Bacterial Complex I

Lead Research Organisation: Medical Research Council

Abstract

Most of the energy required by humans is produced by the respiratory chain in mitochondria, which are ubiquitous cell organelles. This chain consists of five large membrane protein complexes, acting in concert to produce our energy carrier – ATP. To understand how any protein works (or malfunctions in a disease) we need to know its atomic structure. These were known for most components of the respiratory chain, except for its first and largest (~44 subunits) enzyme, complex I. Knowledge of the structure is indispensable for the design of drugs against a number of human neurodegenerative diseases (including Parkinson's), which are associated with malfunction/mutations in complex I.
We mainly study the structure and the mechanism of a simpler (~14 subunits) bacterial complex I as a minimal model of the human enzyme. We have solved the atomic structures of two major parts of the complex and, very recently, of the entire bacterial enzyme. The stucture shows how electrons are transferred via a 'wire' of iron-containing clusters. How exactly this process is coupled to the translocation of protons across the membrane is not yet fully understood. We will focus now on resolving this issue, as well as on solving the structure of the more elaborate mammalian enzyme.

Technical Summary

Our major aim is to determine the structure and to understand the mechanism of NADH:ubiquinone oxidoreductase (complex I). This membrane-embedded enzyme catalyses the transfer of electrons from NADH to ubiquinone, contributing to proton-motive force and energy production in bacteria and mitochondria. Research into complex I is increasing in importance as a growing number of human neurodegenerative diseases are being associated with mutations in its subunits. Human complex I deficiency is the most frequently encountered mitochondrial disorder, leading, for example, to Leigh syndrome and Leber's hereditary optic neuropathy. Dysfunction of complex I may be the central cause of sporadic Parkinson's disease. Also, this enzyme is a major source of reactive oxygen species (ROS) in mitochondria, which may be one of the causes of aging. Complex I is one of the largest known membrane protein complexes - the mammalian enzyme of ~ 1 MDa is formed by 44 subunits. The prokaryotic enzyme performs the same reaction but is approximately half the size, consisting of about 14 ‘core’ subunits, so we can use it as a 'minimal' model of the mammalian complex. The structure of the enzyme is indispensable for understanding its function and for development of drugs for treatment of related diseases.
We have solved, by X-ray crystallography, all known atomic structures of complex I, using enzyme purified from T. thermophilus and E. coli. Initially we have determined the structure of the hydrophilic domain, which consists of eight different subunits, contains all the redox centres and forms the catalytic core of the enzyme. The structure described the unusually long electron transfer pathway from NADH to quinone, via the flavin and seven iron-sulphur clusters. Subsequently we determined the structure of the most of the membrane domain, revealing a novel fold of antiporter-like subunits. Each of the three of these similar subunits contains, uniquely, a single proton-translocation channel formed by two connected symmetry-related half-channels. Finally, very recently we solved the first structure of the entire complex I, showing unusually elongated and out-of-the-membrane quinone-binging site and revealing a forth proton translocation channel at the interface of the two main domains. This is the largest asymmetric membrane protein structure solved to date. We have now solved several structures with quionone-like compounds bound, confirming the identity of the unusual binding site.
Our structures provide the basis for understanding of the organisation of this large molecular machine and allow us to propose a likely mechanism of coupling between electron transfer and proton translocation, based on long-range conformational changes. However, to verify the mechanism it will be necessary in the future to obtain the structures of different redox states. This should allow us to visualise directly any conformational changes during the catalytic cycle. New advanced techniques using X-ray free electron lasers (X-FELs) and caged substrates may even allow time-resolved “movies” of the protein in action to be captured. This analysis will be complemented by MD (molecular dynamics) simulations, site-directed mutagenesis and other functional studies.
The effects of many known human mutations in core subunits of complex I can be already explained on the basis of our structures, confirming that bacterial analogue is a reliable model of human enzyme. However, further analysis will require the determination of the structure of mitochondrial, preferably mammalian, enzyme. It has about 30 additional subunits, which are essential for proper assembly of the complex and can regulate its activity or provide additional functions. We will pursue this major goal using the combination of X-ray crystallography and cryo-electron microscopy.

Publications

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Berrisford JM (2016) Structure of bacterial respiratory complex I. in Biochimica et biophysica acta

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Berrisford JM (2009) Structural basis for the mechanism of respiratory complex I. in The Journal of biological chemistry

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Efremov R (2010) The architecture of respiratory complex I in Nature

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Efremov RG (2011) Respiratory complex I: 'steam engine' of the cell? in Current opinion in structural biology

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Efremov RG (2012) Structure of Escherichia coli OmpF porin from lipidic mesophase. in Journal of structural biology

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Garvin M (2015) Review and meta-analysis of natural selection in mitochondrial complex I in metazoans in Journal of Zoological Systematics and Evolutionary Research

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Holt PJ (2016) Reversible FMN dissociation from Escherichia coli respiratory complex I. in Biochimica et biophysica acta

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Sazanov LA (2015) A giant molecular proton pump: structure and mechanism of respiratory complex I. in Nature reviews. Molecular cell biology

