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1.
Mol Cell ; 83(12): 2137-2147.e4, 2023 Jun 15.
Article in English | MEDLINE | ID: mdl-37244256

ABSTRACT

Biological energy currency ATP is produced by F1Fo-ATP synthase. However, the molecular mechanism for human ATP synthase action remains unknown. Here, we present snapshot images for three main rotational states and one substate of human ATP synthase using cryoelectron microscopy. These structures reveal that the release of ADP occurs when the ß subunit of F1Fo-ATP synthase is in the open conformation, showing how ADP binding is coordinated during synthesis. The accommodation of the symmetry mismatch between F1 and Fo motors is resolved by the torsional flexing of the entire complex, especially the γ subunit, and the rotational substep of the c subunit. Water molecules are identified in the inlet and outlet half-channels, suggesting that the proton transfer in these two half-channels proceed via a Grotthus mechanism. Clinically relevant mutations are mapped to the structure, showing that they are mainly located at the subunit-subunit interfaces, thus causing instability of the complex.


Subject(s)
Adenosine Triphosphate , Proton-Translocating ATPases , Humans , Cryoelectron Microscopy , Adenosine Triphosphate/metabolism , Proton-Translocating ATPases/chemistry , Protein Conformation
2.
Nature ; 2024 Jul 03.
Article in English | MEDLINE | ID: mdl-38961288

ABSTRACT

Bedaquiline (BDQ), a first-in-class diarylquinoline anti-tuberculosis drug, and its analogue, TBAJ-587, prevent the growth and proliferation of Mycobacterium tuberculosis by inhibiting ATP synthase1,2. However, BDQ also inhibits human ATP synthase3. At present, how these compounds interact with either M. tuberculosis ATP synthase or human ATP synthase is unclear. Here we present cryogenic electron microscopy structures of M. tuberculosis ATP synthase with and without BDQ and TBAJ-587 bound, and human ATP synthase bound to BDQ. The two inhibitors interact with subunit a and the c-ring at the leading site, c-only sites and lagging site in M. tuberculosis ATP synthase, showing that BDQ and TBAJ-587 have similar modes of action. The quinolinyl and dimethylamino units of the compounds make extensive contacts with the protein. The structure of human ATP synthase in complex with BDQ reveals that the BDQ-binding site is similar to that observed for the leading site in M. tuberculosis ATP synthase, and that the quinolinyl unit also interacts extensively with the human enzyme. This study will improve researchers' understanding of the similarities and differences between human ATP synthase and M. tuberculosis ATP synthase in terms of the mode of BDQ binding, and will allow the rational design of novel diarylquinolines as anti-tuberculosis drugs.

3.
Proc Natl Acad Sci U S A ; 120(18): e2216713120, 2023 05 02.
Article in English | MEDLINE | ID: mdl-37098072

ABSTRACT

Human complex II is a key protein complex that links two essential energy-producing processes: the tricarboxylic acid cycle and oxidative phosphorylation. Deficiencies due to mutagenesis have been shown to cause mitochondrial disease and some types of cancers. However, the structure of this complex is yet to be resolved, hindering a comprehensive understanding of the functional aspects of this molecular machine. Here, we have determined the structure of human complex II in the presence of ubiquinone at 2.86 Å resolution by cryoelectron microscopy, showing it comprises two water-soluble subunits, SDHA and SDHB, and two membrane-spanning subunits, SDHC and SDHD. This structure allows us to propose a route for electron transfer. In addition, clinically relevant mutations are mapped onto the structure. This mapping provides a molecular understanding to explain why these variants have the potential to produce disease.


Subject(s)
Protein Structure, Quaternary , Humans , Models, Molecular , Mutation , Cryoelectron Microscopy
4.
Proc Natl Acad Sci U S A ; 118(16)2021 04 20.
Article in English | MEDLINE | ID: mdl-33853951

ABSTRACT

Encapsulins containing dye-decolorizing peroxidase (DyP)-type peroxidases are ubiquitous among prokaryotes, protecting cells against oxidative stress. However, little is known about how they interact and function. Here, we have isolated a native cargo-packaging encapsulin from Mycobacterium smegmatis and determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). This encapsulin comprises an icosahedral shell and a dodecameric DyP cargo. The dodecameric DyP consists of two hexamers with a twofold axis of symmetry and stretches across the interior of the encapsulin. Our results reveal that the encapsulin shell plays a role in stabilizing the dodecameric DyP. Furthermore, we have proposed a potential mechanism for removing the hydrogen peroxide based on the structural features. Our study also suggests that the DyP is the primary cargo protein of mycobacterial encapsulins and is a potential target for antituberculosis drug discovery.


