Figure 5: Dephosphorylation effects nucleotide occupancy of the cATPase.

(a) Mass spectra showing the low m/z region of the cATPase in its phosphorylated (untreated) (left-hand side) and its dephosphorylated form (right-hand side). Phosphosites have been identified in subunits α, β, δ, ε and II (red). Incubation with a phosphatase leads to release of additional subunits, assigned to phosphorylated proteins. (b) Complexes of the cATPase formed in solution following dephosphorylation. Peaks are labelled with the same symbols as used in Figs 1 and 2. The peaks reveal splitting with a mass difference that can be attributed to nucleotides. (c) The cATPase was purified with ATP or the non-hydrolysable analogue γS-ATP. When incubated with an excess of ATP (tenfold), three nucleotides are bound (lower spectrum). Both purifications (with ATP and γS-ATP) showed populations with two and three nucleotides bound in the untreated form at an occupancy ratio of 1:1 and 1:2, respectively (middle trace). Dephosphorylated cATPases showed additional peaks corresponding to populations with one or zero nucleotides bound (upper spectrum). The occupancy ratios for these populations are 1:2:4:5 and 2:4:5:4 for ATP and γS-ATP purifications, respectively. (d) The structures of the α/β and β/α interfaces are shown (upper and lower panels respectively; pdb coordinates 1FX0). The catalytic (upper panel) and the non-catalytic (regulatory, lower panel) nucleotide-binding sites (P-loops) are highlighted in red. The phosphosites in the α and β subunits are shown (yellow space filling). Spectra shown represent one experiment from at least three replicates.