Abstract
Changes in the A(3)B(3)CDF-complex of the Methanosarcina mazei Gö1 A(1)-ATPase in response to ligand binding have been studied by small-angle x-ray scattering, protease digestion, fluorescence spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and CuCl(2)-induced disulfide formation. The value of the radius of gyration, R(g), increases slightly when MgATP, MgADP, or MgADP + P(i) (but not MgAMP-PNP) is present. The nucleotide-binding subunits A and B were reacted with N-4[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide, and spectral shifts and changes in fluorescence intensity were detected upon addition of MgAMP-PNP, MgATP, MgADP + P(i), or MgADP. Trypsin treatment of A(1) resulted in cleavage of the stalk subunits C and F, which was rapid in the presence of MgAMP-PNP but slow when MgATP or MgADP were added to the enzyme. When A(1) was supplemented with CuCl(2) a clear nucleotide dependence of an A-A-D cross-linking product was generated in the presence of MgADP and MgATP but not when MgAMP-PNP or MgADP + P(i) was added. The site of cross-link formation was located in the region of the N and C termini of subunit D. The data suggest that the stalk subunits C, D, and F in A(1) undergo conformational changes during ATP hydrolysis.
Highlights
The membrane-integrated archaeal A1AO-ATPase (A3B3CDEFGHIKx, the exact stoichiometry of the subunits is unknown), like the bacterial F1FO-ATPase (␣33␥␦⑀abcx) and the eucaryotic V1VO-ATPase (A3B3CDEFGxHyaczde), possesses an extrinsic domain (A1), containing the catalytic sites, and an intrinsic domain (AO), involved in ion translocation [1,2,3]
Effect of Substrate Binding Studied by X-ray Solution Scattering—Previously, we have characterized the A1-ATPase from M. mazei Go1 by small-angle x-ray scattering (SAXS) and determined the maximum dimension (18.0 Ϯ 0.1 nm) and the radius of gyration (Rg, 5.03 Ϯ 0.1 nm [14])
X-ray solution scattering was used to investigate the influence of nucleotide binding to the quaternary structure of the A1-ATPase from M. mazei Go1
Summary
Materials—All chemicals were at least of analytical grade and were obtained from Biomol (Hamburg, Germany), Merck, Promega (Madison, WI), Sigma, or Serva (Heidelberg, Germany). For solution x-ray scattering experiments (see below) the enzyme was subsequently applied onto a Sephacryl S-300 HR column (10/30, Amersham Biosciences) equilibrated in 50 mM Tris-HCl (pH 6.9), 150 mM NaCl and subjected to gel permeation chromatography (FPLC) in order to isolate a homogeneous and nucleotide-depleted A1 complex [14]. 2 The abbreviations used are: FPLC, fast protein liquid chromatography; CM, N-[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl)]maleimide; AMP-PNP, adenosine 5Ј-(,␥-imino)triphosphate; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; DTT, dithiothreitol; SAXS, by small-angle x-ray scattering. The subunits involved in cross-linking were identified by Western blotting [22] using antisera against subunit A and B as described previously [17] and mass spectrometric analysis For the latter the cross-linked bands were cut out from the SDS-polyacrylamide gel and destained with a solution of 25 mM ammonium bicarbonate and 50% acetonitrile for 12 h.
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call