Structural Insights into the A₁ ATPase from the Archaeon, Methanosarcina mazei Gö1

Abstract

The low-resolution structure and overall dimensions of the A(3)B(3)CDF complex of the A(1) ATPase from Methanosarcina mazei Gö1 in solution is analyzed by synchrotron X-ray small-angle scattering. The radius of gyration and the maximum size of the complex are 5.03 +/- 0.1 and 18.0 +/- 0.1 nm, respectively. The low-resolution shape of the protein determined by two independent ab initio approaches has a knob-and-stalk-like feature. Its headpiece is approximately 9.4 nm long and 9.2 nm wide. The stalk, which is known to connect the headpiece to its membrane-bound A(O) part, is approximately 8.4 nm long. Limited tryptic digestion of the A(3)B(3)CDF complex was used to probe the topology of the smaller subunits (C-F). Trypsin was found to cleave subunit C most rapidly at three sites, Lys(20), Lys(21), and Arg(209), followed by subunit F. In the A(3)B(3)CDF complex, subunit D remained protected from proteolysis.

Three-dimensional Structure of A1A0 ATP Synthase from the Hyperthermophilic Archaeon Pyrococcus furiosus by Electron Microscopy

“…The A subunit contains a 90-amino acid insert near the N terminus, known as the non-homologous region, which makes this subunit with Ϸ66 kDa considerably larger than its homologues (4 -7). The central stalk consists of the C, D, and F subunits, and the D subunit is thought to be the equivalent of the ␥ subunit, responsible for conformational changes in the nucleotide-binding pockets upon rotation (8,9). The transmembrane domain contains a rotor of a number of identical c-subunits and part of the a-subunit.…”

Three-dimensional Organization of the Archaeal A1-ATPase from Methanosarcina mazei Gö1

“…Protein concentrations were determined by the BCA assay (Pierce). ATPase activity was measured as described previously (7).…”

“…The enzyme, as shown by small angle x-ray scattering data, consists of an ϳ10-nm long headpiece and an 8.5-nm high and 6.0-nm diameter stalk (7). A comparison of the central stalk of this A 1 complex with bacterial F 1 -and V 1 -ATPase indicates different lengths of the stalk domain (7)(8)(9).…”

“…The enzyme, as shown by small angle x-ray scattering data, consists of an ϳ10-nm long headpiece and an 8.5-nm high and 6.0-nm diameter stalk (7). A comparison of the central stalk of this A 1 complex with bacterial F 1 -and V 1 -ATPase indicates different lengths of the stalk domain (7)(8)(9). The prevailing view is, however, that ATP synthesis/hydrolysis in the A 1 headpiece is coupled to ion flow in A 0 through rotational movements of the central stalk subunit(s) as demonstrated for the F 1 (reviewed in Refs.…”

Subunit F modulates ATP binding and migration in the nucleotide-binding subunit B of the A1AO ATP synthase of Methanosarcina mazei Gö1

“…This multi subunit enzyme with a proposed stoichiometry of A 3 : B 3 : C: D: E: F: G: H 2 : a: c x (Müller and Grüber 2003) is functionally related to the F 1 F O ATP synthase from eukaryotes and prokaryotes (Lolkema et al 2003;Müller and Grüber 2003;Weber and Senior 2003) where three alternatively arranged subunits A and B contribute towards the water soluble A 1 domain (also referred to as the hexameric head), the primary site for ATP synthesis (Müller and Grüber 2003). The central stalk is made up of subunits C, D and F (Grüber et al 2001). Subunit C has a funnel shaped structure with a central cavity, forming the space for the D and F assembly (Numoto et al 2004).…”