... understanding life in molecular detail

Prof Steve Baldwin

Membrane; Transport; Nucleoside; Protein structure

We are sad to announce that Professor Steve Baldwin passed away in November 2014.

Research in Steve's group centred on gaining a better understanding of the molecular basis for the passage of ions and small molecules across biological membranes and members of his group still continue some of the work. This involves investigation of membrane transporters using structural approaches (X-ray crystallography and electron crystallography), molecular modelling, structure/function analyses and single molecule techniques.

Current major projects include:
  • Active nucleoside transport in bacteria and humans
  • Single molecule dynamics of the bacterial translocon
  • Antibody mimetic binding proteins against membrane proteins
  • Exploitation of transporters to enhance crop plant growth on acidic, n
  • Exploitation of plant transporters for sustainable agriculture

Our laboratory is principally concerned with the structure, function and regulation of membrane transport proteins, both in prokaryotes and in eukaryotes. In addition, we are interested in the structure and function of proteins involved in vesicular trafficking in eukaryotes. Our major approaches include site-directed mutagenesis followed by heterologous expression in bacteria, yeast, Xenopus oocytes and insect cells to allow functional investigation of the mutated proteins. For those proteins, such as prokaryote transporters and eukaryote trafficking proteins, which we are able to express on a large scale, conventional and affinity-purification methods are used produce 10 to 100 mg quantities of pure protein for structural investigations. Methods being employed for such investigations include Fourier transform infrared spectroscopy, Circular Dichroism, Fluorescence Spectroscopy, Solid-State NMR and 2-D and 3-D crystallisation. Structural investigations are complemented by cell biological studies of protein function in cultured mammalian cells, for example using microinjection and bioimaging techniques to follow the trafficking of GFP-labelled proteins. For further information, please see our laboratory website.

Research Projects:

1. Structure of membrane transporters

In those genomes whose complete sequences have been determined, genes encoding putative membrane proteins commonly account for up to one third of the total. These proteins play an enormous variety of physiological roles, but in particular include receptors, channels and transporters. Given this physiological importance, a major goal of our laboratory is to understand how these proteins function at the molecular level, through gaining a better understanding of their structures. Our primary interests centre on two structurally-unrelated families of nucleoside transporters, the equilibrative nucleoside transporters (ENTs) and the concentrative nucleoside transporters (CNTs). These have been chosen because they appear to represent structurally novel families, unrelated to better known transporters such as members of the Major Facilitator Superfamily, and because of the physiological and clinical importance of the transport of adenosine and other nucleosides. Nucleoside uptake via these transporters is essential for synthesis of purine nucleotides by salvage pathways in many mammalian cells that lack de novo biosynthetic pathways. The same is true for protozoan parasites and so purine transporters represent a possible therapeutic target in these organisms.

Nucleoside transporters also represent the route of uptake for a number of cytotoxic analogues used in anti-cancer and anti-viral chemotherapy in man. Gaining a better understanding of their substrate recognition mechanisms should therefore facilitate the development of more selective drugs and lead to improved therapies. Finally, by regulating the accessibility of extracellular adenosine to cell surface receptors, the transporters modulate a wide variety of physiological processes, ranging from cardiac contractility to platelet aggregation.

Equilibrative nucleoside transporters

Our laboratory cloned the first example of an equilibrative nucleoside transporter, hENT1, from human placenta in 1997. This protein proved to be a member of a novel family of transporters, which we have termed the equilibrative nucleoside transporter or ENT family. Subsequent studies in our laboratory and elsewhere have shown that ENTs are distributed widely in mammals, plants, yeasts, insects, nematodes and protozoans, although they are absent from prokaryotes. We have shown, using glycosylation scanning mutagenesis and other approaches, that ENT family members are likely to possess a transmembrane topology of 11 membrane-spanning segments, with a cytoplasmic N-terminus and an extracellular C-terminus.

