Christian Buch Parsbæk:
|
Long-terminal repeat retrotransposons accounts for ~8% of the human genome and originate from ancient viral infections. Some of these endogenous retroviruses have been domesticated in the host genome to adapt to novel functions beneficial to the host organism. The activity-regulated cytoskeleton-associated protein (Arc) originates from the Ty3/Gypsy retrotransposon family and exhibits homology to active exogenous retroviruses.
Arc is highly expressed in neuronal dendrites and functions as a protein interaction hub regulating pathways of neuronal signaling, synaptic plasticity and long-term memory formation. In addition, Arc is capable of self-associating into capsid-like structures for encapsulation of RNA which may be transferred to a neighboring cell reminiscent of the function Gag proteins of active viruses. However, the molecular basis of several of Arc’s functions is not well understood.
To describe the underlying molecular behaviour more accurately, we set out to characterize several structural and functional features of Arc. We characterized the structure of the full C-terminal domain of Arc, the CA domain, revealing a relatively rigid bi-lobar conformation with little interdomain flexibility. Despite the rigidity, we show that Arc CA has retained its ability to self-assemble into capsid-like structures.
Additionally, we investigated the interaction between Arc and the NMDA-receptor subunits GluN2A and GluN2B to detail the specific binding sites for Arc. Interestingly, the interaction inhibited capsid-like structure formation for Arc indicating a functional regulation upon ligand binding. To further pin down the specificity of Arc, we systematically evaluated the binding of alanine-variants of a known binder, TARPG2. We confirm that a proline and tyrosine/phenylalanine at specific positions of the binding motif are crucial for the interaction while we find five additional residues which affect the binding affinity.
Additionally, to work towards a more adequate description of the determinants of ligand binding for Arc, we set up a novel high-throughput screening system to screen for low affinity binders. Finally, we also investigate the interaction of another neuronal protein, PSD-95, and its interaction with TARPG2 and how this interaction influences the phase separation properties of PSD-95.