Structural studies of the yeast type II NADH:quinone oxidoreductase NDI-1
Lead Research Organisation:
Imperial College London
Department Name: Life Sciences
Abstract
Membrane proteins perform a staggering range of biological functions including respiration, signal transduction and molecular transport. The results of various genome projects have shown that up to 30 % of proteins encoded by eukaryotic cells are membrane proteins. A more fundamental understanding of the structure-function relationships of membrane proteins would make invaluable contributions to structural biology, pharmacology and medicine. Recently, we have obtained well-diffracting crystals of a yeast membrane protein NDI-1. This enzyme is a homologue of the mammalian protein and is an essential enzyme for yeast respiration. Structural information for this membrane protein will provide important insights into this respiratory membrane protein, particularly on its substrate (NADH and coenzyme Q10) and cofactor (FAD) binding sites. The structure is also medically important as it can be used for gene therapy of human genetic diseases.
Technical Summary
The oxygen respiratory chain is the final stage of energy catabolism for aerobic organisms. The system exists in the inner membrane of mitochondria and is composed of five membrane protein complexes including Complex I, NADH-ubiquinone oxidoreductase. Many organisms have an alternative enzyme called type II NADH:quinone oxidoreductases, which catalyses the same redox reaction as Complex I without proton translocation. NDI-1 from yeast is a unique type II NADH:quinone oxidoreductase since it is solely responsible for the NADH:quinone redox reaction in yeast, which lacks Complex I. Structural information for this membrane protein will provide important insights into the type II NADH dehydrogenases, particularly on their substrate and cofactor binding sites. The structure is also medically important as it can be used for gene therapy of human genetic diseases caused by defective Complex I. We have well-diffracting crystals of NDI-1 to address these important questions. Very little is known about the structures of eukaryotic membrane proteins, thus the NDI-1 structure will make invaluable contributions to general structural biology and biochemistry.
Publications
Iwata M
(2012)
The structure of the yeast NADH dehydrogenase (Ndi1) reveals overlapping binding sites for water- and lipid-soluble substrates.
in Proceedings of the National Academy of Sciences of the United States of America
Description | Membrane proteins perform a staggering range of biological functions including respiration, signal transduction and molecular transport. The results of various genome projects have shown that up to 30 % of proteins encoded by eukaryotic cells are membrane proteins. A more fundamental understanding of the structure-function relationships of membrane proteins would make invaluable contributions to structural biology, pharmacology and medicine. Recently, we have obtained well-diffracting crystals of a yeast membrane protein NDI-1. This enzyme is a homologue of the mammalian protein and is an essential enzyme for yeast respiration. Structural information for this membrane protein will provide important insights into this respiratory membrane protein, particularly on its substrate (NADH and coenzyme Q10) and cofactor (FAD) binding sites. The structure is also medically important as it can be used for gene therapy of human genetic diseases. |
Exploitation Route | To optimize the expression of membrane proteins including Ndi1 in Saccharomyces cerevisiae, new GFP-fusion based expression system has been developed. This was not only used for Ndi1 but also for many other mammalian membrane protein. The method was published in PNAS and the protocol was released in Nature Protocols. The vector designed has distributed to more than 20 laboratories. This will be used in many other laboratories |
Sectors | Healthcare |
URL | http://www3.imperial.ac.uk/lifesciences/research/membraneproteincrystallography |
Description | N/Structures of Ndi1, substrate-free form, NAD complex and ubiquinol complex will be deposited with PDB. |
First Year Of Impact | 2012 |
Sector | Healthcare |
Impact Types | Cultural |
Description | BBSRC Diamond Professorial fellow |
Amount | £162,012,160 (GBP) |
Funding ID | BB/G023425/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2010 |
End | 12/2014 |
Description | Diamond Membrane Protein Laboratory |
Amount | £1,000,000 (GBP) |
Funding ID | WT099165/Z/12/Z |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2013 |
End | 02/2016 |
Title | NDI |
Description | The coordinates of the NDI-1 structures with and withouts its ligands. Three coordinates 4G9K, 4GAP and 4GAV were deposited with the Protein Data bank. |
Type Of Material | Database/Collection of data |
Year Produced | 2012 |
Provided To Others? | Yes |
Impact | Reveals a unique quinone binding site of this enzyme. |
URL | http://www.rcsb.org |
Description | Diamond Membrane Protein Laboratory |
Organisation | Diamond Light Source |
Country | United Kingdom |
Sector | Private |
PI Contribution | Co-organise Diamond Membrane protein Laboratory |
Collaborator Contribution | Providing the space and the technical assistance |
Impact | We have co-organised the Diamond Membrane Protein Laboratory and our users and ourselves made publications. |
Description | NDI-1 |
Organisation | Scripps Research Institute |
Country | United States |
Sector | Charity/Non Profit |
PI Contribution | Prof. Takao Yagi is our biological partner of the project. |
Collaborator Contribution | He provided clones and samples. He has performed the functional studies of the protein. |
Impact | We have solved the NDI-1 structure and published the results. |
Start Year | 2006 |