Small-angle X-ray scattering reveals the solution structure of the peripheral stalk subunit H of the A1AO ATP synthase from Methanocaldococcus jannaschii and its binding to the catalytic A subunit.

The H subunit of the A1AO ATP synthase is a component of one of the peripheral stalks connecting the A1 and AO domain. Subunit H of the Methanocaldococcus jannaschii A1AO ATP synthase was analyzed by small-angle X-ray scattering (SAXS) in order to determine the first low-resolution structure of this molecule in solution. Independent to the concentration used, the protein is dimeric and has a boomerang-like shape, divided into two arms of 12.0 and 6.8 nm in length. Circular dichroism (CD) spectroscopy revealed that subunit H is comprised of 78% alpha-helix and a coiled-coil arrangement. To understand the orientation of the helices and the localization of the N- and C-termini inside the dimer, three truncated forms of subunit H (H8-104, H1-98, and H8-98) were expressed, purified, and analyzed by CD. SAXS experiments of H1-98 show that the maximum dimension of the truncated protein dropped to 15.1 nm. Comparison of the low-resolution shapes of H and H1-98 indicates that this goes along with structural changes in the C-terminal arm of the boomerang-like structure. Together with the result of a disulfide formation of a fourth truncated form, H1-47, with a cysteine at position 47, the data suggest a parallel alpha-helical interaction. In addition, all four truncated proteins are dimeric in solution. Tryptophan emission spectra showed specific binding of H and H8-104 to the neighboring, catalytic A subunit, which could not be detected in the presence of H1-98. Finally, the arrangement of H within the A1AO ATP synthase is presented.