Structural divergence of paralogous S components from ECF-type ABC transporters

Energy coupling factor (ECF) proteins are ATP-binding cassette transporters involved in the import of micronutrients in prokaryotes. They consist of two nucleotide-binding subunits and the integral membrane subunit EcfT, which together form the ECF module and a second integral membrane subunit that captures the substrate (the S component). Different S components, unrelated in sequence and specific for different ligands, can interact with the same ECF module. Here, we present a high-resolution crystal structure at 2.1 Å of the biotin-specific S component BioY from Lactococcus lactis. BioY shares only 16% sequence identity with the thiamin-specific S component ThiT from the same organism, of which we recently solved a crystal structure. Consistent with the lack of sequence similarity, BioY and ThiT display large structural differences (rmsd = 5.1 Å), but the divergence is not equally distributed over the molecules: The S components contain a structurally conserved N-terminal domain that is involved in the interaction with the ECF module and a highly divergent C-terminal domain that binds the substrate. The domain structure explains howthe S components with large overall structural differences can interact with the same ECF module while at the same time specifically bind very different substrates with subnanomolar affinity. Solitary BioY (in the absence of the ECF module) is monomeric in detergent solution and binds D-biotin with a high affinity but does not transport the substrate across the membrane.

Functional interactions between nitrite reductase and nitric oxide reductase from Paracoccus denitrificans

Denitrification is a microbial pathway that constitutes an important part of the nitrogen cycle on earth. Denitrifying organisms use nitrate as a terminal electron acceptor and reduce it stepwise to nitrogen gas, a process that produces the toxic nitric oxide (NO) molecule as an intermediate. In this work, we have investigated the possible functional interaction between the enzyme that produces NO; the cd1 nitrite reductase (cd1NiR) and the enzyme that reduces NO; the c-type nitric oxide reductase (cNOR), from the model soil bacterium P. denitrificans. Such an interaction was observed previously between purified components from P. aeruginosa and could help channeling the NO (directly from the site of formation to the side of reduction), in order to protect the cell from this toxic intermediate. We find that electron donation to cNOR is inhibited in the presence of cd1NiR, presumably because cd1NiR binds cNOR at the same location as the electron donor. We further find that the presence of cNOR influences the dimerization of cd1NiR. Overall, although we find no evidence for a high-affinity, constant interaction between the two enzymes, our data supports transient interactions between cd1NiR and cNOR that influence enzymatic properties of cNOR and oligomerization properties of cd1NiR. We speculate that this could be of particular importance in vivo during metabolic switches between aerobic and denitrifying conditions

Structural diversity of ABC transporters

ATP-binding cassette (ABC) transporters form a large superfamily of ATP-dependent protein complexes that mediate transport of a vast array of substrates across membranes. The 14 currently available structures of ABC transporters have greatly advanced insight into the transport mechanism and revealed a tremendous structural diversity. Whereas the domains that hydrolyze ATP are structurally related in all ABC transporters, the membrane-embedded domains, where the substrates are translocated, adopt four different unrelated folds. Here, we review the structural characteristics of ABC transporters and discuss the implications of this structural diversity for mechanistic diversity.</p

The ABC of ECF transporters : discovery and initial characterization of ECF-type ATP-binding casette (ABC) importers

