Rebuilt 3D structure of the chloroplast f1 ATPase–tentoxin complex

Abstract

The F1 part of the chloroplast H+ adenosine triphosphate (ATP)-synthase (CF1) strongly interacts with tentoxin, a natural fungous cyclic tetrapeptide known to inhibit the chloroplast enzyme and not the mammalian mitochondrial enzyme. Whereas the synthesis or the hydrolysis of ATP requires the stepwise rotation of the protein rotor gamma within the (alphabeta)3 crown, only one molecule of tentoxin is needed to fully inhibit the complex. With the help of an original homology modeling technique, based on robust distance geometry protocols, we built a tridimensional model of the alpha3beta3gamma CF1) subcomplex (3200 esidues), in which we introduced three different nucleotide occupancies to check their possible influence on the tentoxin binding site. Simultaneous comparison of three available high-resolution X-ray structures of F1, performed with a local structural alignment search tool, led to characterizing common structural blocks and the distorsions experienced by the complex during the catalytic turnover. The common structural blocks were used as a starting point of the spinach CF1 structure rebuilding. Finally, tentoxin was docked into its putative binding site of the reconstructed structure. The docking method was initially validated in the mitochondrial enzyme by its ability to relocate nucleotides into their original position in the crystal. Tentoxin binding was found possible to the two alpha/beta interfaces associated with the empty and adenosine diphosphate (ADP)-loaded catalytic sites, but not to the one associated with the ATP-loaded site. These results suggest a mechanism of CF1 inhibition by one molecule of tentoxin, by the impossibility of the alpha/beta interface bearing tentoxin to pass through the ATP-loaded state.

Functional Anthology of Intrinsic Disorder. 2. Cellular Components, Domains, Technical Terms, Developmental Processes, and Coding Sequence Diversities Correlated with Long Disordered Regions

“…These enzymes couple a downhill proton flow with ATP synthesis through adenosine diphosphate (ADP) photophosphorylation. , The ATP synthases can be biochemically fragmented into two different subcomplexes, known as F 0 and F 1 (CF 0 and CF 1 in chloroplasts). The CF 0 complex is membranous and acts as the proton channel, whereas the CF 1 moiety is a soluble α 3 β 3 γδε complex which is capable of ATP hydrolysis . Crystal structures determined for several F 1 ATPases and corresponding subcomplexes revealed that these multichain enzymes are structured (ordered) and highly organized entities. , …”

“…The CF 0 complex is membranous and acts as the proton channel, whereas the CF 1 moiety is a soluble α 3 β 3 γδε complex which is capable of ATP hydrolysis . Crystal structures determined for several F 1 ATPases and corresponding subcomplexes revealed that these multichain enzymes are structured (ordered) and highly organized entities. , …”

“…147 Crystal structures determined for several F1 ATPases and corresponding subcomplexes revealed that these multichain enzymes are structured (ordered) and highly organized entities. 147,148 Reaction Center. Photosynthetic reaction center is a membrane proteinaceous machine, which is the site of the lightmodulated reactions associated with photosynthesis.…”

Metabolism of N‐methyl‐amide by cytochrome P450s

“…Here, we show, through the example of tentoxin (TTX), that such intermediates may occur more regularly than reported in the metabolism of natural N ‐methylated cyclopeptides. TTX [cyclo‐( l ‐ N‐ MeAla 1 ‐ l ‐Leu 2 ‐ N‐ Me(Δ Z )Phe 3 ‐Gly 4 ] (Scheme 2) is a natural hydrophobic cyclotetrapeptide which acts in certain plant species as a noncompetitive inhibitor of chloroplast ATP‐synthase [29–31], provoking chlorosis. We have previously shown that TTX is efficiently metabolized through N ‐demethylation by mammal cytochrome P450 [32].…”

An Insight into the Mechanism of Inhibition and Reactivation of the F1-ATPases by Tentoxin

“…Using a photoactivatable TTX analogue, we found that TTX specifically binds the R subunit of CF 1 -. We then docked TTX in its putative site located at the three possible R/β interfaces of a rebuilt 3D structure of CF 1 R 3 β 3 γ subcomplex, made asymmetrical after insertion of γ subunit (28). Kinetic experiments performed on CF 1 -using well-characterized TTX analogues as competitors revealed how the inhibitory site influences the reactivatory site, whereas chase experiments using 14 C-labeled TTX showed that the reactivatory site has no influence on the binding properties of the inhibitory site.…”