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Nitrate transporter structure paves the way for exciting new areas of work

Crystal structure of nitrate bound NRT1.1 (PDB 4CL5)

Crystal structure of nitrate bound NRT1.1 (PDB 4CL5) (Click to enlarge)

The elucidation of the structure of a key nitrate transporter in plants has revealed details that will help us understand how a family of physiologically important transporters in plants and humans work.

The research by Dr Jo Parker and Dr Simon Newstead, published in Nature [1], provides the first crystal structure for a plant transporter and is one of only four crystal structures of a eukaryotic membrane transporter to be solved. It reveals how the transporter NRT1.1 from Arabidopsis thaliana binds nitrate and is regulated by phosphorylation.

The work will inform studies of related mammalian transporters that are attractive targets for improving the efficiency of drug delivery in the human body. It also provides the first step towards the possible design of crop plant variants with improved nitrogen use efficiency.

NRT1.1 belongs to a family of proton-coupled transporters found in many organisms. In bacteria and mammals, the transporters are important for uptake of peptides. But in plants, NRT1.1 and other members of this family have evolved to transport nitrate as well as hormones and metabolites –a remarkably chemically diverse group of substrates.

A notable feature of NRT1.1 is that it is able to adapt to changing levels of nitrate in the soil. Functioning as a ‘dual affinity transporter’, it can switch its ability to recognise nitrate depending on soil availability. In conditions of high external nitrate, NRT1.1 displays low affinity for nitrate; when nitrate levels fall, intracellular phosphorylation switches it to a high affinity state, able to scavenge the last remains of the nutrient for growth.

Dr Jo Parker took a two-pronged approach to probe the molecular features of NRT1.1, combining X-ray crystallography with an in vitro functional assay for the transporter.

The researchers were able to describe the overall architecture of the protein and identify two key sites of interest: the site of phosphorylation that switches the transporter between the low and high affinity states, and the location of nitrate binding.

Their findings suggest that phosphorylation increases structural flexibility of the protein and in turn the rate of transport, as Dr Newstead explains: ‘Increased nitrate uptake is likely to be the result of the transporter working at a faster rate - essentially moving up a gear to move more nitrate into the root cell.’

A group in the US has also determined the crystal structure of NRT1.1 at the same time [2]. In their study they show that phosphorylation switches NRT1.1 from being a dimer in the membrane to a monomer, a mechanism consistent with the findings from the Biochemistry Department researchers.

By comparing bacterial and plant transporters, Dr Parker and Dr Newstead went on to identify the changes in the binding pocket that have switched the transporter from recognising peptides to recognising nitrate. They also identified the elements that are retained between the different transporters and are an integral part of the conserved coupling mechanism.

Model for nitrate uptake in plants via NRT1.1 (reproduced with permission Nature Publishing group)

Model for nitrate uptake in plants via NRT1.1 (reproduced with permission Nature Publishing group) (Click to enlarge)

Dr Newstead comments: ‘Our study also informs our understanding of the molecular features that enable these transporters to recognise substrates of very different chemical make-up. The remarkable thing is that there appears to be only a few changes to switch it from one to the other.’

The results are exciting because they shed light on how transporters may be regulated in biology and provide molecular details essential for future manipulation of mammalian and plant proteins.

‘This is the first structure of a transporter that has the dual-affinity characteristic that is switched by phosphorylation,’ says Dr Newstead. ‘That’s a key point because we don’t know how transporters are regulated very well other than through trafficking.’

‘Our work reveals the molecular basis for that switch, something that we may be able to apply to understanding similar switches in the mammalian proteins which almost certainly exist. The fact that we could replicate the same kind of switch in the bacterial proteins indicates that there is some common fundamental mechanism there that we can tap into.’

Another possible application of the findings is to inform the engineering of a plant transporter with enhanced characteristics. A transporter that takes up nitrate more efficiently, for example, would alleviate the use of nitrogen-based fertilisers for crop production [3].

Our understanding of the regulatory networks that control nitrate uptake is not sufficiently advanced for this at present. But a strength of the study is that it focuses on NRT1.1 protein from Arabidopsis, a model plant organism and a member of the brassica family. This increases the relevance of the results to crop plants and facilitates the translation of the research from the lab to the field.

The identification of the molecular basis for the different transporters’ specificities, including the pinpointing of conserved or variable elements of the binding site, also has direct implications for the study of mammalian transporters PepT1 and PepT2.

The work will not only guide biochemical investigations on these transporters. In addition, because absorption of many commonly prescribed beta-lactam antibiotics in the body is facilitated by the transporters, it will help in efforts to engineer other drugs to be taken up via this route.

A recently awarded Wellcome Trust Investigator Award to Dr Newstead will enable his group to focus on understanding how PepT1 and PepT2 carry out their functions in the human body (


  1. Parker, J. & Newstead, S. Molecular basis of nitrate uptake by the plant nitrate transporter NRT1.1. Nature 507, 68-72 (2014).
  2. Sun et al. Crystal structure of the plant dual-affinity transporter NRT1.1. Nature 507, 73-77 (2014).
  3. Yi-Fang Tsay. Nature News & Views ‘How to switch affinity’. Nature 507, 44-45 (2014).






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