Cross-talk in the A1-ATPase from Methanosarcina mazei Go1 due to nucleotide binding.
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.
Coskun
Unal
U
Universität des Saarlandes, Fachrichtung 2.5-Biophysik, D-66421 Homburg, Germany.
Grüber
Gerhard
G
Koch
Michel H J
MH
Godovac-Zimmermann
Jasminka
J
Lemker
Thorsten
T
Müller
Volker
V
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D003300:Copper / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008024:Ligands; D008274:Magnesium / Q000378:metabolism; D017020:Methanosarcina / Q000201:enzymology; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D012542:Scattering, Radiation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013053:Spectrophotometry; D014357:Trypsin / Q000378:metabolism; D014965:X-Rays
D003432:Cross-Linking Reagents; D008024:Ligands; D009711:Nucleotides; D000244:Adenosine Diphosphate; D003300:Copper; D000255:Adenosine Triphosphate; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; C029892:cupric chloride