Ammonium Sensor Proteins in Signal Transduction

• Applicant: Professorin Dr. Susana Andrade
• Subject Area: Biochemistry
• Term: from 2012 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 226824845

Ammonium transport proteins (Amt) play a central role in the uptake of ammonium ions as a source of nitrogen for biosyntheses in microorganisms and plants. The orthologous Rhesus proteins of animals are required for the regulation of urine concentration and the homeostasis of important electrolytes. Database searches now increasingly produce Amt family members that contain further cytoplasmatic or extracellular domains in addition to the conserved intramembrane Amt domain. In many cases these are typicial signal transducer domains that suggest a role in signal recognition and transduction for these membrane proteins. Within the present proposal we will characterize two selected members of these ammonium sensor proteins with respect to structure and function, in order to elucidate the nature of the signal and the molecular mechanisms of signal binding and transducer domain activation.Kuenenia stuttgartiensis, a model organism for anaerobic ammonium oxidation (anammox) contains the protein Amt-5, an Amt-based sensor-transducer system with a C-terminal domain that belongs to the class of histidine kinases and as such, upon activation, phosphorylates a downstream partner in the transduction chain under ATP hydrolysis. We have already produced and crystallized Amt-5, but while these crystals yielded a high-resolution structure for the membrane domain, the transducer domain was disordered. The second system, Amt-1 from Shewanella denitrificans, shows a similar architecture, but its cytoplasmatic domain belongs to the GGDEF family of diguanylate cyclases. These domains produce cyclic di-GMP as a second messenger, and their activation is commonly triggered by multimerization. In the context of an Amt sensor this indicates a structural rearrangement in the sensor part, the Amt domain. We have also produced Amt-1 with good yields and have obtained single crystals of high quality. The sensor and transducer domains of both proteins were also produced separately, and initial functional studies show that the transducer domains are active and that they are activated in the presence of ammonium ions in the full-length proteins.The proposed work aiming at understanding the functional properties of ammonium sensor proteins will not only lead to the first quantitative description of these systems, but will ideally also provide important information concerning the basic functional principles of coupled sensor-transducer systems in cellular signal transduction. By using various structural methods such as SAXS and X-ray crystal diffractometry as well as functional studies on individual domains and full-length proteins we aim for a description at unprecedented precision.

• DFG Programme: Research Grants