Josy ter Beek heeft een nieuwe klasse transporteiwitten in de celmembraan ontdekt en gekarakteriseerd. Aangezien deze transporter alleen door bacteriën wordt gebruikt en voor het transport van verscheidene belangrijke stoffen zorgt, kan informatie over deze nieuwe klasse transporters in de toekomst worden gebruikt in het ontwerp van nieuwe antiobiotica die specifiek deze transporter kunnen blokkeren. Alle levende wezens zijn opgebouwd uit cellen. Alle cellen zijn omgeven door een membraan, die de inhoud van de cel scheidt van de omgeving. Elke cel moet, om te kunnen overleven, (groei-)stoffen opnemen uit de omgeving en andere (afval-)stoffen uitscheiden. Daarom zijn er in de membraan allerlei transporteiwitten aanwezig. De nieuwe klasse transporters die Ter Beek heeft ontdekt en gekarakteriseerd maakt deel uit van een bekende groep transporters die stoffen over het membraan pompen met energie uit adenosinetrifosfaat (ATP). Transporters van deze groep bestaan altijd uit twee eiwitten in het membraan en twee eiwitten aan de binnenkant van de cel, die samenwerken. De nieuwe klasse komt alleen voor in bacteriën en is belangrijk voor de import van vitamines. Het verschil met deze nieuwe klasse en de twee klasses van importers die al bekend waren is dat ze geen gebruik maken van een extra eiwit aan de buitenkant van de cel om de te transporteren stof te binden. In plaats daarvan is één van de eiwitten in het membraan erin gespecialiseerd om de te transporteren stof te binden. Wat deze transporters bovendien bijzonder maakt, is dat het eiwit dat de stof bindt, kan worden uitgewisseld voor een ander eiwit dat een andere stof bindt. Hierdoor kan de module van de overige drie eiwitten verschillende stoffen transporteren. De energiemodule uit Lactococcus lactis kan samenwerken met wel acht verschillende eiwitten. Welke van deze verschillende eiwitten gebonden is aan de energiemodule, kan in de tijd veranderen. Eiwitten die de te transporteren vitamine gebonden hebben, binden effectiever aan de energiemodule dan eiwitten zonder gebonden vitamine. Ook vond Ter Beek dat de verschillende eiwitten, die verschillende stoffen binden, dezelfde eiwitvouwing blijken te hebben. Dit verklaart hoe ze met dezelfde module kunnen samenwerken. Door, in samenwerking met andere onderzoekers uit de groep, de eiwitvouwing van twee verschillende eiwitten op te helderen en te vergelijken kon zij een hypothese opstellen over de verwachte bindingsplaats met de energiemodule en een mogelijk transportmechanisme. Jozy ter Beek has discovered and characterized a new class of transporters. All living creatures are built up from cells. All cells are surrounded by a membrane that protects the inside of the cell from the outside. To survive, every cell has to take up (growth-)substances from its surroundings and excrete other (waste-)substances. To accomplish this many transport-proteins are present in the membrane. The new class of transporters Ter Beek has discovered and characterized is part of a large group of transporters that uses the energy from adenosine triphosphate to pump substances across the cell membrane. Transporters of this group always consist of two proteins in the membrane and two proteins on the inside of the cell that work together. The new class of transporters only exists in bacteria and is important for the import of vitamins. The difference between the newly discovered class and the two other known classes of importers is that they do not use an additional protein at the outside of the cell to bind the substance that has to be transported. Instead, one of the proteins in the membrane is specialized to bind the substance. The other thing that makes these transporters stand out is that the protein that binds the substance can be exchanged by another protein that binds another substance. In this way a variety of substances can be transported. Since these transporters only exist in bacteria and are used to transport various important substances, information on these transporters can in the future be used for the design of antibiotics.

Structural and functional characterization of TraI from pKM101 reveals basis for DNA processing

Type 4 secretion systems are large and versatile protein machineries that facilitate the spread of antibiotic resistance and other virulence factors via horizontal gene transfer. Conjugative type 4 secretion systems depend on relaxases to process the DNA in preparation for transport. TraI from the well-studied conjugative plasmid pKM101 is one such relaxase. Here, we report the crystal structure of the trans-esterase domain of TraI in complex with its substrate oriT DNA, highlighting the conserved DNA-binding mechanism of conjugative relaxases. In addition, we present an apo structure of the trans-esterase domain of TraI that includes most of the flexible thumb region. This allows us for the first time to visualize the large conformational change of the thumb subdomain upon DNA binding. We also characterize the DNA binding, nicking, and religation activity of the trans-esterase domain, helicase domain, and full-length TraI. Unlike previous indications in the literature, our results reveal that the TraI trans-esterase domain from pKM101 behaves in a conserved manner with its homologs from the R388 and F plasmids

<i>c</i>NOR structure with the suggested proton pathway and the investigated residues.

