Insights into monovalent cation binding aided by the poisoner’s poison

Monovalent cations are important components of many biological macromolecular assemblies but are typically challenging to study by structural biology techniques. Through our studies of Kbp, a potential regulator of cellular homeostasis in E. coli whose structure is K+ dependent, we have explored strategies to discover and characterise the monovalent cation binding site leveraging the K+ analogue Tl+.
Potassium is key to cellular homeostasis. E. coli possesses several specific K+ influx and efflux systems that maintain the intracellular K+ concentration over a broad range of external pH, osmolarity and K+ concentrations. Although regulatory proteins and sensor domains have been identified for several K+ ion transport systems, the exact mechanism by which K+ concentration is sensed in the cell and therefore how these systems are regulated remains unknown.
Expression of the cytoplasmic protein Kbp is strongly upregulated in response to osmotic stress. We discovered that Kbp is an unprecedented, highly selective, soluble K+ binding protein and determined its K+-bound structure. Kbp undergoes a conformational change upon K+ binding that we and others have leveraged to generate novel genetically encoded fluorescent K+ sensors. We have used a range of techniques to explore the conformational repertoire of Kbp and identify the ion binding site.
Our latest results include progress towards an NMR method for the direct observation of the interaction of proteins with monovalent cations backed by quantum mechanical calculations.