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abstracts
Sodium/proton (Na+/H+) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis. In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets. The best understood model system for Na+/H+ antiport is NhaA from Escherichia coli, for which both electron microscopy and crystal structures are available. NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein. Like many Na+/H+ antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, at which point a conformational change is thought to occur. The only reported NhaA crystal structure so far is of the low pH inactivated form. Here we describe the active-state structure of a Na+/H+ antiporter, NapA from Thermus thermophilus, at 3 Å resolution, solved from crystals grown at pH 7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding directly, a role supported here by molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 200 against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second, Na+/H+ antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.
abstracts
Nitrogen uptake and assimilation is a key limiting factor for plant growth and crop productivity and also acts a major signaling molecule, controlling many aspects of plant development. Many plants obtain nitrogen through the uptake of nitrate from the soil via specific membrane transporters. Two families of nitrate transporter have been identified that act within the root cell, termed NRT1 and NRT2. NRT1 members are predominantly low affinity transporters, with KM values in the millimolar range, whereas NRT2 members are high affinity transporters, with KM values in the low micromolar range. Dual affinity transporter systems are used in biology to allow the cell to respond to changes in an external nutrient supply. In the case of nitrate transport in plants, decreasing levels of external nitrate cause an increase in the expression of NRT2 family transporter genes, in particular NRT2.1, allowing the cell to take up more of the available nitrate. However, plants have evolved a faster way of responding to nitrate levels involving post-translational control of nitrate uptake. NRT1.1 also known as CHL1, is a dual affinity nitrate transporter. In response to decreasing levels of nitrate, NRT1.1 is capable of switching between low and high KM modes, a switch achieved through the post-translational phosphorylation of an intracellular threonine. Here I will present our recently determined crystal structures of NRT1.1 in both the apo and nitrate bound forms. Together with in vitro binding and transport data we identify key residues involved in nitrate recognition and provide the first biochemical explanation for the phosphorylation controlled `dual affinity' switch observed in NRT1.1. Finally we present our model for the molecular basis of nitrate uptake via this transporter.
