The results gave Kd values with 95% confidence intervals of 2.0 μM (0.18–2.27 μM) for E156A, 6.7 μM (6.4–7.1 μM) for L163A, and 13.0 μM (10–15 μM) for the S165G/T168A double mutant. The I164A mutation produced a much greater disruption, such that we can set only a lower limit on the Kd of 200 μM or larger. selleck products We also screened two additional sets of KA2 mutants in regions of the structure which might be anticipated to affect heterodimer formation. The first set tested, KA2 C64S/C315S, targeted the disulfide bond which holds loop three in place (Figure 5A). The second set targeted Lys148 and Glu150 at

the N terminus of α-helix E, which were candidates for mediating contacts with His105, Ser108 and Asp109 in domain R1 of the GluR6 protomer. However, both the C64S/C315S and K148A/E150A KA2 double mutants produced no change in oligomerization when mixed with either GluR6Δ2 Erastin cell line or GluR6Δ2 F58A and analyzed by UV/RI/MALS-SEC (Figure S5). The lack of effect of

loop 3 disulfide bond disruption likely occurs because, in a heterodimer assembly with GluR6, loop 3 of the KA2 subunit is held in place by other contacts such as the hydrogen bond between the main chain carbonyl oxygen of Cys315 and the side chain of Lys62 in the GluR6 subunit. We were unable to test the effect of the GluR6 C65S/C316S mutant, because this construct could not be expressed at levels sufficient for biochemical analysis, possibly due to misfolding. The interactions made by Lys148 and Glu150 with

the GluR6 subunit are formed in solvent exposed loops with weak electron density, and it is likely that this region is quite mobile, since our results reveal that it does not contribute to dimer stability. To estimate the strength of the interactions underlying dimer formation we purified a series of 15 mutant ATD proteins and measured their Kd for homodimer and heterodimer formation using SV experiments. To select mutant combinations suitable for analysis by SV we performed SEC-UV/RI/MALS experiments to assay for either depletion of the Protein kinase N1 monomer KA2 peak when mixed with GluR6Δ2, or an increase in dimer peak when mixed with GluR6Δ2F58A (Figures 1C and S5A; Table S1). Out of 30 combinations tested, 13 pairs were selected for analysis by SV; examples of isotherms for weighted-average sedimentation coefficients for KA2 mutants mixed with GluR6Δ2F58A and GluR6Δ2 are shown in Figures 4C and S5C, respectively. To calculate ΔΔG values we used SV measurements for the GluR6Δ2 homodimer Kd (250 nM), the KA2 homodimer Kd (350 μM), and the GluR6Δ2/KA2 heterodimer Kd (11 nM), as reference values (Table S1). The formation of GluR6Δ2/KA2 heterodimers is favored by 6.04 kcal/mol compared to the KA2 subunit homodimer Kd.