Three-dimensional organization of the archaeal A1-ATPase from Methanosarcina mazei Gö1.
A modified isolation procedure provides a homogeneous A(1)-ATPase from the archaeon Methanosarcina mazei Gö1, containing the five subunits in stoichiometric amounts of A(3):B(3):C:D:F. A(1) obtained in this way was characterized by three-dimensional electron microscopy of single particles, resulting in the first three-dimensional reconstruction of an A(1)-ATPase at a resolution of 3.2 nm. The A(1) consists of a headpiece of 10.2 nm in diameter and 10.8 nm in height, formed by the six elongated subunits A(3) and B(3). At the bottom of the A(3)B(3) complex, a stalk of 3.0 nm in length can be seen. The A(3)B(3) domain surrounds a large cavity that extends throughout the length of the A(3)B(3) barrel. A part of the stalk penetrates inside this cavity and is displaced toward an A-B-A triplet. To investigate further the topology of the stalk subunits C-F in A(1), cross-linking has been carried out by using dithiobis[sulfosuccinimidylpropionate] (DSP) and 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (EDC). In experiments where DSP was added the cross-linked products B-F, A(x)-D, A-B-D, and A(x)-B(x)-D were formed. Subunits B-F, A-D, A-B-D, and A-B-C-D could be cross-linked by EDC. The subunit-subunit interaction in the presence of DSP was also studied as a function of nucleotide binding, demonstrating movements of subunits C, D, and F during ATP cleavage. Finally, the three-dimensional organization of this A(1) complex is discussed in terms of the relationship to the F(1)- and V(1)-ATPases at a resolution of 3.2 nm.
Universität des Saarlandes, Fachrichtung 2.5-Biophysik, D-66421 Homburg, Germany.
Unal
U
Coskun
Michael
M
Radermacher
Volker
V
Müller
Teresa
T
Ruiz
Gerhard
G
Grüber
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D001105:Archaea / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry*; D007091:Image Processing, Computer-Assisted; D017020:Methanosarcina / Q000201:enzymology*; D008854:Microscopy, Electron; D017434:Protein Structure, Tertiary
D003432:Cross-Linking Reagents; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go