Binding Proteins and Receptor Binding Domains as Sensor Elements for Biological and Artificial Nanopores

Abstract

Miniaturized electrical biosensors have become a promising tool for monitoring and analyzing biologically relevant substances by converting a biological signal into an electric current. Despite great progress in the analysis of macromolecules, the detection of small molecules that are of particular interest for medical and environmental analytics still remains a challenge. Here, the functional connection of a sensing element to an electrical switch is a bottleneck in biosensor design. Bacterial substrate binding proteins (SBPs) and ligand gated ion channels (LGICs) evolved over billions of years to recognize a variety of biologically relevant molecules with high selectivity and sensitivity. While SBPs are involved in the uptake of substances across bacterial cell membranes, LGICs mediate neuronal excitation in the central nervous system of vertebrates. The ancient binding modules of these two protein families share a conserved clamshell-like structure and entrap the ligand in their inter-lobe cleft by inducing a large conformational transition between the open- and closed- cleft states in a venus flytrap-like mechanism. In this work, i) the underlying mechanisms of ligand recognition and functional adaptability of LGICs and SBPs are investigated and exploited to ii) couple SBPs as sensor domains to biological and solid-state nanopores to build new types of electrical biosensors for the specific detection of biologically relevant small molecules. Despite their intrinsic capability to convert a chemical signal into an electrical signal, LGICs are only now gradually being used for biosensor design since core aspects in the mechanistic understanding of ligand recognition, modulation and activation in different receptor subtypes are poorly understood. Here we investigated the structural impact and mechanism(s) of full and partial agonism in glycine receptors (GlyRs) in its native lipid environment and the modulatory role of the amino terminal domain (NTD) in GluN1/GluN3 NMDA receptor auto-inhibition after glycine binding to the low affinity GluN1 subunit. We show that the full agonist glycine and the partial agonist taurine induce different conformational transitions of the α1 GlyR. In addition, we show that the expression system dependent variability of agonist affinity in HEK293 cells and Xenopus oocytes is not mediated by an altered conformational change. Furthermore, we report that the GluN3A NTD has a major role in GluN1/GluN3A receptor regulation by reducing the efficacy of glycine-depended receptor activation by agonist-evoked auto-inhibition. This effect is possibly mediated by the subunit interface and the NTD-LBD linkers of the GluN3A NTD. These insights into the conformational changes and structural adaptability have been further exploited to use bacterial SBPs and SBDs from LGICs as a molecule detector when connected to an electrical switch by coupling the ectoine binding protein EhuB to the channel pore of the ionotropic glutamate receptor GluR0 to design receptor-based biosensor and by coupling the phosphonate binding protein PhnD inside a single track-etched solid-state nanopore that combines the high affinity and selectivity of SBPs with the robustness of artificial nanopores. These new classes of electrical biosensors are characterized by a high ligand-affinity and specificity with concentration-dependent changes in the (nanopore) current after Ligand binding. The results in this work provide an excellent foundation for the use of SBPs and LGICs as sensor domains to developme new classes of electric biosensors with high specificity and affinity for detection of biologically relevant small molecules. Moreover, our approaches and insights into ligand recognition and modulation enhance the repertoire of biophysical methods and may deepen the understanding of the functions of LGICs at the molecular, synaptic and systemic level.