Rapid purification of membrane extrinsic F<sub>1</sub>‐domain of chloroplast ATP synthase in monodisperse form suitable for 3D‐crystallization

Groth, Georg; Schirwitz, Katja

doi:10.1046/j.1432-1327.1999.00101.x

Cited by 10 publications

(9 citation statements)

References 38 publications

(40 reference statements)

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“…This complex, however, was assembled from individual subunits overexpressed in E. coli (44), and the significance of the nucleotide occupancy in this complex might be questioned. The chloroplast ␣ 3 ␤ 3 ␥⑀ complex crystallized in this study, however, was isolated from its natural source and retained catalytic activity (12,28). Thus, the closed conformation might also exist in the absence of nucleotides in the native F 1 complex.…”

Section: The Structure Of the Chloroplast F 1 -Atpasementioning

confidence: 89%

“…Crystallization and Data Collection-Purification and crystallization of the membrane extrinsic F 1 domain of the chloroplast ATP synthase have been described previously (28). Briefly, CF 1 was released from thylakoid membranes of spinach plants in a medium containing 300 mM sucrose, 2 mM Tricine, pH 7.8, 2 mM dithiothreitol, 0.875 mM EDTA, and 0.002% (w/v) phenylmethylsulfonyl fluoride.…”

Section: Methodsmentioning

confidence: 99%

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The Structure of the Chloroplast F1-ATPase at 3.2 Å Resolution

Groth¹,

Pohl²

2001

Journal of Biological Chemistry

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119

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The structure of the F 1 -ATPase from spinach chloroplasts was determined to 3.2 Å resolution by molecular replacement based on the homologous structure of the bovine mitochondrial enzyme. The crystallized complex contains four different subunits in a stoichiometry of ␣ 3 ␤ 3 ␥⑀. Subunit ␦ was removed before crystallization to improve the diffraction of the crystals. The overall structure of the noncatalytic ␣-subunits and the catalytic ␤-subunits is highly similar to those of the mitochondrial and thermophilic subunits. However, in the crystal structure of the chloroplast enzyme, all ␣-and ␤-subunits adopt a closed conformation and appear to contain no bound adenine nucleotides. The superimposed crystallographic symmetry in the space group R32 impaired an exact tracing of the ␥-and ⑀-subunits in the complex. However, clear electron density was present at the core of the ␣ 3 ␤ 3 -subcomplex, which probably represents the C-terminal domain of the ␥-subunit. The structure of the spinach chloroplast F 1 has a potential binding site for the phytotoxin, tentoxin, at the ␣␤-interface near ␤Asp 83 and an insertion from ␤Gly 56 -Asn 60 in the N-terminal ␤-barrel domain probably increases the thermal stability of the complex. The structure probably represents an inactive latent state of the ATPase, which is unique to chloroplast and cyanobacterial enzymes.The membrane-bound proton translocating ATP synthase of chloroplasts, CF 1 F 0 , 1 catalyzes ATP synthesis and ATP hydrolysis coupled to proton translocation across the thylakoid membrane (for a recent review see Ref.

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Section: The Structure Of the Chloroplast F 1 -Atpasementioning

confidence: 89%

Section: Methodsmentioning

confidence: 99%

The Structure of the Chloroplast F1-ATPase at 3.2 Å Resolution

Groth¹,

Pohl²

2001

Journal of Biological Chemistry

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119

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“…The samples were centrifuged for 24 h at 75,000 ϫ g at 4°C. The fractions were analyzed by SDS-polyacrylamide gel electrophoresis, and those containing CF 1 F 0 were pooled and further purified by anion exchange chromatography on POROS HQ20 (Applied Biosystems Inc., Foster City, CA) as described (47), using a buffer containing 50 mM Tricine, pH 8.0, 10% (v/v) glycerol, 4% (w/v) glycine, 5 mM magnesium chloride, 5 mM DTT, 0.002% (w/v) PMSF, and 0.1% (w/v) DDM. Purity of the protein was checked by SDS gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

Structure of the c14 Rotor Ring of the Proton Translocating Chloroplast ATP Synthase

Vollmar

Schlieper

Winn

et al. 2009

Journal of Biological Chemistry

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119

114

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The structure of the membrane integral rotor ring of the proton translocating F 1 F 0 ATP synthase from spinach chloroplasts was determined to 3.8 Å resolution by x-ray crystallography. The rotor ring consists of 14 identical protomers that are symmetrically arranged around a central pore. Comparisons with the c 11 rotor ring of the sodium translocating ATPase from Ilyobacter tartaricus show that the conserved carboxylates involved in proton or sodium transport, respectively, are 10.6 -10.8 Å apart in both c ring rotors. This finding suggests that both ATPases have the same gear distance despite their different stoichiometries. The putative proton-binding site at the conserved carboxylate Glu 61 in the chloroplast ATP synthase differs from the sodium-binding site in Ilyobacter. Residues adjacent to the conserved carboxylate show increased hydrophobicity and reduced hydrogen bonding. The crystal structure reflects the protonated form of the chloroplast c ring rotor. We propose that upon deprotonation, the conformation of Glu 61 is changed to another rotamer and becomes fully exposed to the periphery of the ring. Reprotonation of Glu 61 by a conserved arginine in the adjacent a subunit returns the carboxylate to its initial conformation.