<p>Structure of <i>c</i>NOR from <i>Ps</i>. <i>aeruginosa</i> (Proton Data Bank code 3O0R [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152745#pone.0152745.ref005" target="_blank">5</a>]). The NorB (light grey) and NorC (dark grey) subunits are shown in surface and cartoon representation on the left. Hemes and side-chains of the discussed residues are shown in stick representation. All Fe³⁺ and Ca²⁺ ions are shown as spheres. The residues of proton pathway 1 are shown in cyan, the investigated residues of pathway 2 are shown in pink. The residues that are predicted to lead the proton to the active site are shown in dark green. Small red spheres indicate crystallographic waters within 3.5 Å of the shown residues, heme propionates and metal sites. The A and D-propionate of the <i>b</i>_<i>3</i> heme are also indicated. Blue (dotted) lines indicate the suggested proton pathway.</p

Investigating the Proton Donor in the NO Reductase from <i>Paracoccus denitrificans</i>

<div><p>Variant nomenclature: the variants were made in the NorB subunit if not indicated by the superscript ^c, which are variants in the NorC subunit (e.g. E122A = exchange of Glu-122 in NorB for an Ala, E71^cD; exchange of Glu-71 in NorC for an Asp).</p><p>Bacterial NO reductases (NORs) are integral membrane proteins from the heme-copper oxidase superfamily. Most heme-copper oxidases are proton-pumping enzymes that reduce O₂ as the last step in the respiratory chain. With electrons from cytochrome <i>c</i>, NO reductase (<i>c</i>NOR) from <i>Paracoccus (P</i>.<i>) denitrificans</i> reduces NO to N₂O via the following reaction: 2NO+2e^-+2H⁺→N₂O+H₂O. Although this reaction is as exergonic as O₂-reduction, <i>c</i>NOR does not contribute to the electrochemical gradient over the membrane. This means that <i>c</i>NOR does not pump protons and that the protons needed for the reaction are taken from the periplasmic side of the membrane (since the electrons are donated from this side). We previously showed that the <i>P</i>. <i>denitrificans c</i>NOR uses a single defined proton pathway with residues Glu-58 and Lys-54 from the NorC subunit at the entrance. Here we further strengthened the evidence in support of this pathway. Our further aim was to define the continuation of the pathway and the immediate proton donor for the active site. To this end, we investigated the region around the calcium-binding site and both propionates of heme <i>b</i>₃ by site directed mutagenesis. Changing single amino acids in these areas often had severe effects on <i>c</i>NOR function, with many variants having a perturbed active site, making detailed analysis of proton transfer properties difficult. Our data does however indicate that the calcium ligation sphere and the region around the heme <i>b</i>₃ propionates are important for proton transfer and presumably contain the proton donor. The possible evolutionary link between the area for the immediate donor in <i>c</i>NOR and the proton loading site (PLS) for pumped protons in oxygen-reducing heme-copper oxidases is discussed.</p></div

Energy Coupling Factor-Type ABC Transporters for Vitamin Uptake in Prokaryotes

Energy coupling factor (ECF) transporters are a subgroup of ATP-binding cassette (ABC) transporters involved in the uptake of vitamins and micronutrients in prokaryotes. In contrast to classical ABC importers, ECF transporters do not make use of water-soluble substrate binding proteins or domains but instead employ integral membrane proteins for substrate binding (named S-components). S-components form active translocation complexes with the ECF module, an assembly of two nucleotide-binding domains (NBDs, or EcfA) and a second transmembrane protein. In some cases, the ECF module is dedicated to a single S-component, but in many cases, the ECF module can interact with several different S-components that are unrelated in sequence and bind diverse substrates. The modular organization with exchangeable S-components on a single ECF module allows the transport of chemically different substrates via a common route. The recent determination of the crystal structures of the S-components that recognize thiamin and riboflavin has provided a first clue about the mechanism of S-component exchange. This review describes recent advances and the current views of the mechanism of transport by ECF transporters.