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

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

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

Structural insight in the toppling mechanism of an energy-coupling factor transporter

Energy-coupling factor (ECF) transporters mediate uptake of micronutrients in prokaryotes. The transporters consist of an S-component that binds the transported substrate and an ECF module (EcfAA′T) that binds and hydrolyses ATP. The mechanism of transport is poorly understood but presumably involves an unusual step in which the membrane-embedded S-component topples over to carry the substrate across the membrane. In many ECF transporters, the S-component dissociates from the ECF module after transport. Subsequently, substrate-bound S-components out-compete the empty proteins for re-binding to the ECF module in a new round of transport. Here we present crystal structures of the folate-specific transporter ECF–FolT from Lactobacillus delbrueckii. Interaction of the ECF module with FolT stabilizes the toppled state, and simultaneously destroys the high-affinity folate-binding site, allowing substrate release into the cytosol. We hypothesize that differences in the kinetics of toppling can explain how substrate-loaded FolT out-competes apo-FolT for association with the ECF module

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

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

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

A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency

Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif.Fil: Partipilo, Michele. University of Groningen; Países BajosFil: Yang, Guang. University of Groningen; Países BajosFil: Mascotti, María Laura. University of Groningen; Países Bajos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Wijma, Hein J.. University of Groningen; Países BajosFil: Slotboom, Dirk Jan. University of Groningen; Países BajosFil: Fraaije, Marco Wilhelmus. University of Groningen; Países Bajo

Iminoboronates as Dual-Purpose Linkers in Chemical Probe Development

Chemical probes that covalently modify proteins of interest are powerful tools for the research of biological processes. Important in the design of a probe is the choice of reactive group that forms the covalent bond, as it decides the success of a probe. However, choosing the right reactive group is not a simple feat and methodologies for expedient screening of different groups are needed. We herein report a modular approach that allows easy coupling of a reactive group to a ligand. α-Nucleophile ligands are combined with 2-formylphenylboronic acid derived reactive groups to form iminoboronate probes that selectively label their target proteins. A transimination reaction on the labeled proteins with an α-amino hydrazide provides further modification, for example to introduce a fluorophore.</p