Subject(s)
Bacterial Proteins/ultrastructure , Mycobacterium smegmatis/ultrastructure , Peroxidases/ultrastructure , Bacterial Proteins/metabolism , Cryoelectron Microscopy/methods , Mycobacterium smegmatis/metabolism , Mycobacterium smegmatis/pathogenicity , Organelles/metabolism , Organelles/physiology , Peroxidases/metabolism
5.
Water Res ; 225: 119127, 2022 Oct 15.
Article in English | MEDLINE | ID: mdl-36155007

ABSTRACT

Expanding knowledge about new types of antibiotic resistance genes is of great significance in dealing with the global antibiotic resistance crisis. Herein, a novel oxidoreductase capO was discovered to be responsible for oxidative inactivation of chloramphenicol and thiamphenicol. The antibiotic resistance mechanism was comprehensively deciphered using multi-omics and multiscale computational approaches. A 66,383 bp DNA fragment carrying capO was shared among four chloramphenicol-resistant strains, and the co-occurrence of capO with a mobile genetic element cluster revealed its potential mobility among different taxa. Metagenomic analysis of 772 datasets indicated that chloramphenicol was the crucial driving factor for the development and accumulation of capO in activated sludge bioreactors treating antibiotic production wastewater. Therefore, we should pay sufficient attention to its possible prevalence and transfer to pathogens, especially in some hotspot environments contaminated with high concentrations of chloramphenicols. This finding significantly expands our knowledge boundary about chloramphenicols resistance mechanisms.


Subject(s)
Sewage , Thiamphenicol , Wastewater , Metagenomics , Anti-Bacterial Agents , Chloramphenicol/pharmacology , Oxidoreductases , Oxidative Stress , Genes, Bacterial
6.
Nat Commun ; 12(1): 4621, 2021 07 30.
Article in English | MEDLINE | ID: mdl-34330928

ABSTRACT

Cytochromes bd are ubiquitous amongst prokaryotes including many human-pathogenic bacteria. Such complexes are targets for the development of antimicrobial drugs. However, an understanding of the relationship between the structure and functional mechanisms of these oxidases is incomplete. Here, we have determined the 2.8 Å structure of Mycobacterium smegmatis cytochrome bd by single-particle cryo-electron microscopy. This bd oxidase consists of two subunits CydA and CydB, that adopt a pseudo two-fold symmetrical arrangement. The structural topology of its Q-loop domain, whose function is to bind the substrate, quinol, is significantly different compared to the C-terminal region reported for cytochromes bd from Geobacillus thermodenitrificans (G. th) and Escherichia coli (E. coli). In addition, we have identified two potential oxygen access channels in the structure and shown that similar tunnels also exist in G. th and E. coli cytochromes bd. This study provides insights to develop a framework for the rational design of antituberculosis compounds that block the oxygen access channels of this oxidase.


Subject(s)
Bacterial Proteins/ultrastructure , Cryoelectron Microscopy/methods , Cytochrome b Group/ultrastructure , Electron Transport Chain Complex Proteins/ultrastructure , Mycobacterium smegmatis/enzymology , Oxidoreductases/ultrastructure , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cytochrome b Group/chemistry , Cytochrome b Group/metabolism , Electron Transport , Electron Transport Chain Complex Proteins/chemistry , Electron Transport Chain Complex Proteins/metabolism , Heme/chemistry , Heme/metabolism , Models, Molecular , Mycobacterium smegmatis/genetics , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Oxygen/metabolism , Protein Conformation , Protein Subunits/chemistry , Protein Subunits/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Substrate Specificity
7.
Elife ; 102021 11 25.
Article in English | MEDLINE | ID: mdl-34819223

ABSTRACT

Pathogenic mycobacteria pose a sustained threat to global human health. Recently, cytochrome bcc complexes have gained interest as targets for antibiotic drug development. However, there is currently no structural information for the cytochrome bcc complex from these pathogenic mycobacteria. Here, we report the structures of Mycobacterium tuberculosis cytochrome bcc alone (2.68 Å resolution) and in complex with clinical drug candidates Q203 (2.67 Å resolution) and TB47 (2.93 Å resolution) determined by single-particle cryo-electron microscopy. M. tuberculosis cytochrome bcc forms a dimeric assembly with endogenous menaquinone/menaquinol bound at the quinone/quinol-binding pockets. We observe Q203 and TB47 bound at the quinol-binding site and stabilized by hydrogen bonds with the side chains of QcrBThr313 and QcrBGlu314, residues that are conserved across pathogenic mycobacteria. These high-resolution images provide a basis for the design of new mycobacterial cytochrome bcc inhibitors that could be developed into broad-spectrum drugs to treat mycobacterial infections.


Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/chemistry , Cytochromes/chemistry , Imidazoles/pharmacology , Mycobacterium tuberculosis/drug effects , Piperidines/pharmacology , Pyridines/pharmacology , Cryoelectron Microscopy , Drug Development
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