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

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Henderson P (2012) Comprehensive Biophysics

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Henderson PJ (2013) This is about the in and the out. in Nature structural & molecular biology

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Jones LN (2010) Defining topological features of membrane proteins by nanoelectrospray ionisation mass spectrometry. in Rapid communications in mass spectrometry : RCM

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Roberts, Gordon; European Biophysical Societies' Association (EBSA) (2012) Encyclopedia of Biophysics

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Roberts, Gordon; European Biophysical Societies' Association (EBSA) (2012) Encyclopedia of Biophysics

 
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 10/2012 
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