Structure-function relationships of the extended families of NCS-1 NSS and SSS membrane transport proteins
Lead Research Organisation:
University of Leeds
Department Name: Institute of Membrane & Systems Biology
Abstract
Bacteria and yeasts have been used for millennia to produce desirable products for man. Examples are yeasts for bread and alcohol, bacteria for vinegar as a preservative, and bacterial conversion of milk to yoghourts. Recently the huge potential of such organisms for making useful chemicals has started to be realised in commercial developments. Examples are production of ethanol and methane for biofuels, and also for uses in the chemical and pharmaceutical industries; penicillins and many other antibiotics; citric acid for drinks; and monosodium glutamate for food preservation and flavour. One of these desirable products is a family of molecules called hydantoins, which can be utilized for production of individual amino acids for human nutrition, and also for pharmaceutical feedstocks. They are produced as a result of metabolism of nucleic acids by the microbial cells, which are surrounded by an impermeable membrane, usually reinforced by a 'cell wall'. The uptake and efflux of the hydantoins and amino acids through the otherwise impermeable membrane is effected by special proteins called membrane transport proteins. These are very important in all organisms, since up to15% of the genetic information is used to encode them. In 2000 Henderson was approached by the Ajinomoto Company of Japan, who needed to characterize such a protein that they thought might transport hydantoins and would have commercial potential. Mr Suzuki from Japan spent two years working with Henderson in a highly confidential project, and they successfully characterized the protein, the exploitation of which is now protected by patents in Japan and USA, and the results published. Since the protein, now called Mhp1, was purified Ajinomoto and BBSRC funded a further joint project with Iwata to elucidate its 3-dimensional structure, a major scientific undertaking that has now reached fruition. The structure reveals not only intimate details of its molecular mechanism, but also an unexpected similarity to bacterial sugar and amino acid transport proteins and to human proteins involved in functions of gut, nerve and brain. [Henderson and Baldwin were the first to realise that transport proteins in bacteria can be similar to those in man]. Most transport proteins are difficult to study. They have a low abundance in the membrane, they are difficult to solubilise in aqueous solutions, and then they are difficult to purify. We have solved these problems by genetically manipulating the genes so that the protein products are overexpressed in host cells of an easy-to-grow bacterium called Escherichia coli. We devised methods for extracting, solubilising, and purifying the protein. The whole process is done in a large scale of 30-100 litres by growing the bacteria in fermenters, itself a specialised technology. As a result we can produce purified transport proteins in the large amounts required to crystallise the proteins. We can deduce the 3D structure of the protein through measuring the diffraction pattern of these crystals when they are exposed to X-rays. Since crystals of membrane proteins diffract only weakly the X-ray source needs to be very bright. Synchrotrons, such as the 'Diamond Light Source' near Oxford, provide such radiation. Iwata is an expert in the demanding techniques of crystallising membrane proteins and X-ray diffraction, and he has set up a 'Membrane Protein Laboratory' there. He and his colleagues, using protein produced in Leeds, have just determined the 3D structure of the Mhp1 protein. This is an important discovery. It is a major step towards the next stage of the research to find out how the protein actually works as a tiny machine, moving important nutrients across the cell membrane. Such a first structure is a template to understand how many related proteins in other microbes and man actually work. Also, from knowing their structures we can manipulate the activities of the proteins to enhance their commercial potential.
Technical Summary
Membrane proteins comprise up to about 30% of the genome capacity in all organisms, yet the 3D structures of less than 200 are known, compared to over 10,000 independent structures (>50,000 total) of soluble proteins. So far, over 800 Nucleoside-Cation-Symporter-1 (NCS-1) transport proteins are identified in eubacteria, fungi and plants, which play important roles in salvage pathways for capture of nutrients. We discovered that the NCS-1 family has a similar structural fold to the Neurotransmitter-Sodium-Symport (NSS) and Solute-Sodium-Symport (SSS) sub-families, which occur in most life forms from microbe to man, so recognition of substrates by these three related transport families is particularly wide ranging, including nucleobases, amino acids, sugars and numerous metabolites. In man the SSS and NSS transporters play key roles in tissue nutrition and differentiation, related for example to the onset of epilepsy, depression, pain and addiction to hard drugs, when their roles are subverted in disease. Our aim is to define the structure-function relationships and establish the complete molecular mechanism of NCS-1 transport proteins, by exploiting our recent breakthrough in obtaining a crystal structure of the stereotypical transporter Mhp1 coupled with knowledge from elsewhere of the structure of the NSS sodium-leucine transporter, LeuT, and of the SSS sodium-galactose transporter, vSGLT. The biochemical and biophysical characterizations of the Mhp1 protein and homologues will be carried out in the laboratories of Henderson, Baldwin and Ashcroft at Leeds. The amplified expression, purification and large-scale production of the proteins is achieved in both Henderson and Iwata's laboratories using 30-100 litre fermentation equipment. The strategies for crystallisation and structure determination will be devised in Iwata's laboratory using automated high throughput equipment. Baldwin provides models of target proteins to guide and interpret mutagenesis experiments.
