Periodic Reporting for period 1 - GlutaMature (Engineering and structure of transient ionotropic glutamate receptor complexes)
Reporting period: 2023-09-01 to 2025-08-31
Communication between neuronal cells is performed by cellular junctions called synapses. A hallmark of synapses is their ability to change their potency of signal transmission in response to patterns of neuronal activity, which is known as “synaptic plasticity”, a process that is important for allowing long-term information storage. Signal transmission through synapses is mediated by ligand-gated ion channels at the postsynapse, called ionotropic glutamate receptors (iGluRs), that are activated by the neurotransmitter glutamate. Multiple regulatory mechanisms control the magnitude of this postsynaptic response, such as the channel properties as well as the number and spatial positioning of the receptors. An important but not well studied mechanism is the regulation of iGluRs interact by extracellular proteins, notably adhesion molecules and synaptic organizer proteins, which modulate iGluR location and function. Despite their capital importance, the mechanism of iGluR modulation by synaptic organizer proteins remains highly unexplored.
The goal of this proposal was to elucidate the assembly of two iGluR subclasses (AMPA and GluD receptors) with extracellular scaffolding proteins. To this end, we aimed to determine the structure of AMPARs in complex with ESPs via protein engineering and biophysical/structural methods.
This integrative structural biology approach should reveal general structural principles of iGluR regulation by ESPs which will aid in understanding the role of ESPs in synaptic plasticity and could uncover novel therapeutic avenues for neurodegenerative diseases.
The goal of this proposal was to elucidate the assembly of two iGluR subclasses (AMPA and GluD receptors) with extracellular scaffolding proteins. To this end, we aimed to determine the structure of AMPARs in complex with ESPs via protein engineering and biophysical/structural methods.
This integrative structural biology approach should reveal general structural principles of iGluR regulation by ESPs which will aid in understanding the role of ESPs in synaptic plasticity and could uncover novel therapeutic avenues for neurodegenerative diseases.
During the project we have achieved several advances on the biophysical and structural characterisation of AMPAR:ESP complexes.
- We established an appropriate protein production and purification platform where we can rapidly generate stable cell lines using a lentiviral plasmid suite.
-We performed full biophysical profiling of the AMPAR ATD : ESP interaction.
- We enabled affinity maturation of the AMPARATD : ESP interaction, by establishing a protein engineering pipeline relying on mutant library generation via error-prone PCR followed by yeast display.The selection of high affinity mutants from the yeast display library was based on magnetic and fluorescence-enabled cell sorting and mutants were validated by BLI.
- The increased affinity enabled purification of stable complexes amenable for structural studies. Affinity matured complexes could be produced and purified to high yield and purity enabling X-ray crystallography and cryo-EM experiments. Via X-ray crystallography we obtained a low-resolution structure already revealing the binding epitopes. A higher resolution structure was achieved via cryo-EM which showed the detailed molecular interactions of the AMPARATD : ESP interface. The cryo-EM structure also revealed the structural basis for the affinity enhancing mutations.
- We validated the observed interface by generating structure-based mutants designed to abrogate key interactions. The effect of these mutants was validated by BLI showing the interaction could be completely blocked by structure-guided interface mutations.
- Further validation of affinity enhancing as well as blocking mutations was performed in cellular in collaboration with the Daniel Choquet team at IINS, these experiments confirmed the effect of the different mutations in an in vitro cellular model.
- We established an appropriate protein production and purification platform where we can rapidly generate stable cell lines using a lentiviral plasmid suite.
-We performed full biophysical profiling of the AMPAR ATD : ESP interaction.
- We enabled affinity maturation of the AMPARATD : ESP interaction, by establishing a protein engineering pipeline relying on mutant library generation via error-prone PCR followed by yeast display.The selection of high affinity mutants from the yeast display library was based on magnetic and fluorescence-enabled cell sorting and mutants were validated by BLI.
- The increased affinity enabled purification of stable complexes amenable for structural studies. Affinity matured complexes could be produced and purified to high yield and purity enabling X-ray crystallography and cryo-EM experiments. Via X-ray crystallography we obtained a low-resolution structure already revealing the binding epitopes. A higher resolution structure was achieved via cryo-EM which showed the detailed molecular interactions of the AMPARATD : ESP interface. The cryo-EM structure also revealed the structural basis for the affinity enhancing mutations.
- We validated the observed interface by generating structure-based mutants designed to abrogate key interactions. The effect of these mutants was validated by BLI showing the interaction could be completely blocked by structure-guided interface mutations.
- Further validation of affinity enhancing as well as blocking mutations was performed in cellular in collaboration with the Daniel Choquet team at IINS, these experiments confirmed the effect of the different mutations in an in vitro cellular model.
The final results of the project are the engineering of the AMPAR:ESP interaction and the ensuing biophysical and structural characterisation. These results will have implications for further research on AMPAR regulation during synaptic plasticity as well as development of therapeutics for neurodegenerative diseases.
- The affinity enhancing mutations obtained from the affinity maturation pipeline could be used in the design of therapeutics for enhancing synaptic connectivity in neurodegenerative diseases.
- The novel structural information ons AMPAR:ESP complexes provides a framework for the results of future neurobiology research.
- The affinity enhancing mutations obtained from the affinity maturation pipeline could be used in the design of therapeutics for enhancing synaptic connectivity in neurodegenerative diseases.
- The novel structural information ons AMPAR:ESP complexes provides a framework for the results of future neurobiology research.