Metal sensing properties of the disordered loop from the Arabidopsis metal transceptor IRT1

The plant IRT1 iron transporter is a plasma membrane protein that takes up iron in root upon iron-limited conditions. Besides its primary metal substrate iron, IRT1 transports other divalent metals that overaccumulate in plants when soil iron is low and IRT1 is highly expressed. We previously reported that the intracellular regulatory loop between transmembrane helices TM4 and TM5 is involved in the post-translational regulation of IRT1 by its non-iron metal substrates. Upon excess of zinc, IRT1 undergoes phosphorylation by CIPK23 followed by its ubiquitination by IDF1 to target IRT1 for vacuolar degradation. This zinc-dependent downregulation of IRT1 requires the presence of four histidine ( residues in the IRT1 loop, that directly bind zinc. However, how selective metal binding is achieved and how this allows downstream regulation to take place is largely unknown. Here, we characterized the metal binding properties and structure of the IRT1 loop to better understand the molecular basis of non-iron metal sensing and signaling. Using a combination of circular dichroism and NMR, we reveal that zinc and manganese bind to the IRT1 loop with nanomolar range affinity, and that metal binding does not trigger structuration of the loop. We validate that zinc and manganese binding is mediated by four residues and identify aspartic acid (D) residue D173 as helping in metal coordination and participating to metal sensing and metal-dependent degradation of IRT1 in plants. Altogether, our data provide further understanding about how IRT1 regulatory loop senses high cytosolic divalent metalconcentrations to regulate metal uptake in plants.