Interactions of rotor subunits in the chloroplast ATP synthase modulated by nucleotides and by Mg 2+

Abstract

ATP synthases – rotary nano machines – consist of two major parts, F O and F 1, 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 F O to F 1, inducing conformational changes of the catalytic centers in F 1. 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 Mg 2+ 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 Mg 2+ 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.