Our current research is concentrating on hENT1 and on a second homologue, hENT2, also cloned in our laboratory from human placenta. These transporters are not only responsible for the cellular uptake of natural nucleosides and nucleoside drugs but are also the targets for coronary vasodilator drugs such as dipyridamole. In addition, in collaboration with Glenn McConkey at Leeds, we are investigating the homologous transporter PfENT1 from the protozoan parasite Plasmodium falciparum as a potential target for novel antimalarial drugs.

Although we have been able to achieve limited over-expression of mammalian representatives of the ENT family in the yeast Saccharomyces cerevisiae, purification of the expressed protein on a sufficient scale for biophysical studies has not yet proven possible. Consequently, our current approach to investigation of the proteins? structure and function relies on non-crystallographic techniques and small-scale expression, particularly in Xenopus oocytes. This work, funded by the Wellcome Trust and MRC, is being performed in collaboration with Carol Cass and Jim Young at the University of Alberta in Edmonton, Canada. As an example of our approach, we have taken advantage of differences in substrate and inhibitor specificity between different members of the mammalian transporter family to identify regions and residues involved in the recognition of coronary vasodilator drugs and nucleoside analogues. This has been achieved by production of chimaeras and site-directed mutants, followed by expression in oocytes and examination of function. Armed with this preliminary information and simple models generated from analysis of aligned sequences of the transporter family, we are now embarked on a programme of site-directed mutagenesis aimed at delineating the substrate-binding site(s) of the transporters. Clearly, in order to understand the mechanism of transport in detail, in the longer term we need to be able to produce amounts of protein sufficient for crystallographic approaches to structure determination. Consequently, in parallel we are exploring other systems for the large-scale expression of the transporters, including the baculovirus system. At present, this has yielded higher levels of functional protein than yeast, but accompanied by a considerable amount of incorrectly folded protein. Optimisation of expression systems for eukaryotic membrane proteins represents a major goal of the laboratory.

In parallel with structure/function studies on mammalian and protozoan transporters, we are also investigating the biological roles of nucleoside transporters in a model metazoan, the nematode Caenorhabditis elegans. This organism was the first metazoan to have its complete genome sequenced, and contains two genes encoding CNT homologues and five genes encoding ENT homologues. In collaboration with the groups of Elwyn Isaac, David Coates and Ian Hope at Leeds we have already cloned and expressed all of the putative ENT genes and shown them to encode genuine nucleoside transporters. Their patterns of expression are currently being investigated using green fluorescent protein reporter constructs, and their biological roles are being assessed by disrupting the expression of individual transporters using RNA interference and gene knockouts. By these means we are beginning to bridge the gap between structural studies on individual transporters and an understanding of transporter function at the level of whole organism physiology.

Concentrative nucleoside transporters

Concentrative nucleoside transporters (CNTs) are found both in mammals and in prokaryotes, where they catalyse the symport of nucleosides with sodium ions and protons respectively. Despite this difference in ion-selectivity, they form part of a single gene family that is unrelated to the ENT family or to other known transporter families. Our MRC-funded studies of these proteins are being performed in collaboration with Simon Phillips, Peter Henderson and Richard Herbert at Leeds. Key partners in this work are also Maurice Gallagher at the University of Edinburgh, who cloned the first example of a prokaryote nucleoside transporter, NupC from Escherichia coli, and Jim Young and Carol Cass from the University of Alberta, who cloned the first example of a mammalian active transporter, rCNT1 from rat jejunum. Mammals possess three major types of CNT which differ in their substrate selectivities: cit-type transporters are pyrimidine-selective, cif-type transporters are purine-selective and cib-type transporters are of broad substrate specificity. The cit- and cif-types of transport activity correspond to two transporters, CNT1 and CNT2 respectively, that were cloned from various mammalian species several years ago. More recently, our collaboration with Canada has resulted in the cloning of the transporter corresponding to cib-type activity, which we have designated CNT3. Interestingly, CNT3 appears to transport 2 sodium ions per nucleoside, rather than 1 as is the case for CNT1 and CNT2. We are currently exploiting this difference in coupling ratio and the different substrate selectivities of the CNT family members in our attempts to gain an understanding of the mechanism of active transport by these proteins.

