter capsulatus (Steimle et al ., 2021) and Vigna radiata (Maldonado et al ., 2021) revealed subpopulations with the FeS domain in either the b or c positions. Collectively, these data suggest that the FeS head domain stochastically adopts different conformations when the Q P site is unoccupied by ligands (Berry and Huang, 2003; Sarewicz and Osyczka, 2015).…”

Section: Discussionmentioning

confidence: 99%

“…While it is likely that both the Inz-5-bound and drug-free datasets include CIII particles with both occupied and empty Q P sites, the weak UQ density in the drug-free map suggests that Q P sites are mostly empty in that dataset. CryoEM structures of CIII 2 from other organisms have also found empty Q P sites (Hartley et al ., 2019; Rathore et al ., 2019; Maldonado et al ., 2021; Steimle et al ., 2021). In an S. cerevisiae CIII 2 structure where the Q P site was empty, the Rieske head domain was observed in an intermediate position (Hartley et al ., 2019; Rathore et al ., 2019).…”

Section: Discussionmentioning

confidence: 99%

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Rieske head domain dynamics and indazole-derivative inhibition of Candida albicans complex III

Trani

Liu

Whitesell

et al. 2021

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During cellular respiration, electron transfer between the integral membrane protein complexes of the electron transport chain is coupled to proton translocation across the inner mitochondrial membrane, which in turn powers synthesis of ATP and transmembrane transport processes. The homodimeric electron transport chain Complex III (CIII2) oxidizes ubiquinol (UQH2) to ubiquinone (UQ), transferring electrons to cytochrome c, and translocating protons through a mechanism known as the Q cycle. The Q cycle involves UQH2 oxidation and UQ reduction at two different sites within each CIII monomer, as well as movement of the head domain of the Rieske subunit. We used cryoEM to determine the structure of CIII2 from Candida albicans, revealing density for endogenous UQ in the structure and allowing us to directly visualize the continuum of conformations of the Rieske head domain. Analysis of these conformations does not indicate cooperativity in the position of the Rieske head domains or binding of ligands in the two CIIIs of the CIII2 dimer. CryoEM with the indazole derivative Inz-5, which inhibits fungal CIII2 and is fungicidal when administered with fungistatic azole drugs, showed that inhibition by Inz-5 alters the equilibrium of the Rieske head domain positions.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Rieske head domain dynamics and indazole-derivative inhibition of Candida albicans complex III

Trani

Liu

Whitesell

et al. 2021

Preprint

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“…OM, outer membrane; IM, inner (cytoplasmic) membrane. (Steimle et al, 2021). The presence of CcoZ in the same high molecular weight band resolved by BN-PAGE as the subunits of both the CcoNOP and QcrABC proteins is consistent with such an association in C. jejuni, but clearly further work will be needed to confirm this.…”

Section: Discussionmentioning

confidence: 73%

“…The presence of CcoZ in the same high molecular weight band resolved by BN-PAGE as the subunits of both the CcoNOP and QcrABC proteins is consistent with such an association in C. jejuni, but clearly further work will be needed to confirm this. Such a complex may be unstable and difficult to isolate, as obtaining the cryo-EM structure of the Rhodobacter supercomplex required artificial linking of the constituent Qcr and oxidase complexes (Steimle et al, 2021). Unlike the obligate Qcr-Cco interaction in actinobacteria (where soluble c-type cytochromes are absent), in Gram-negative bacteria there may be regulation of the association of these complexes (e.g., under different growth conditions or oxygen supply) and it would be informative to examine how electron transfer from Qcr to the Cco complex in C. jejuni is altered in the absence of CcoXYZ as well as how Qcr-oxidase interactions are changed.…”

Section: Discussionmentioning

confidence: 99%

Genes Linking Copper Trafficking and Homeostasis to the Biogenesis and Activity of the cbb3-Type Cytochrome c Oxidase in the Enteric Pathogen Campylobacter jejuni

