Ammonium is a vital source of nitrogen for bacteria, fungi and plant, and is a toxic waste product of metabolism for animals, including human. Hence, the transport of ammonium across biological membranes is a process of fundamental importance in all living organisms. In procaryotes and plant, ammonium uptake is mediated by Amt proteins whereas mammals excrete it using Amt orthologs, the Rhesus proteins. Despite their primary sequence homologies, the functional context of Amt and Rhesus is diverse: bacteria, fungi and plant use Amt proteins to scavenge ammonium from their environment, whereas mammals use the Rhesus proteins for detoxification in erythrocytes kidney and liver tissues. Although the structural insights and an increasing number of functional studies have greatly increased our knowledge of the Amt/Rh protein family over the past few years, important mechanistic questions are still largely unresolved. The most pressing are
What are the functional features that are important for the ion specificity and ion permeation?
What are the structural features that explain the mechanistic difference between Amt (transporter) and Rh (channel)?
What are the conformational changes associated with the transport cycle?
Defects in Rhesus factors are related to human pathologies affecting red blood cells, kidney function and male fertility. Hence, answering these questions may lead to an understanding of these disorders with the prospect of potential therapeutic interventions.
We have developed the Escherichia coli ammonium transport protein (EcAmtB) has the model system of choice for analysing ammonium uptake by the Amt protein. Genes encoding Rh proteins have been identified in some bacterial genomes. We have studied one of these from Nitrosomonas europaea (NeRh50). NeRh50 is a homotrimer structurally very similar to AmtB. Hence the EcAmt and NeRh50 represent the perfect system for our study. The overall aim of this project is to use an ambitious cross-disciplinary and multi-faceted approach calling upon expertise in genetics, biochemistry, biophysics and molecular dynamic simultion to gain valuable functional information on the important and ubiquitous Amt/Rhesus family of proteins.