Figure 6.

Structural basis for balanced assembly. (A) Top: Crystal structure of the GluA2-N54A NTD shows subtle alterations in the dimeric packing. Structural alignment of the N54A (green) and the WT (grey) crystal structures are shown. As expected, disrupting the hydrogen bond between N54 and the L310 main-chain carbonyl resulted in a different top loop conformation, but the L310 side chain was not shifted from its interface location. 2F_o−F_c omit map of A54 contoured to 1.0σ is shown for the relevant residue (blue) and the neighbouring residues (grey) in N54A. Bottom: In the WT context (PDB 3HSY), N54 makes a cross-dimer polar contact only on one side of the dimer interface (circled in blue). The result is unsatisfied hydrogen bonding potential at the dimer interface (circled in red), a destabilizing component of the WT interface. (B) Model of the thermodynamic effect of NTD mutations and their effects on whole-channel assembly. Whereas WT GluA2 NTD (top) is capable of balancing homo- and heteromerization at the level of the NTD and the whole receptor, N54A and T78A (bottom), which have opposite effects on NTD dimer stability, preferentially heteromerize the whole channel. Free energy changes of dimer dissocation (ΔΔG) in kcal mol⁻¹ are denoted. (C) Two interconnected steps dictate dimer assembly: homodimer dissociation (step I) and optimal homo- and heterodimer association (step II). Hotspots (stars) in the GluA2 NTD (red) upper lobe interface affect both these processes. Whereas the T78A mutation facilitates homomer dissociation, the N54A mutation stabilizes both homomerization and heteromerization (K_ds of dimer dissociation measured by AU-FDS are shown below the relevant assemblies). As heterodimerization requires both homodimer dissociation and re-association of heterodimers, the functional outcome for both mutations in the context of the full receptor is, therefore, greater heteromerization. The GluA1 NTD is shown in green for reference.