Mutation of site 1 produced a −4.5 kcal/mol loss of binding energy for the GluR6Δ2F58A homodimer, but only a −2.9 kcal/mol loss for the GluR6Δ2F58A/KA2Y57A heterodimer, with equal contributions by the F58A and Y57A mutants, of −1.4 and −1.5 kcal/mol, respectively

(Table S1). The excess total dimerization energy, totaling 3.1 kcal/mol, must come from other sites in the heterodimer interface. The mutation E156A produced a loss of only −0.43 kcal/mol; for L163A the loss was −1.31 kcal/mol. The S165G/T168A double mutant produced a loss of binding energy of −1.74 kcal/mol. Strikingly, Ser165 and Thr168 do not make contacts with the GluR6 subunit, and instead merely serve to stabilize the loop conformation which positions Leu163 and Ile164 in the dimer interface. To examine whether the binding Pexidartinib order mechanism for heterodimer formation was an additive Buparlisib or cooperative process we performed a mutant cycle analysis looking at interactions between sites in domains R1 and R2 with both the GluR6Δ2 and GluR6Δ2F58A mutant used as heterodimer partners. Mutant cycles were calculated as shown in Figure 5B, where coupling coefficients (Ω) greater than one indicate positive cooperativity (Carter

et al., 1984 and Hidalgo and MacKinnon, 1995). The analysis yielded coupling coefficients (Ω values) of only 0.8–1.7 and reveals clearly that heterodimer assembly is an additive process with little cooperativity between domains R1 and R2 (Supplemental Experimental Procedures).

Sodium butyrate The much larger disruption of heterodimer assembly observed for the I164A mutant likely reflects conformational changes resulting from destabilization of the hydrophobic patch formed by the loop rearrangement in the KA2 subunit. Of note, amino acid sequence alignments (Figure 5C) reveal that in other iGluR subunits Ile164 is replaced by charged or polar residues, consistent with a unique role for Ile164 in mediating heterodimer assembly for KA1 and KA2. This alignment also reveals exchange of Glu167 (Asp165 in KA1) by Trp/Ile/Leu in other iGluR subunits (Figure 5C). The residues exchanged are in a flexible loop region connecting helix F and strand 7, the conformation of which differs in individual iGluR families. In GluR1–4 and GluR5–7 the Trp/Ile/Leu residues form part of the hydrophobic core of domain 2, while in the KA2 subunit the polar residues are surface exposed, and make intersubunit contacts in the heterodimer assembly. In the KA1 and KA2 subunits, Phe160/162 fills the space in the hydrophobic core which in other iGluR subunits is occupied by the Trp/Ile/Leu residues which align with Asp165/Glu167 in KA1 and KA2. At the corresponding position in the AMPA receptors and GluR5–7 the Phe residue is replaced by smaller Ala or Pro side chains. In order to elucidate the structure of the GluR6/KA2 ATD tetramer, we crystallized a complex of wt GluR6 and KA2.