Biochemie und Chemie
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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.
Cryo-EM structures of KdpFABC suggest a K+ transport mechanism via two inter-subunit half-channels
(2018)
P-type ATPases ubiquitously pump cations across biological membranes to maintain vital ion gradients. Among those, the chimeric K+ uptake system KdpFABC is unique. While ATP hydrolysis is accomplished by the P-type ATPase subunit KdpB, K+ has been assumed to be transported by the channel-like subunit KdpA. A first crystal structure uncovered its overall topology, suggesting such a spatial separation of energizing and transporting units. Here, we report two cryo-EM structures of the 157 kDa, asymmetric KdpFABC complex at 3.7 Å and 4.0 Å resolution in an E1 and an E2 state, respectively. Unexpectedly, the structures suggest a translocation pathway through two half-channels along KdpA and KdpB, uniting the alternating-access mechanism of actively pumping P-type ATPases with the high affinity and selectivity of K+ channels. This way, KdpFABC would function as a true chimeric complex, synergizing the best features of otherwise separately evolved transport mechanisms.
Ribosome recycling orchestrated by the ATP binding cassette (ABC) protein ABCE1 can be considered as the final—or the first—step within the cyclic process of protein synthesis, connecting translation termination and mRNA surveillance with re-initiation. An ATP-dependent tweezer-like motion of the nucleotide-binding domains in ABCE1 transfers mechanical energy to the ribosome and tears the ribosome subunits apart. The post-recycling complex (PRC) then re-initiates mRNA translation. Here, we probed the so far unknown architecture of the 1-MDa PRC (40S/30S·ABCE1) by chemical cross-linking and mass spectrometry (XL-MS). Our study reveals ABCE1 bound to the translational factor-binding (GTPase) site with multiple cross-link contacts of the helix–loop–helix motif to the S24e ribosomal protein. Cross-linking of the FeS cluster domain to the ribosomal protein S12 substantiates an extreme lever-arm movement of the FeS cluster domain during ribosome recycling. We were thus able to reconstitute and structurally analyse a key complex in the translational cycle, resembling the link between translation initiation and ribosome recycling.
Electron transfer in respiratory chains generates the electrochemical potential that serves as energy source for the cell. Prokaryotes can use a wide range of electron donors and acceptors and may have alternative complexes performing the same catalytic reactions as the mitochondrial complexes. This is the case for the alternative complex III (ACIII), a quinol:cytochrome c/HiPIP oxidoreductase. In order to understand the catalytic mechanism of this respiratory enzyme, we determined the structure of ACIII from Rhodothermus marinus at 3.9 Å resolution by single-particle cryo-electron microscopy. ACIII presents a so-far unique structure, for which we establish the arrangement of the cofactors (four iron–sulfur clusters and six c-type hemes) and propose the location of the quinol-binding site and the presence of two putative proton pathways in the membrane. Altogether, this structure provides insights into a mechanism for energy transduction and introduces ACIII as a redox-driven proton pump.