A New Solution Structure of ATP Synthase Subunit c from Thermophilic Bacillus PS3, Suggesting a Local Conformational Change for H+-Translocation (original) (raw)
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Conformational Transitions of Subunit ɛ in ATP Synthase from Thermophilic Bacillus PS3
Biophysical Journal, 2010
Subunit epsilon of bacterial and chloroplast F(O)F(1)-ATP synthase is responsible for inhibition of ATPase activity. In Bacillus PS3 enzyme, subunit epsilon can adopt two conformations. In the "extended", inhibitory conformation, its two C-terminal alpha-helices are stretched along subunit gamma. In the "contracted", noninhibitory conformation, these helices form a hairpin. The transition of subunit epsilon from an extended to a contracted state was studied in ATP synthase incorporated in Bacillus PS3 membranes at 59 degrees C. Fluorescence energy resonance transfer between fluorophores introduced in the C-terminus of subunit epsilon and in the N-terminus of subunit gamma was used to follow the conformational transition in real time. It was found that ATP induced the conformational transition from the extended to the contracted state (half-maximum transition extent at 140 microM ATP). ADP could neither prevent nor reverse the ATP-induced conformational change, but it did slow it down. Acid residues in the DELSEED region of subunit beta were found to stabilize the extended conformation of epsilon. Binding of ATP directly to epsilon was not essential for the ATP-induced conformational change. The ATP concentration necessary for the half-maximal transition (140 microM) suggests that subunit epsilon probably adopts the extended state and strongly inhibits ATP hydrolysis only when the intracellular ATP level drops significantly below the normal value.
Journal of Biological Chemistry, 2000
V-type ATPase (V o V 1 ) capable of ATP-driven H ؉ pumping and of H ؉ gradient driven ATP synthesis was isolated from a thermophilic eubacterium, Thermus thermophilus. When the enzyme was analyzed by gel electrophoresis in the presence of sodium dodecyl sulfate, it showed eight polypeptide bands of which four were subunits of V 1 . We also isolated the V o V 1 operon, containing nine genes in the order of atpG-I-L-E-X-F-A-B-D, which encoded proteins with molecular sizes of 13, 43, 10, 20, 35, 11, 64, 53, and 25 kDa, respectively. The last four genes were identified as those for V 1 subunits; atpA, B, D, and F encoded the A, B, ␥, and ␦ subunits, respectively. The first five genes, atpG-atpX, were identified as genes for the V o subunits. The product of atpL, the proteolipid subunit, lacked a 19-amino acid presequence and, unlike V-type ATPases, contained two membranespanning domains rather than four. The hydrophobic 43-kDa product of atpI is the smallest member so far found of the eukaryotic 100-kDa subunit family. Its electrophoretic band overlapped with the band of the A subunit. Therefore, all the gene products were found in our purified V o V 1 . We isolated the A 3 B 3 subcomplex reconstituted from the isolated subunits and the A 3 B 3 ␥ subcomplex from subunit-expressing Escherichia coli. Electron microscopic observation of these subcomplexes revealed that the ␥ subunit of V 1 filled the central cavity of A 3 B 3 and might be central subunit, similar to the ␥ subunit of F 1 -ATPase.
Journal of Bacteriology, 2003
We describe here purification and biochemical characterization of the F 1 F o-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F 1 F o-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F 1 subunits ␣, , ␥, ␦, and and F o subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F 1 F o holoenzyme or the isolated F 1 moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F 1. After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (⌬). A transmembrane proton gradient or sodium ion gradient in the absence of ⌬ was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na ؉ /H ؉ antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na ؉ ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.
J Biomol Nmr, 2004
The structure of the 30 KDa subunit a of the membrane component (F 0) of E. coli ATP synthase is investigated in a mixture of chloroform, methanol and water, a solvent previously used for solving the structure of another integral membrane protein, subunit c. Near complete backbone chemical shift assignments were made from a set of TROSY experiments including HNCO, HNCA, HN(CA)CB, HN(CO)CACB and 4D HNCOCA and HNCACO. Secondary structure of subunit a was predicted from the backbone chemical shifts using TALOS program. The protein was found to consist of multiple elongated α-helical segments. This finding is generally consistent with previous predictions of multiple transmembrane α-helices in this polytopic protein.
Journal of Biological Chemistry, 1998
An affinity resin for the F 1 sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader. b 24-156 , a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity resin for binding of F 1. Truncated forms of b 24-156 , in which one or four residues from the C terminus were removed, competed poorly for F 1 binding, suggesting that these residues play an important role in b-F 1 interactions. Sedimentation velocity analytical ultracentrifugation revealed that removal of these C-terminal residues from b 24-156 resulted in a disruption of its association with the purified ␦ subunit of the enzyme. To determine whether these residues interact directly with ␦, cysteine residues were introduced at various C-terminal positions of b and modified with the heterobifunctional cross-linker benzophenone-4-maleimide. Cross-links between b and ␦ were obtained when the reagent was incorporated at positions 155 and 158 (two residues beyond the normal C terminus) in both the reconstituted b 24-156-F 1 complex and the membranebound F 1 F 0 complex. CNBr digestion followed by peptide sequencing showed the site of cross-linking within the 177-residue ␦ subunit to be C-terminal to residue 148, possibly at Met-158. These results indicate that the b and ␦ subunits interact via their C-terminal regions and that this interaction is instrumental in the binding of the F 1 sector to the b subunit of F 0 .
MGG Molecular & General Genetics, 1991
The atp operon from the extreme alkaliphile Bacillus firmus OF4 was cloned and sequenced, and shown to contain genes for the eight structural subunits of the ATP synthase, preceded by a ninth gene predicted to encode a 14 kDa hydrophobic protein. The arrangement of genes is identical to that of the atp operons from Escherichia coli, Bacillus megaterium, and thermophilic Bacillus PS3. The deduced amino acid sequences of the subunits of the enzyme are also similar to their homologs in other ATP synthases, except for several unusual substitutions, particularly in the a and c subunits. These substitutions are in domains that have been implicated in the mechanism of proton translocation through Fo-ATPase, and therefore could contribute to the gating properties of the alkaliphile ATP synthase or its capacity for proton capture.