Abstract
A key structural element in the ion translocating F-, A-, and V-ATPases is the peripheral stalk, an assembly of two polypeptides that provides a structural link between the ATPase and ion channel domains. Previously, we have characterized the peripheral stalk forming subunits E and H of the A-ATPase from Thermoplasma acidophilum and demonstrated that the two polypeptides interact to form a stable heterodimer with 1:1 stoichiometry (Kish-Trier, E., Briere, L. K., Dunn, S. D., and Wilkens, S. (2008) J. Mol. Biol. 375, 673-685). To define the domain architecture of the A-ATPase peripheral stalk, we have now generated truncated versions of the E and H subunits and analyzed their ability to bind each other. The data show that the N termini of the subunits form an alpha-helical coiled-coil, approximately 80 residues in length, whereas the C-terminal residues interact to form a globular domain containingalpha- and beta-structure. We find that the isolated C-terminal domain of the E subunit exists as a dimer in solution, consistent with a recent crystal structure of the related Pyrococcus horikoshii A-ATPase E subunit (Lokanath, N. K., Matsuura, Y., Kuroishi, C., Takahashi, N., and Kunishima, N. (2007) J. Mol. Biol. 366, 933-944). However, upon the addition of a peptide comprising the C-terminal 21 residues of the H subunit (or full-length H subunit), dimeric E subunit C-terminal domain dissociates to form a 1:1 heterodimer. NMR spectroscopy was used to show that H subunit C-terminal peptide binds to E subunit C-terminal domain via the terminal alpha-helices, with little involvement of the beta-sheet region. Based on these data, we propose a structural model of the A-ATPase peripheral stalk.
Highlights
MAY 1, 2009 VOLUME 284 NUMBER 18 whereas two heterodimeric EH complexes are thought to form the peripheral stalk attachment to A0 seen in electron microscopy reconstructions [5, 6]
In the A0 domain, the K subunits form a ring that is linked to the central stalk by the C subunit, whereas the cytoplasmic N-terminal domain of the I subunit probably mediates the binding of the EH peripheral stalks to A0, as suggested for the bacterial A/V-type enzyme [7]
The data show that the EH complex is comprised of two distinct domains, one that contains both N termini interacting via a coiled-coil and a second that contains both C termini folded in a globular structure containing mixed secondary structure
Summary
MAY 1, 2009 VOLUME 284 NUMBER 18 whereas two heterodimeric EH complexes are thought to form the peripheral stalk attachment to A0 seen in electron microscopy reconstructions [5, 6]. Characterization of ENT2HNT by CD Spectroscopy—The EH N-terminal coiled-coil domain was reconstituted by mixing individually expressed ENT2 (residues 2– 83) and HNT (residues 2–91), and the resulting ENT2HNT complex was purified by ion exchange and size exclusion chromatography as described with the HNCACB experiment supplemented with 15N HSQC- under “Experimental Procedures.” Fig. 2A shows the elution nuclear Overhauser enhancement spectroscopy/total correla- profile of ENT2HNT and intact EH.
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