Structural basis of proton-coupled potassium transport in the KUP family

Potassium homeostasis is vital for all organisms, but is challenging in single-celled organisms like bacteria and yeast and immobile organisms like plants that constantly need to adapt to changing external conditions. KUP transporters facilitate potassium uptake by the co-transport of protons. Here, we uncover the molecular basis for transport in this widely distributed family. We identify the potassium importer KimA from Bacillus subtilis as a member of the KUP family, demonstrate that it functions as a K+/H+ symporter and report a 3.7 Å cryo-EM structure of the KimA homodimer in an inward-occluded, trans-inhibited conformation. By introducing point mutations, we identify key residues for potassium and proton binding, which are conserved among other KUP proteins

High-resolution cryo-EM structures of respiratory complex I : Mechanism, assembly, and disease

Respiratory complex I is a redox-driven proton pump, accounting for a large part of the electrochemical gradient that powers mitochondrial adenosine triphosphate synthesis. Complex I dysfunction is associated with severe human diseases. Assembly of the one-megadalton complex I in the inner mitochondrial membrane requires assembly factors and chaperones. We have determined the structure of complex I from the aerobic yeast Yarrowia lipolytica by electron cryo-microscopy at 3.2-angstrom resolution. A ubiquinone molecule was identified in the access path to the active site. The electron cryo-microscopy structure indicated an unusual lipid-protein arrangement at the junction of membrane and matrix arms that was confirmed by molecular simulations. The structure of a complex I mutant and an assembly intermediate provide detailed molecular insights into the cause of a hereditary complex I-linked disease and complex I assembly in the inner mitochondrial membrane.Peer reviewe

High-resolution cryo-EM structures of respiratory complex I : Mechanism, assembly, and disease

Respiratory complex I is a redox-driven proton pump, accounting for a large part of the electrochemical gradient that powers mitochondrial adenosine triphosphate synthesis. Complex I dysfunction is associated with severe human diseases. Assembly of the one-megadalton complex I in the inner mitochondrial membrane requires assembly factors and chaperones. We have determined the structure of complex I from the aerobic yeast Yarrowia lipolytica by electron cryo-microscopy at 3.2-angstrom resolution. A ubiquinone molecule was identified in the access path to the active site. The electron cryo-microscopy structure indicated an unusual lipid-protein arrangement at the junction of membrane and matrix arms that was confirmed by molecular simulations. The structure of a complex I mutant and an assembly intermediate provide detailed molecular insights into the cause of a hereditary complex I-linked disease and complex I assembly in the inner mitochondrial membrane.Peer reviewe

Structure of alcohol oxidase from pichia pastoris by cryo-electron microscopy

The first step in methanol metabolism in methylotrophic yeasts, the oxidation of methanol and higher alcohols with molecular oxygen to formaldehyde and hydrogen peroxide, is catalysed by alcohol oxidase (AOX), a 600-kDa homo-octamer containing eight FAD cofactors. When these yeasts are grown with methanol as the carbon source, AOX forms large crystalline arrays in peroxisomes. We determined the structure of AOX by cryo-electron microscopy at a resolution of 3.4 Å. All residues of the 662-amino acid polypeptide as well as the FAD are well resolved. AOX shows high structural homology to other members of the GMC family of oxidoreductases, which share a conserved FAD binding domain, but have different substrate specificities. The preference of AOX for small alcohols is explained by the presence of conserved bulky aromatic residues near the active site. Compared to the other GMC enzymes, AOX contains a large number of amino acid inserts, the longest being 75 residues. These segments are found at the periphery of the monomer and make extensive inter-subunit contacts which are responsible for the very stable octamer. A short surface helix forms contacts between two octamers, explaining the tendency of AOX to form crystals in the peroxisomes

Functional asymmetry and electron flow in the bovine respirasome

Respirasomes are macromolecular assemblies of the respiratory chain complexes I, III and IV in the inner mitochondrial membrane. We determined the structure of supercomplex I1III2IV1 from bovine heart mitochondria by cryo-EM at 9 Å resolution. Most protein-protein contacts between complex I, III and IV in the membrane are mediated by supernumerary subunits. Of the two Rieske iron-sulfur cluster domains in the complex III dimer, one is resolved, indicating that this domain is immobile and unable to transfer electrons. The central position of the active complex III monomer between complex I and IV in the respirasome is optimal for accepting reduced quinone from complex I over a short diffusion distance of 11 nm, and delivering reduced cytochrome c to complex IV. The functional asymmetry of complex III provides strong evidence for directed electron flow from complex I to complex IV through the active complex III monomer in the mammalian supercomplex

Model of the AOX monomer.

<p>(a) Structural domains. The domain assignment for the GMC family [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159476#pone.0159476.ref002" target="_blank">2</a>]) is as follows: FAD-binding domain (red); FAD covering lid (pink); extended FAD-binding domain (yellow); flavin attachment loop (green) and intermediate region (light green); substrate-binding domain (cyan). Helix H12, which forms crystal contacts, is shown in orange. The longest AOX-specific insert 481–548 is shown in dark blue, other AOX-specific sequences are colored purple. The FAD cofactor is blue. The location of the five β-sheets A-E and some α-helices (nomenclature from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159476#pone.0159476.s001" target="_blank">S1 Fig</a>) and sequence mentioned in the text is indicated. The view of the monomer is along the fourfold axis from inside the complex. (b) Conservation of residues. Residues conserved in the GMC family (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159476#pone.0159476.s001" target="_blank">S1 Fig</a>) are shown in red and residues conserved in AOX are green; unconserved residues are white. Structural elements unique for AOX are yellow. Note that the FAD binding domain (right; red in (a)) has high conservation in the GMC family, while the substrate binding domain centered around β-sheet C (left; cyan in (a)) is conserved between AOX species.</p

A slice of the density near the fourfold surface.

<p>A salt bridge between Glu656 and Arg214 of different subunits (gold and light blue) may contribute to intersubunit contacts.</p