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“…Spinach chloroplast F 1 -ATPase was isolated by EDTA extraction and anion exchange chromatography from thylakoid membranes as described (11,15). Subunit ␦ was removed from the catalytic F 1 -domain according to (11) and the remaining ␣ 3 ␤ 3 ␥-complex was incubated with 0.05 mM tentoxin in a buffer containing 25 mM Hepes pH 7.5, 1 mM DTT, 0.002% (wt͞vol) PMSF, and 0.01% (wt͞vol) sodium azide.…”

Section: Methodsmentioning

confidence: 99%

Structure of spinach chloroplast F ₁ -ATPase complexed with the phytopathogenic inhibitor tentoxin

Groth

2002

Proc. Natl. Acad. Sci. U.S.A.

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Tentoxin, a natural cyclic tetrapeptide produced by phytopathogenic fungi from the Alternaria species affects the catalytic function of the chloroplast F1-ATPase in certain sensitive species of plants. In this study, we show that the uncompetitive inhibitor tentoxin binds to the ␣␤-interface of the chloroplast F1-ATPase in a cleft localized at ␤Asp-83. Most of the binding site is located on the noncatalytic ␣-subunit. The crystal structure of the tentoxininhibited CF1-complex suggests that the inhibitor is hydrogen bonded to Asp-83 in the catalytic ␤-subunit but forms hydrophobic contacts with residues Ile-63, Leu-65, Val-75, Tyr-237, Leu-238, and Met-274 in the adjacent ␣-subunit. Except for minor changes around the tentoxin-binding site, the structure of the chloroplast ␣ 3 ␤ 3 -core complex is the same as that determined with the native chloroplast ATPase. Tentoxin seems to act by inhibiting intersubunit contacts at the ␣␤-interface and by blocking the interconversion of binding sites in the catalytic mechanism.]} produced by various phytopathogenic fungi from the Alternaria species, acts as a selective energy transfer inhibitor of the chloroplast F 1 -ATPase in certain sensitive species of plants but shows no effect on the homologous mitochondrial or bacterial enzymes. In the soluble, isolated CF 1 -complex, the phytopathogen inhibits ATP hydrolysis, whereas both catalytic reactions, ATP synthesis and hydrolysis, are blocked in the membrane-bound CF 1 . Binding studies indicate that the inhibition of the ATPase by tentoxin is uncompetitive with nucleotides (1-3) and suggest that the phytopathogen prevents the cooperative release of tightly bound nucleotides from the enzyme (4). However, the precise number and location of the tentoxin-binding site(s) and the mechanism by which the phytopathogen affects the catalytic activity of the chloroplast ATPase are still unknown. Labeling studies suggest at least one high-affinity inhibitory binding site (K d Ͻ 10 Ϫ8 M), probably located at the ␣␤-interface, and 1-2 low-affinity binding sites (K d Ͼ 10 Ϫ6 M), which cause a reactivation of the enzyme (5-6). Analysis of tentoxin-sensitive and -resistant Nicotiana species indicated that residue ␤Asp-83 located at the interface of the N-terminal ␤-barrel domains in the ␣-and ␤-subunits is crucial for tentoxin binding and͞or sensitivity (7). Substitution of this residue by glutamate, alanine, or leucine caused tentoxin resistance (8), whereas substitution of the native glutamate in the tentoxin-resistant Chlamydomonas reinhardtii F 1 -ATPase by aspartate induced tentoxin sensitivity (9). On the other hand, tentoxin-resistant F 1 -ATPases from thermophilic Bacillus PS3 and from E. coli (10) contain aspartate in the equivalent position of the ␤-subunit as well. Hence, additional structural elements in the ␣-and͞or ␤-subunits are probably required for tentoxin binding and sensitivity in F 1 -ATPases. More detailed information on these critical residues was obtained from the crystal structure of the native spinach chloroplast F ...

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Rapid purification of membrane extrinsic F₁‐domain of chloroplast ATP synthase in monodisperse form suitable for 3D‐crystallization

Cited by 10 publications

References 38 publications

The Structure of the Chloroplast F1-ATPase at 3.2 Å Resolution

The Structure of the Chloroplast F1-ATPase at 3.2 Å Resolution

Structure of the c14 Rotor Ring of the Proton Translocating Chloroplast ATP Synthase

Structure of spinach chloroplast F ₁ -ATPase complexed with the phytopathogenic inhibitor tentoxin

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Rapid purification of membrane extrinsic F1‐domain of chloroplast ATP synthase in monodisperse form suitable for 3D‐crystallization

Cited by 10 publications

References 38 publications

The Structure of the Chloroplast F1-ATPase at 3.2 Å Resolution

The Structure of the Chloroplast F1-ATPase at 3.2 Å Resolution

Structure of the c14 Rotor Ring of the Proton Translocating Chloroplast ATP Synthase

Structure of spinach chloroplast F 1 -ATPase complexed with the phytopathogenic inhibitor tentoxin

Contact Info

Product

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Rapid purification of membrane extrinsic F₁‐domain of chloroplast ATP synthase in monodisperse form suitable for 3D‐crystallization

Structure of spinach chloroplast F ₁ -ATPase complexed with the phytopathogenic inhibitor tentoxin