Gephyrin is a dynamic receptor-associated protein that plays a crucial role in the clustering and anchoring as well as trafficking of inhibitory neuronal receptors. Gephyrin is composed of three regions, an N-terminal G-domain, a C-terminal E-domain, and a presumably unstructured linker region connecting the two domains. The isolated G- and E-domains form trimers and dimers, respectively, and the two independent oligomerization interfaces led to the proposal that gephyrin oligomers can assemble into a hexagonal lattice below the postsynaptic membrane providing binding sites for the inhibitory neurotransmitter receptors on one side and components of the cytoskeleton on the opposite side, thereby anchoring the receptors either directly or indirectly.

The interactions with the light chains of the dynein motor (DYNLL1 and DYNLL2) mediate the retrograde transport of glycine receptor-gephyrin complexes via the dynein motor. A short peptide (residues 203-214) within the central linker of gephyrin has been iv identified as the DYNLL-binding site. I determined the crystal structures of DYNLL1 and DYNLL2 in their apo-state and in complex with a peptide derived from gephyrin's linker. The peptide is present in an extended conformation and binds as an additional β-strand to either DYNLL. The analysis of the binary complexes revealed the contribution of individual amino acids to this interaction and revealed that Tyr65, His68 and Phe73 of DYNLL and Gln211 of gephyrin are critical for this interaction. Comparisons with other DYNLL-peptide complexes demonstrate a remarkable plasticity in the ligand-binding pocket.

The gephyrin linker region revealed that it contains the binding sites of additional gephyrin interacting proteins besides DYNLL, but its structural properties are not well characterized. The functional significance of gephyrin linker region which is presumably unstructured has been analyzed here. A thermostablility analysis revealed that the C-terminal end of the linker region stabilizes the E-domain. The oligomerization potential of the linker fused to either the G- or E-domain has been biochemically analyzed since the isolated linker cannot be analyzed due to its proteolytic sensitivity. These studies revealed that the linker of gephyrin contains an independent oligomerization site. The N-terminal half of linker region including the C-terminal end of the G-domain was deduced as an important region for oligomerization of the linker. The binding on G- or E-domain fused linkers to DYNLL revealed that interactions involving on the linker region can modulate the oligomerization of gephyrin.