 
Description Textbooks
Geographic Reach Multiple continents/international 
Policy Influence Type Citation in systematic reviews
 
Description Textbooks
Geographic Reach Multiple continents/international 
Policy Influence Type Citation in systematic reviews
 
Description EMBO long-term fellowship
Amount £53,000 (GBP)
Organisation European Molecular Biology Organisation 
Sector Charity/Non Profit
Country Germany
Start 01/2013 
End 12/2014
 
Description C Robinson 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution Sharing of knowledge/expertise, provision of samples
Collaborator Contribution Mass spectrometric analysis of sub-complexes of complex I
Impact Prelimiary results indicate that masses of several hydrophobic sub-complexes of complex I can be measured reproducibly. Further data analysis is necessary before deciding whether to proceed with additional samples. Currently collaboration is on hold, as most of the scientific questions were resolved by structural data.
 
Description D Saunders 
Organisation The Garvan Institute for Medical Research
Department Cancer Research Program
Country Australia 
Sector Hospitals 
PI Contribution Structural analysis of novel mutations in complex I subunits discovered in cancer patients.
Collaborator Contribution Genetic analyses of samples from cancer patients.
Impact Joint paper published.
Start Year 2013
 
Description E Nakamaru-Ogiso 
Organisation University of Pennsylvania
Department Johnson Research Foundation
Country United States 
Sector Academic/University 
PI Contribution Characterisation/crystallisation of mutants
Collaborator Contribution Generation of site-directed mutants in E. coli complex I
Impact Collaboration just started, currently at the level of designing mutations of interest in the membrane domain of complex I. In 2011, characterisation of mutants has been finished and the manuscript is in preparation. Joint paper published in 2016.
Start Year 2010
 
Description G Cook 
Organisation University of Otago
Department Department of Microbiology & Immunology
Country New Zealand 
Sector Academic/University 
PI Contribution Help in solving the structure of NDH-2 protein.
Collaborator Contribution Purification, crystallisation and structure determination of NDH-2 protein.
Impact Structure of NDH-2 from Caldalkalibacillus thermarum solved. Joint paper published.
Start Year 2012
 
Description L Kussmaul 
Organisation F. Hoffmann-La Roche AG
Department Roche Diagnostics
Country Global 
Sector Private 
PI Contribution Sharing of knowledge/expertise, provision of purified bacterial complex I
Collaborator Contribution Testing potential compounds limiting ROS production by bovine and human complex I
Impact Prelimiary results suggest that the compounds interact also with bacterial enzyme. Currently collaboration is on hold due to financial situation at Boehringer.
 
Description M Garvin 
Organisation University of Alaska
Department School of Fisheries and Ocean Sciences
Country United States 
Sector Academic/University 
PI Contribution Structural analysis of the mutations in complex I subunits discovered by bioinformatic analyses.
Collaborator Contribution Bioinformatic analysis of evolutionary trends in complex I subunits.
Impact Joint paper published.
Start Year 2012
 
Description M Verkhovskaya and M Wikstrom 
Organisation University of Helsinki
Country Finland 
Sector Academic/University 
PI Contribution Sharing of knowledge/expertise, provision of purified bacterial complex I
Collaborator Contribution EPR measurements with redox titrations
Impact Fe-S clusters in complex I from T. thermophilus have been characterised. Publication is in preparation.
Start Year 2008
 
Description T Yagi and T Ohnishi 
Organisation University of Pennsylvania
Department Johnson Research Foundation
Country United States 
Sector Academic/University 
PI Contribution Sharing of knowledge/expertise, characterisation of mutants
Collaborator Contribution Mutagenesis of E. coli complex I for functional and structural studies
Impact Analysis of mutants created by collaborators is currently in progress. In 2011, most of mutants have been studied and the manuscript is in preparation.
 
Description Article for MRC newsletter and homepage 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Prepared an article on our major scientific breakthrough, published in Science, for the MRC newsletter and homepage.

N/A
Year(s) Of Engagement Activity 2006
 
Description Articles for MRC newsletter and other publications 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Type Of Presentation Paper Presentation
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Prepared articles on our recent research breakthroughs, published in Nature in 2010, 2011 and 2013 for MRC newsletter and homepage, as well as for such publications as "The scientist", ESRF highlights, SLS highlights and Symmetry Magazine.

Public informed about our research.
Year(s) Of Engagement Activity 2010,2011,2012,2013
 
Description Big Bang Science Fair, London 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact A member of my research group presented the crystallography exhibit at the Big Bang Science Fair.

Interest from further afield in the Unit's work.
Year(s) Of Engagement Activity 2013,2014
 
Description Cambridge Science Festival 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Three members of my research group participated in the Unit's contribution to the Cambridge Science Festival.

Greater awareness of the Unit's work.
Year(s) Of Engagement Activity 2013,2014,2015
 
Description ESRF Highlights publication 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Summary of the latest research for ESRF HIghlights and News publications.

.
Year(s) Of Engagement Activity 2013,2014
 
Description Media interview 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Interview with Symmetry Magazine (USA) about the group's latest research.

.
Year(s) Of Engagement Activity 2013