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

Abstract

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 ...

ATP Synthase and the Actions of Inhibitors Utilized To Study Its Roles in Human Health, Disease, and Other Scientific Areas

“…In the crystal structure of the CF 1 -tentoxin complex, a tentoxin molecule is bound at the high-affinity binding site located in a cleft at an ␣␤ subunit interface. Here, it blocks the contact between ␣Arg-297 and ␤Asp-83 (153,155), restrains the movements of these residues, and also restrains conformational changes at the catalytic interface. This may arrest the catalytic ␣␤ interface in the closed conformation and thereby hinder its transformation into the open conformation (153,155 (316,351) (Fig.…”

Structural and Functional Assays of AtTLP18.3 Identify Its Novel Acid Phosphatase Activity in Thylakoid Lumen

“…The most abundant protein complexes, such as PSII and PSI, cytochrome b 6 f, and ATP synthase, are photosynthetic apparatuses located at the thylakoid membrane for biogenesis. Intensive research into the photosynthetic protein complexes resolved the crystal structures of PSII/I, cytochrome b 6 f, and ATP synthase (Jordan et al, 2001;Zouni et al, 2001;Groth, 2002;Stroebel et al, 2003;Loll et al, 2005;Amunts et al, 2007;Umena et al, 2011). Combined with biochemical and functional analyses, structural studies have provided the detailed orientation of the cofactors and the molecular mechanism for photosynthesis.…”

Molecular Processes of Inhibition and Stimulation of ATP Synthase Caused by the Phytotoxin Tentoxin

“…This means that a single tentoxin molecule bound to one binding site acts like a clamp as shown in Fig. 4A and inhibits the transition of the related ␤ subunit from a closed to an open conformation as suggested by the crystal structure of the spinach chloroplast F 1 complexed with tentoxin (11). This particular feature could be useful to mark a native enzyme at one position within a complete 360°turn.…”

“…The tentoxin-binding site is located at the contact surface between an ␣ and a ␤ subunit close to the N-terminal ␤-barrel structure on the ␣ 3 ␤ 3 hexagon. The crystal structure of spinach chloroplast F 1 complexed with tentoxin showed that the catalytic site on the ␣␤ pair with bound toxin is in a closed conformation (11). Thus, it was deduced that tentoxin inhibits the activity of the enzyme by blocking the conformational shifts at the catalytic sites.…”

“…The magnitude of inhibition and stimulation agrees well with data obtained for spinach chloroplast F 1 (15) but showed a higher K i (0.8 Ϯ 0.5 M) as reported for other cyanobacterial F 1 complexes (16). The observed difference in the inhibition constant might be attributed to a Ser-Ala substitution in the tentoxin-binding site of the cyanobacterial enzymes, so that the hydrogen bonding with ␤ D83 weakens the tentoxin-enzyme interaction (8,11).…”