Solution Structure, Determined by Nuclear Magnetic Resonance, of the b30-82 Domain of Subunit b of Escherichia coli F ₁ F _o ATP Synthase

Abstract

Subunit b, the peripheral stalk of bacterial F 1 F o ATP synthases, is composed of a membrane-spanning and a soluble part. The soluble part is divided into tether, dimerization, and ␦-binding domains. The first solution structure of b30-82, including the tether region and part of the dimerization domain, has been solved by nuclear magnetic resonance, revealing an ␣-helix between residues 39 and 72. In the solution structure, b30-82 has a length of 48.07 Å. The surface charge distribution of b30-82 shows one side with a hydrophobic surface pattern, formed by alanine residues. Alanine residues 61, 68, 70, and 72 were replaced by single cysteines in the soluble part of subunit b, b22-156. The cysteines at positions 61, 68, and 72 showed disulfide formation. In contrast, no cross-link could be formed for the A70C mutant. The patterns of disulfide bonding, together with the circular dichroism spectroscopy data, are indicative of an adjacent arrangement of residues 61, 68, and 72 in both ␣-helices in b22-156.ATP synthesis by oxidative phosphorylation or photophosphorylation is a multistep membrane-located process that provides the bulk of cellular energy in eukaryotes and many prokaryotes. The majority of ATP synthesis is accomplished by the enzyme ATP synthase (EC 3.6.1.34), also called F 1 F o ATP synthase, which, in its simplest form, as in bacteria, is composed of eight different subunits (␣ 3 , ␤ 3 , ␥, ␦, ε, a, b 2 , and c 9-12 ). This multisubunit complex is divided into the F 1 headpiece, ␣ 3 :␤ 3 , and a membrane-embedded ion-translocating part known as F o , to which F 1 is attached by a central and a peripheral stalk (1,5,25). ATP is synthesized or hydrolyzed on the ␣ 3 :␤ 3 hexamer, and the energy provided for or released during that process is transmitted to the membrane-bound F o sector, consisting of subunits a and c and part of subunit b (30, 31). The energy coupling between the two active domains occurs via the stalk part(s) (6). The central stalk is made of subunits ␥ and ε, and the peripheral stalk is formed by subunits ␦ and b. The peripheral stalk, which lies at the edge of the multisubunit assembly of the F 1 F o ATP synthase, acts as a stator to counter the tendency of the ␣ 3 :␤ 3 hexamer to follow the rotation of the central stalk and the attached c-ring, and to anchor the membrane-embedded a subunit (17, 36).In Escherichia coli, subunit b with its 156 residues extends with its soluble part (b sol ; b21-156) from the top of the F 1 sector down, into, and across the membrane, where it is associated with subunit a (2, 15, 32, 34). The 156-residue b subunit has been divided into four functional domains (28). They are, in order from the N to the C terminus; the membrane domain, the tether region, the dimerization domain, and the ␦-binding

Variations of Subunit ε of the Mycobacterium tuberculosis F 1 F o ATP Synthase and a Novel Model for Mechanism of Action of the Tuberculosis Drug TMC207

“…Steady-state CD spectra were measured in far-UV light (185 to 260 nm) using a Chirascan spectropolarimeter (Applied Photophysics) as described previously (23). Circular dichroism (CD) spectroscopy of Mtε, Mtε-T19A, and Mtε-R37G (1.0 mg/ ml) and the peptide ε 103-120 (2.0 mg/ml) was performed in buffer A (50 mM Tris-HCl, pH 8.5, 200 mM NaCl, 10% glycerol) and buffer B (25 mM phosphate, pH 6.5, 50% TFE [4-chlorobutanol and 2,2,2-trifluoroethanol]), respectively.…”

Structure of ATP synthase from Paracoccus denitrificans determined by X-ray crystallography at 4.0 Å resolution

“…Structures have been described of the F 1 domains of the enzymes from Escherichia coli (10, 11), Caldalkalibacillus thermarum (12), and Geobacillus stearothermophilus (formerly Bacillus PS3) (13); of the α 3 β 3 -subcomplex of the F 1 domain from G. stearothermophilus (14); and of isolated c-rings from the rotors of several species (15)(16)(17)(18)(19). There is also structural information on the peripheral stalk region of the F-ATPase from E. coli and on the N-terminal domain of the δ-subunit and its interaction with the N-terminal region of the α-subunit (20) and segments of the b-subunit (21)(22)(23).…”

Membrane Invaginations in Bacteria and Mitochondria: Common Features and Evolutionary Scenarios

“…These basic residues are commonly found in the interface regions of monotopic membrane proteins, like . The E. coli subunit b dimer model ( a ) was built using the solution NMR structures of the membrane domain (residues 1-34) (1b9u.pdb) [Dmitriev et al 1999], the tether domain (residues 30-82) (2khk.pdb) [Priya et al 2009] and the X-ray structure of the dimerization coiled-coil domain (residues 62-122) (1ld2.pdb) [Del Rizzo et al 2002]. The δ-binding domain (δBD; residues 122-155) is not shown, but SAXS analysis [Priya et al 2008] revealed a boomerang-like shape in solution, similar to the mitochondrial F 1 F O -ATP synthase subunit b and the related H subunit of A 1 A O -ATP synthase.…”