Interactions of rotor subunits in the chloroplast ATP synthase modulated by nucleotides and by Mg2+

ATP synthases – rotary nano machines – consist of two major parts, FO and F1, connected by two stalks: the central and the peripheral stalk. In spinach chloroplasts, the central stalk (subunits γ, ε) forms with the cylinder of subunits III the rotor and transmits proton motive force from FO to F1, inducing conformational changes of the catalytic centers in F1. The ε subunit is an important regulator affecting adjacent subunits as well as the activity of the whole protein complex. Using a combination of chemical cross-linking and mass spectrometry, we monitored interactions of subunit ε in spinach chloroplast ATP synthase with III and γ. Onto identification of interacting residues in subunits ε and III, one cross-link defined the distance between ε-Cys6 and III-Lys48 to be 9.4 Å at minimum. ε-Cys6 was competitively cross-linked with subunit γ. Altered cross-linking yields revealed the impact of nucleotides and Mg2+ on cross-linking of subunit ε. The presence of nucleotides apparently induced a displacement of the N-terminus of subunit ε, which separated ε-Cys6 from both, III-Lys48 and subunit γ, and thus decreasing the yield of the cross-linked subunits ε and γ as well as ε and III. However, increasing concentrations of the cofactor Mg2+ favoured cross-linking of ε-Cys6 with subunit γ instead of III-Lys48 indicating an approximation of subunits γ and ε and a separation from III-Lys48.