et al. 2021

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Bacterial C-type haem-copper oxidases in the cbb3 family are widespread in microaerophiles, which exploit their high oxygen-binding affinity for growth in microoxic niches. In microaerophilic pathogens, C-type oxidases can be essential for infection, yet little is known about their biogenesis compared to model bacteria. Here, we have identified genes involved in cbb3-oxidase (Cco) assembly and activity in the Gram-negative pathogen Campylobacter jejuni, the commonest cause of human food-borne bacterial gastroenteritis. Several genes of unknown function downstream of the oxidase structural genes ccoNOQP were shown to be essential (cj1483c and cj1486c) or important (cj1484c and cj1485c) for Cco activity; Cj1483 is a CcoH homologue, but Cj1484 (designated CcoZ) has structural similarity to MSMEG_4692, involved in Qcr-oxidase supercomplex formation in Mycobacterium smegmatis. Blue-native polyacrylamide gel electrophoresis of detergent solubilised membranes revealed three major bands, one of which contained CcoZ along with Qcr and oxidase subunits. Deletion of putative copper trafficking genes ccoI (cj1155c) and ccoS (cj1154c) abolished Cco activity, which was partially restored by addition of copper during growth, while inactivation of cj0369c encoding a CcoG homologue led to a partial reduction in Cco activity. Deletion of an operon encoding PCuAC (Cj0909) and Sco (Cj0911) periplasmic copper chaperone homologues reduced Cco activity, which was partially restored in the cj0911 mutant by exogenous copper. Phenotypic analyses of gene deletions in the cj1161c–1166c cluster, encoding several genes involved in intracellular metal homeostasis, showed that inactivation of copA (cj1161c), or copZ (cj1162c) led to both elevated intracellular Cu and reduced Cco activity, effects exacerbated at high external Cu. Our work has therefore identified (i) additional Cco subunits, (ii) a previously uncharacterized set of genes linking copper trafficking and Cco activity, and (iii) connections with Cu homeostasis in this important pathogen.

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“…This type of Cox is widely distributed in bacteria, but absent in eukaryotes ( Ekici et al, 2012 ). The cbb 3 -type Cox of the facultative phototrophic bacterium Rhodobacter capsulatus has developed into a well-studied model system for dissecting bacterial Cu homeostasis, because cbb 3 -Cox is the only Cox present in this organism and contains only one Cu ion in its catalytic heme b -Cu B center of subunit CcoN ( Gray et al, 1994 ; Steimle et al, 2021 ). This is different from the universally conserved aa 3 -type Cox, which contains a second Cu center in the electron-accepting subunit II ( Thompson et al, 2012 ).…”

Section: Introductionmentioning

confidence: 99%

The CopA2-Type P1B-Type ATPase CcoI Serves as Central Hub for cbb3-Type Cytochrome Oxidase Biogenesis

et al. 2021

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Copper (Cu)-transporting P1B-type ATPases are ubiquitous metal transporters and crucial for maintaining Cu homeostasis in all domains of life. In bacteria, the P1B-type ATPase CopA is required for Cu-detoxification and exports excess Cu(I) in an ATP-dependent reaction from the cytosol into the periplasm. CopA is a member of the CopA1-type ATPase family and has been biochemically and structurally characterized in detail. In contrast, less is known about members of the CopA2-type ATPase family, which are predicted to transport Cu(I) into the periplasm for cuproprotein maturation. One example is CcoI, which is required for the maturation of cbb3-type cytochrome oxidase (cbb3-Cox) in different species. Here, we reconstituted purified CcoI of Rhodobacter capsulatus into liposomes and determined Cu transport using solid-supported membrane electrophysiology. The data demonstrate ATP-dependent Cu(I) translocation by CcoI, while no transport is observed in the presence of a non-hydrolysable ATP analog. CcoI contains two cytosolically exposed N-terminal metal binding sites (N-MBSs), which are both important, but not essential for Cu delivery to cbb3-Cox. CcoI and cbb3-Cox activity assays in the presence of different Cu concentrations suggest that the glutaredoxin-like N-MBS1 is primarily involved in regulating the ATPase activity of CcoI, while the CopZ-like N-MBS2 is involved in Cu(I) acquisition. The interaction of CcoI with periplasmic Cu chaperones was analyzed by genetically fusing CcoI to the chaperone SenC. The CcoI-SenC fusion protein was fully functional in vivo and sufficient to provide Cu for cbb3-Cox maturation. In summary, our data demonstrate that CcoI provides the link between the cytosolic and periplasmic Cu chaperone networks during cbb3-Cox assembly.

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Cryo-EM structures of engineered active bc1-cbb3 type CIII2CIV super-complexes and electronic communication between the complexes

Cited by 22 publications

References 101 publications

Rieske head domain dynamics and indazole-derivative inhibition of Candida albicans complex III

Rieske head domain dynamics and indazole-derivative inhibition of Candida albicans complex III

Genes Linking Copper Trafficking and Homeostasis to the Biogenesis and Activity of the cbb3-Type Cytochrome c Oxidase in the Enteric Pathogen Campylobacter jejuni

The CopA2-Type P1B-Type ATPase CcoI Serves as Central Hub for cbb3-Type Cytochrome Oxidase Biogenesis

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