ITC measurements were performed with a low-volume Nano ITC (TA Instruments). PKA-CWT and PKA-CL205R were dialyzed into 20 mM Mops, 90 mM KCl, 10 mM dithiothreitol (DTT), 10 mM MgCl2, and 1 mM NaN3 (pH 6.5). PKA-C concentrations for ITC measurements were between 100 and 130 μM as confirmed by A280 = 53,860 M−1 cm−1. All measurements with ATPγN-saturated PKA-CWT and PKA-CL205R were performed at 2 mM ATPγN and 4 mM ATPγN, respectively. ITC measurements were performed at 300 K in triplicate. Approximately 300 μl of PKA-C was used for each experiment, as well as 50 μl of 2 to 4 mM ATPγN, 0.6 to 4 mM PKI, or 2 mM VPS36 in the titrant syringe. The heat of dilution of the ligand into the buffer was taken into account for all experiments and subtracted accordingly. Binding was assumed to be 1:1, and curves were analyzed with the NanoAnalyze software (TA Instruments) using the Wiseman isotherm (40)d[MX]d[Xtot]=H°V0[12+11r2Rm/2(Rm22Rm(1r)+(1+r)2)1/2](1)where d[MX] is the change in total complex with respect to change in total protein concentration and d[Xtot] is dependent on r (the ratio of Kd with respect to the total protein concentration) and RM (the ratio between total ligand and total protein concentration). The free energy of binding was determined using the followingG=RTlnKdwhere R is the universal gas constant and T is the temperature at measurement (300 K). The entropic contribution to binding was calculated using the followingTS=HG

Calculations for the cooperativity constant (σ) were calculated as followsσ=Kd ApoKd Nucleotidewhere Kd Apo is the Kd of PKI5–24 binding to the apoenzyme and Kd Nucleotide is the Kd of PKI5–24 binding to the nucleotide-bound enzyme.

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