Publications
Kalverda AP
(2014)
TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor.
in Molecular membrane biology
Henderson PJ
(2013)
This is about the in and the out.
in Nature structural & molecular biology
Weyand S
(2011)
The alternating access mechanism of transport as observed in the sodium-hydantoin transporter Mhp1.
in Journal of synchrotron radiation
Henderson PJ
(2012)
Structural biology: Bundles of insights into sugar transporters.
in Nature
Patching SG
(2018)
Recent developments in nucleobase cation symporter-1 (NCS1) family transport proteins from bacteria, archaea, fungi and plants.
in Journal of biosciences
Patching S
(2018)
Recent developments in nucleobase cation symporter-1 (NCS1) family transport proteins from bacteria, archaea, fungi and plants
in Journal of Biosciences
Bill RM
(2011)
Overcoming barriers to membrane protein structure determination.
in Nature biotechnology
Simmons KJ
(2014)
Molecular mechanism of ligand recognition by membrane transport protein, Mhp1.
in The EMBO journal
Shimamura T
(2010)
Molecular basis of alternating access membrane transport by the sodium-hydantoin transporter Mhp1.
in Science (New York, N.Y.)
Roberts, Gordon; European Biophysical Societies' Association (EBSA)
(2012)
Encyclopedia of Biophysics
Roberts, Gordon; European Biophysical Societies' Association (EBSA)
(2012)
Encyclopedia of Biophysics
Roberts, Gordon; European Biophysical Societies' Association (EBSA)
(2012)
Encyclopedia of Biophysics
Roberts, Gordon; European Biophysical Societies' Association (EBSA)
(2012)
Encyclopedia of Biophysics
Jones LN
(2010)
Defining topological features of membrane proteins by nanoelectrospray ionisation mass spectrometry.
in Rapid communications in mass spectrometry : RCM
Henderson P
(2012)
Comprehensive Biophysics
Ma P
(2016)
Allantoin transport protein, PucI, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity.
in Microbiology (Reading, England)
Description | Three-dimensional structures of four conformations of a membrane transport protein, Mhp1 Four structures of Mhp1 with different bound ligands At least ten novel ligands Advanced kinetic analyses identifying conformational intermediates Discovery of a novel inhibitor |
Exploitation Route | Conversion of hydantoins to added-value products including amino acids Extended range of substrates for a protein of commercial interest |
Sectors | Agriculture Food and Drink Pharmaceuticals and Medical Biotechnology |
Description | Researchers in agricultural sciences are using the information to understand metabolism of nucleobases by plants |
First Year Of Impact | 2013 |
Sector | Agriculture, Food and Drink,Chemicals,Education |
Impact Types | Economic |
Description | Impact of research papers on understanding of membrane transport |
Geographic Reach | Multiple continents/international |
Policy Influence Type | Citation in systematic reviews |
Description | Wellcome Trust Studentship |
Amount | £20,000 (GBP) |
Organisation | Wellcome Trust |
Department | Wellcome Trust Research Training Fellowship |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2012 |
End | 09/2015 |
Title | Determination of specificities of membrane proteins avoiding use of radioisotopes. Elucidation of topology of membrane proteins by mass spectrometry |
Description | See above |
Type Of Material | Technology assay or reagent |
Year Produced | 2016 |
Provided To Others? | Yes |
Impact | None yet |
Title | Structures of Mhp1 |
Description | Protein Data Bank known as the PDB |
Type Of Material | Database/Collection of data |
Year Produced | 2010 |
Provided To Others? | Yes |
Impact | The structures of the Na+-hydantoin transport protein we deposited in the database have been widely used and referenced in the field of research into membrane transport |
Description | Dr Alex Cameron of Imperial College, Diamond Light Source and Warwick University |
Organisation | Diamond Light Source |
Country | United Kingdom |
Sector | Private |
PI Contribution | Expertise in protein production and membrane transport |
Collaborator Contribution | Expertise in X-ray crystallography |
Impact | See authorship of joint publications |
Start Year | 2007 |
Description | Dr Alex Cameron of Imperial College, Diamond Light Source and Warwick University |
Organisation | Imperial College London |
Department | Imperial College Trust |
Country | United Kingdom |
Sector | Charity/Non Profit |
PI Contribution | Expertise in protein production and membrane transport |
Collaborator Contribution | Expertise in X-ray crystallography |
Impact | See authorship of joint publications |
Start Year | 2007 |
Description | Dr Arwen Pearson of Leeds University and Hamburg |
Organisation | University of Hamburg |
Country | Germany |
Sector | Academic/University |
PI Contribution | Expertise in membrane transport |
Collaborator Contribution | Expertise in X-ray crystallography |
Impact | PhD Thesis of Dr Anna Polyakova Publications in preparation |
Start Year | 2013 |
Title | Structures of the membrane transport protein Mhp1 with substrates bound |
Description | Structures of the membrane transport protein Mhp1 with substrates bound |
IP Reference | |
Protection | Protection not required |
Year Protection Granted | |
Licensed | No |
Impact | Much interest in the field of membrane transport |
Description | Conferences and seminars (Australia, China, Sweden, Estonia, Germany, Netherlands, Portugal, Spain, UK, USA) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | Conferences of peer group scientists, workshops for postgraduate students, lectures to undergraduates |
Year(s) Of Engagement Activity | 2011,2012,2013,2014,2015 |