Small-Angle X-ray Scattering Reveals the Solution Structure of the Peripheral Stalk Subunit H of the A₁A_O ATP Synthase from Methanocaldococcus jannaschii and Its Binding to the Catalytic A Subunit

Abstract

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.

Stoichiometry and Localization of the Stator Subunits E and Gin Thermus thermophilus H+-ATPase/Synthase

“…6D). A recent small angle x-ray scattering study of the isolated subunit from Methanocaldococcus jannaschii confirmed an elongated structure consistent with this hypothesis (41), although the homodimeric nature of the rod shaped subunit G from M. jannaschii is not consistent with our findings for the T. thermophilus subunit.…”

The Stator Complex of the A1A0-ATP Synthase—Structural Characterization of the E and H Subunits

“…For example, isolated subunit H from M. jannaschii A-ATPase was shown to exist as a dimer in solution, a structural model of which was determined by small-angle X-ray scattering. 13 The low-resolution model showed an elongated, elbowlike structure and it was speculated in that study that one of the peripheral stalks is constituted by the subunit H dimer alone. In the case of subunit E, dimer formation has been observed in a crystal structure obtained for the C-terminal domain of the subunit from the Pyrococcus horikoshii A-ATPase.…”

New insights into structure‐function relationships between archeal ATP synthase (A1A0) and vacuolar type ATPase (V1V0)

“…In subunit B, this region forms a crosslink with the C terminus of subunit F. (7) The residue Trp118 of A subunit, which is shown to have interaction with the H subunit, is shown as stick representation (yellow). (39) This Trp residue is part of the non-homologous region (blue) of subunit A, which comprises eight b-strands and is folded into a compact b-sandwich structure with internal two-fold symmetry. (25) The peptide Gly13 to Pro23 of subunit B, homologue to the sequence of the actin-binding site in subunit B of V-ATPases, is highlighted in brown.…”

“…(7,8,22,38) Building up the structure of the peripheral stalk and the collar domain The subunit H of the Methanocaldococcus jannaschii A-ATP synthase in solution forms an elongated dimer with a boomerang-like shape. (39) The protein has a high helical content (78%) and a high degree of coiling, whereby the helices inside the dimer of H are in a parallel and in-register arrangement. (39) Connected via its C-terminal arm to the A subunit ( Fig.…”

“…(39) The protein has a high helical content (78%) and a high degree of coiling, whereby the helices inside the dimer of H are in a parallel and in-register arrangement. (39) Connected via its C-terminal arm to the A subunit ( Fig. 1A,B), H exceeds the total distance of the A 1 headpiece and the central stalk (39) and becomes oriented with its N-terminal arm close to the collar-like structure of the enzyme complex, predicted to be formed by subunit E. (6,39) The most-recent NMR titration and FCS experiments demonstrated that the amino acid residues 41-60 of M. jannaschii subunit E bind to the N terminus (region 1-24) of H. (6) The crystallographic structure of the C-terminal half of subunit E (E 81 -198 ) from Pyrococcus horikoshii OT3 A-ATP synthase consists of six a-helices and four antiparallel b-strands, forming a dimer.…”