Section 2: Analysis
Five workpackages dedicated to functional and structural analysis
In workpackage 5, structural data of the acetylcholine binding proteins (AChBPs), co-crystalized with various ligands, was obtained in order to understand the fundamentals of the structure of the CLR ligand-binding domain. Also, AChBP was used to probe for new classes of ligands.
Due to the work in workpackage 6 Neurocypres is able, in its first year, to celebrate the first structure solved of an entire cys-loop receptor by one of its partners (PJ Corringer, partner 7). Read the article >
This workpackage is devoted to prokaryotic CLRs. A combined genomic search, functional expression and X-ray crystallography yielded the discovery and atomic structural resolution of (several) bacterial homologs of LGICs. The homolog from the cyanobacterium Gloeobacter violaceus (GLIC) revealed that it functions as a proton-gated ion channel. Its X-ray structure was solved at 2.9 Å resolution in an apparently open conformation.
Structural comparative analysis of GLIC and ELIC, a bacterial homologue from Erwinia chrysanthemi was solved in a presumed closed conformation, reveals a rotation of each extracellular b-sandwich domain as a rigid body, interface rearrangements, and a reorganization of the transmembrane domain, involving a tilt of the M2 and M3 a-helices away from the pore axis. These data are consistent with a model of pore opening based on both quaternary twist and tertiary deformation.
Within the Neurocypres project we expect to substantially expand our the knowledge of the bacterial receptors.
Workpackage 7 is devoted to high-throughput analysis of CLRs. These include, for instance, high-throughput electrophysiological analyses of receptor expression in oocytes. In particular, new technological advancements have been made in the lab of Bertrand (partner 12), enabling the screening of compounds on receptors expressed in oocytes at a previously unprecedented scale. Also, fluorescence based assays were developed that will be used for the screening of ligands for the 5HT3R. Beactica (partner 20) has been able to set-up and affinity measurements for ligands on the AChBP proteins using surface plasmon resonance.
In workpackage 8 we further explored the use of CLRs to probe neuronal function. This work on so-called biosensors, has now progressed to the first studies in neurons. Further experiments in the context of the mouse brain are underway using transgenic expression and knockin mice.
Workpackage 9 is devoted to analysing CLR subunit stoichiometry. In particular to reveal the subunit stoichiometries for native receptors in the CNS and to enable the identification of potential receptor-associated proteins, several approaches and chemistries to pull-down receptors were developed by Gotti (partner 13) and ProteoSys (partner 18).