Through the use of glycosylation scanning mutagenesis and site-directed antibodies, we have established that the mammalian members of the concentrative nucleoside transporter family are likely to possess 13 transmembrane segments. Their bacterial counterparts appear to lack the first three such segments. By approaches similar to those described for the equilibrative transporters, we have been able to identify regions of the transporters putatively involved in substrate recognition, and are currently engaged in a programme of mutagenesis to delineate the binding sites more precisely. In parallel, we are attempting structural studies. The latter have become possible because of the existence of bacterial members of the family, which are potentially easier to express on a large scale than their eukaryotic counterparts. Indeed, working in collaboration with Maurice Gallagher and Peter Henderson, we have been able to over-express NupC to levels equivalent to approximately 25% of the total membrane protein in Escherichia coli. Excitingly, such amounts have proven sufficient for examination of the nucleoside binding site by solid-state NMR approaches, which are being undertaken in collaboration with Richard Herbert, Peter Henderson and Adrian Brough at Leeds, together with David Middleton at UMIST and Tony Watts and Paul Spooner at the National Biological Solid State NMR Facility, University of Oxford. In addition, we have recently been able to affinity purify the expressed protein on a sufficiently large scale (tens of mg) to embark upon 2-D and 3-D crystallisation trials.

2. Structure of membrane trafficking proteins

In addition to our investigations of membrane transporter structure, our group is also working on the protein machinery involved in vesicular trafficking in eukaryotic cells, in collaboration with Simon Phillips, Steve Homans and John Trinick at Leeds. Our interest in this area stems from the finding that trafficking of transporters between intracellular compartments and the cell surface is commonly involved in regulation of transport. For example, the hormone insulin stimulates sugar uptake in adipocytes and muscle cells by increasing the rate of exocytotic insertion into the plasma membrane of vesicles containing the glucose transporter GLUT4. Our particular current interest is the structure of the exocyst, a large complex of at least 8 different proteins that plays essential roles in the trafficking of vesicles from the Golgi apparatus to the plasma membrane in eukaryotic organisms ranging from yeast to man. We have recently been funded by the BBSRC as part of the MASIF Initiative to investigate the structure of this complex by applying a combination of X-ray crystallography, NMR spectroscopy and cryo-electron microscopy to the study of native and recombinant protein subunits, domains and the intact complex. This work is being complemented by cell biological studies of exocyst function, in collaboration with Vas Ponnambalam at Leeds.

Detailed research programme                  Close ▲

Selected Publications

  1. De Marcos Lousa, C., van Roermund, C.W., Postis, V.L.G., Dietrich, D., Kerr, I.D., Wanders, R.J.A., Baldwin, S.A., Baker, A. & Theodoulou, F.L. (2013) Intrinsic acyl-CoA thioesterase activity of a peroxisomal ABC transporter is required for transport and metabolism of fatty acids. Proc. Natl. Acad. Sci. USA, 110, 1279-1284.

  2. Huysmans, G.H., Chan, N., Baldwin, J.M., Postis, V.L., Tzokov, S.B., Deacon, S.E., Yao, S.Y., Young, J.D., McPherson, M.J., Bullough, P.A. & Baldwin, S.A. (2012) A urea channel from Bacillus cereus reveals a novel hexameric structure. Biochem. J. 445, 157-166.

  3. Newstead, S., Drew, D., Cameron, A.D., Postis, V.L., Xia, X., Fowler, P.W., Ingram, J.C., Carpenter, E.P., Sansom, M.S., McPherson, M.J., Baldwin, S.A. & Iwata, S. (2011) Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2. EMBO J. 30, 417-426.

  4. Deville, K., Gold, V.A., Robson, A., Whitehouse, S., Sessions, R.B., Baldwin, S.A., Radford, S.E. & Collinson, I. (2011) The oligomeric state and arrangement of the active bacterial translocon. J. Biol. Chem. 286, 4659-4669.