Figure 4

Trapping DGK in nucleotide- and/or lipid-bound states. (a) Competitive inhibition assay proves the binding of Mg*AMP-PCP to the active sites of DGK. DGK proteoliposomes were incubated with 4 to 16 mM of Mg*AMP-PCP, which equates 4 to 16- fold molar excess compared to DGK. A 3-fold molar excess of Mg*ATP (3 mM) compared to DGK was present in each sample. A concentration of at least 10 mM of Mg*AMP-PCP (10-fold molar excess) is necessary to reduce the activity of DGK below 10%, leading to a fully saturated system. 100% activity corresponds to the rate recorded with wtDGK in 90 mol% DMPC/10 mol% DMPA of 90 (±9.9) µmol min⁻¹ mg⁻¹. Experiments were repeated three times. The activity was calculated as the mean value. Error bars correspond to standard deviations. (b) Binding of AMP-PCP is confirmed by ³¹P-CP MAS. Proteoliposomes were incubated with a 14-fold molar excess of Mg*AMP-PCP (pH 7.2). (c) DGK was reconstituted into 80 mol% DMPC/DMPA and 20 mol% DOG and incubated with a 14-fold molar excess of Mg*ATP (pH 7.2). DGK phosphorylates DOG to DOG-PA, which can be detected by ³¹P-MAS NMR, both by cross- and direct polarization (in green) as described before²¹. Both nucleotide and DOG-PA have bound and non-bound populations. The CP experiment emphasizes the bound fraction. The spectra verify that DOG can reach the active site of DGK in our preparations. (d) ³¹P-CP spectrum of DGK reconstituted into 80 mol% DMPC/DMPA and 20 mol% DOG and incubated with a 14-fold molar excess of Mg*AMP-PCP (pH 7.2), verifying a binding of AMP-PCP to DGK by showing ³¹P species of the bound AMP-PCP.