Crystal structure of potato 14-3-3 protein St14f revealed the importance of helix I in StFDL1 recognition.

In potato (Solanum tuberosum L.), 14-3-3 protein forms a protein complex with the FLOWERING LOCUS T (FT)-like protein StSP6A and the FD-like protein StFDL1 to activate potato tuber formation. Eleven 14-3-3 isoforms were reported in potato, designated as St14a-k. In this study, the crystal structure of the free form of St14f was determined at 2.5 Å resolution. Three chains were included in the asymmetric unit of the St14f free form crystal, and the structural deviation among the three chain structures was found on the C-terminal helix H and I. The St14f free form structure in solution was also investigated by nuclear magnetic resonance (NMR) residual dipolar coupling analysis, and the chain B in the crystal structure was consistent with NMR data. Compared to other crystal structures, St14f helix I exhibited a different conformation with larger B-factor values. Larger B-factor values on helix I were also found in the 14-3-3 free form structure with higher solvent contents. The mutation in St14f Helix I stabilized the complex with StFDL1. These data clearly showed that the flexibility of helix I of 14-3-3 protein plays an important role in the recognition of target protein.

Monomeric structure of an active form of bovine cytochrome c oxidase.

Cytochrome c oxidase (CcO), a membrane enzyme in the respiratory chain, catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme with a proton pump across the membrane. In all crystals reported to date, bovine CcO exists as a dimer with the same intermonomer contacts, whereas CcOs and related enzymes from prokaryotes exist as monomers. Recent structural analyses of the mitochondrial respiratory supercomplex revealed that CcO monomer associates with complex I and complex III, indicating that the monomeric state is functionally important. In this study, we prepared monomeric and dimeric bovine CcO, stabilized using amphipol, and showed that the monomer had high activity. In addition, using a newly synthesized detergent, we determined the oxidized and reduced structures of monomer with resolutions of 1.85 and 1.95 Å, respectively. Structural comparison of the monomer and dimer revealed that a hydrogen bond network of water molecules is formed at the entry surface of the proton transfer pathway, termed the K-pathway, in monomeric CcO, whereas this network is altered in dimeric CcO. Based on these results, we propose that the monomer is the activated form, whereas the dimer can be regarded as a physiological standby form in the mitochondrial membrane. We also determined phospholipid structures based on electron density together with the anomalous scattering effect of phosphorus atoms. Two cardiolipins are found at the interface region of the supercomplex. We discuss formation of the monomeric CcO, dimeric CcO, and supercomplex, as well as their role in regulation of CcO activity.

Lipid-induced conformational changes within the cytochrome b6f complex of oxygenic photosynthesis.

Cytochrome b6f catalyzes quinone redox reactions within photosynthetic membranes to generate a transmembrane proton electrochemical gradient for ATP synthesis. A key step involves the transfer of an electron from the [2Fe-2S] cluster of the iron-sulfur protein (ISP) extrinsic domain to the cytochrome f heme across a distance of 26 Å, which is too large for competent electron transfer but could be bridged by translation-rotation of the ISP. Here we report the first crystallographic evidence of significant motion of the ISP extrinsic domain. It is inferred that extensive crystallographic disorder of the ISP extrinsic domain indicates conformational flexibility. The ISP disorder observed in this structure, in contrast to the largely ordered ISP structure observed in the b6f complex supplemented with neutral lipids, is attributed to electrostatic interactions arising from anionic lipids.