Elevator-type mechanisms of membrane transport

Membrane transporters are integral membrane proteins that mediate the passage of solutes across lipid bilayers. These proteins undergo conformational transitions between outward- and inward-facing states, which lead to alternating access of the substrate-binding site to the aqueous environment on either side of the membrane. Dozens of different transporter families have evolved, providing a wide variety of structural solutions to achieve alternating access. A sub-set of structurally diverse transporters operate by mechanisms that are collectively named 'elevator-type'. These transporters have one common characteristic: they contain a distinct protein domain that slides across the membrane as a rigid body, and in doing so it 'drags" the transported substrate along. Analysis of the global conformational changes that take place in membrane transporters using elevator-type mechanisms reveals that elevator-type movements can be achieved in more than one way. Molecular dynamics simulations and experimental data help to understand how lipid bilayer properties may affect elevator movements and vice versa

Literatuurscan oorzaken geweld tegen kinderen en jongeren in afhankelijkheidsrelaties

Naar aanleiding van het rapport van de Commissie Seksueel misbruik van minderjarigen in de Rooms-Katholieke Kerk (Commissie Deetman), heeft de Minster van Veiligheid en Justitie, mede namens de Staatssecretaris van VWS, een onderzoek naar dieperliggende oorzaken van seksueel geweld en andere vormen van geweld in afhankelijkheidsrelaties toegezegd aan de Tweede Kamer. Dit onderzoek heeft betrekking op de eerste fase van het onderzoek. Het betreft een literatuurscan die de stand van de wetenschappelijke kennis op hoofdlijnen in kaart moet brengen met betrekking tot de etiologie van seksueel geweld en fysiek geweld tegen kinderen en jongeren, binnen afhankelijkheidsrelaties. De volgende deelvragen staan daarbij centraal: - Wat is de stand van de kennis over de etiologie van fysieke kindermishandeling en seksueel misbruik? - Waar liggen grofweg de mogelijkheden om te interveniëren? - Op welke thema's zou nader, al dan niet empirisch, onderzoek in Nederland wenselijk zijn en hoe zou dat er idealiter uitzien

Insights into the bilayer-mediated toppling mechanism of a folate-specific ECF transporter by cryo-EM

Energy-coupling factor (ECF)–type transporters are small, asymmetric membrane protein complexes (∼115 kDa) that consist of a membrane-embedded, substrate-binding protein (S component) and a tripartite ATP-hydrolyzing module (ECF module). They import micronutrients into bacterial cells and have been proposed to use a highly unusual transport mechanism, in which the substrate is dragged across the membrane by a toppling motion of the S component. However, it remains unclear how the lipid bilayer could accommodate such a movement. Here, we used cryogenic electron microscopy at 200 kV to determine structures of a folate-specific ECF transporter in lipid nanodiscs and detergent micelles at 2.7- and 3.4-Å resolution, respectively. The structures reveal an irregularly shaped bilayer environment around the membrane-embedded complex and suggest that toppling of the S component is facilitated by protein-induced membrane deformations. In this way, structural remodeling of the lipid bilayer environment is exploited to guide the transport process

Muscle glycogen recovery after exercise measured by13C-magnetic resonance spectroscopy in humans: effect of nutritional solutions

The rate of glycogen resynthesis in human skeletal muscle after glycogen-depleting exercise is known to depend on carbohydrate intake and is reported to reach a platean after an adequate amount of carbohydrate (CHO) consumption. Efforts to maximize the rate of glycogen storage by changing the type and form of CHO, as well as by adding proteins or lipids have yielded inconsistent results. The objective of this study was to assess whether isocaloric addition of proteins and arginine to a CHO diet in the first 4 h after an endurance exercise would increase the rate of glycogen synthesis. The CHO solution, given twice at a 2 h interval according to earlier optimized protocols, contained 1.7 g CHO kgbody weight. The effects of this solution were compared to those of an isocaloric solution containing 1.2 g CHO/kgbody weight plus 0.5 g protein/kgbody weight (including 5 g arginine). Glycogen was measured in quadriceps muscle in vivo with natural abundance13C-magnetic resonance spectroscopy before exercise and twice after exercise, before and at the end of a 4-h period following the intake of one of the solutions. Eight subjects took part in a randomized cross-over trial separated by at least 1 week. Glycogen synthesis was found to be significantly increased with both regimes compared to a zero-caloric placebo diet, but no significant difference in glycogen resynthesis was found between the CHO-only diet and the one supplemented by proteins and arginine. It is estimated that significance would have been reached for an increase of 34%, while the effectively measured synthesis rates only differed by 5

Bacterial multi-solute transporters

Bacterial membrane proteins of the SbmA/BacA family are multi-solute transporters that mediate the uptake of structurally diverse hydrophilic molecules, including aminoglycoside antibiotics and antimicrobial peptides. Some family members are full-length ATP-binding cassette (ABC) transporters, whereas other members are truncated homologues that lack the nucleotide-binding domains and thus mediate ATP-independent transport. A recent cryo-EM structure of the ABC transporter Rv1819c from Mycobacterium tuberculosis has shed light on the structural basis for multi-solute transport and has provided insight into the mechanism of transport. Here, we discuss how the protein architecture makes SbmA/BacA family transporters prone to inadvertent import of antibiotics and speculate on the question which physiological processes may benefit from multi-solute transport

Correction to:On the Role of a Conserved Methionine in the Na+-Coupling Mechanism of a Neurotransmitter Transporter Homolog (Neurochemical Research, (2022), 47, 1, (163-175), 10.1007/s11064-021-03253-w)

After publication, the authors realized that the version of the supplementary information that was originally submitted was incomplete in that it omitted results examining alternative NBFIX corrections to the force field. Those data have now been added as Supplementary Fig. S3 and they reaffirm the conclusions of the manuscript. In addition, the legend to Fig. 3b should read: “Using the NBFIX correction of Na+- methionine interactions, all Na+ ions and the substrate remain stably bound throughout the trajectory. See also Fig. S3.

Minimal Pathway for the Regeneration of Redox Cofactors

[Image: see text] Effective metabolic pathways are essential for the construction of in vitro systems mimicking the biochemical complexity of living cells. Such pathways require the inclusion of a metabolic branch that ensures the availability of reducing equivalents. Here, we built a minimal enzymatic pathway confinable in the lumen of liposomes, in which the redox status of the nicotinamide cofactors NADH and NADPH is controlled by an externally provided formate. Formic acid permeates the membrane where a luminal formate dehydrogenase uses NAD(+) to form NADH and carbon dioxide. Carbon dioxide diffuses out of the liposomes, leaving only the reducing equivalents in the lumen. A soluble transhydrogenase subsequently utilizes NADH for reduction of NADP(+) thereby making NAD(+) available again for the first reaction. The pathway is functional in liposomes ranging from a few hundred nanometers in diameter (large unilamellar vesicles) up to several tens of micrometers (giant unilamellar vesicles) and remains active over a period of 7 days. We demonstrate that the downstream biochemical process of reduction of glutathione disulfide can be driven by the transfer of reducing equivalents from formate via NAD(P)H, thereby providing a versatile set of electron donors for reductive metabolism

Biochemical and structural insight into the chemical resistance and cofactor specificity of the formate dehydrogenase from Starkeya novella

Formate dehydrogenases (Fdhs) mediate the oxidation of formate to carbon dioxide and concomitant reduction of nicotinamide adenine dinucleotide (NAD + ). The low cost of the substrate formate and importance of the product NADH as a cellular source of reducing power make this reaction attractive for biotechnological applications. However, the majority of Fdhs are sensitive to inactivation by thiol-modifying reagents. In this study, we report a chemically resistant Fdh (Fdh SNO ) from the soil bacterium Starkeya novella strictly specific for NAD + . We present its recombinant overproduction, purification and biochemical characterization. The mechanistic basis of chemical resistance was found to be a valine in position 255 (rather than a cysteine as in other Fdhs) preventing the inactivation by thiol-modifying compounds. To further improve the usefulness of Fdh SNO as for generating reducing power, we rationally engineered the protein to reduce the coenzyme nicotinamide adenine dinucleotide phosphate (NADP + ) with better catalytic efficiency than NAD + . The single mutation D221Q enabled the reduction of NADP + with a catalytic efficiency k CAT /K M of 0.4 s -1 mM -1 at 200 mM formate, while a quadruple mutant (A198G/D221Q/H379K/S380V) resulted in a 5-fold increase in catalytic efficiency for NADP + compared to the single mutant. We determined the cofactor-bound structure of the quadruple mutant to gain mechanistic evidence behind the improved specificity for NADP + . Our efforts to unravel the key residues for the chemical resistance and cofactor specificity of Fdh SNO may lead to wider use of this enzymatic group in a more sustainable (bio)manufacture of value-added chemicals, as for instance the biosynthesis of chiral compounds. </p

On the Role of a Conserved Methionine in the Na+-Coupling Mechanism of a Neurotransmitter Transporter Homolog

Excitatory amino acid transporters (EAAT) play a key role in glutamatergic synaptic communication. Driven by transmembrane cation gradients, these transporters catalyze the reuptake of glutamate from the synaptic cleft once this neurotransmitter has been utilized for signaling. Two decades ago, pioneering studies in the Kanner lab identified a conserved methionine within the transmembrane domain as key for substrate turnover rate and specificity; later structural work, particularly for the prokaryotic homologs Glt(Ph) and Glt(Tk), revealed that this methionine is involved in the coordination of one of the three Na(+) ions that are co-transported with the substrate. Albeit extremely atypical, the existence of this interaction is consistent with biophysical analyses of Glt(Ph) showing that mutations of this methionine diminish the binding cooperativity between substrates and Na(+). It has been unclear, however, whether this intriguing methionine influences the thermodynamics of the transport reaction, i.e., its substrate:ion stoichiometry, or whether it simply fosters a specific kinetics in the binding reaction, which, while influential for the turnover rate, do not fundamentally explain the ion-coupling mechanism of this class of transporters. Here, studies of Glt(Tk) using experimental and computational methods independently arrive at the conclusion that the latter hypothesis is the most plausible, and lay the groundwork for future efforts to uncover the underlying mechanism. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s11064-021-03253-w) contains supplementary material, which is available to authorized users