Dirk Slotboom

University of Groningen | RUG · Department of Biochemistry

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...

Energy-coupling factor (ECF) transporters belong to the ATP-binding cassette (ABC)-transporter family and mediate the uptake of essential micronutrients in many prokaryotic species. Two crystal structures of bacterial ECF transporters have recently been obtained and suggest that transport involves an unprecedented re-orientation of a membrane prote...

Excitatory amino acid transporters (EAATs) are secondary transport proteins that mediate the uptake of glutamate and other amino acids. EAATs fulfil an important role in neuronal signal transmission by clearing the excitatory neurotransmitters from the synaptic cleft after depolarization of the postsynaptic neuron. An intensively studied model syst...

PnuC transporters catalyze cellular uptake of the NAD(+) precursor nicotinamide riboside (NR) and belong to a large superfamily that includes the SWEET sugar transporters. We present a crystal structure of Neisseria mucosa PnuC, which adopts a highly symmetrical fold with 3 + 1 + 3 membrane topology not previously observed in any protein. The high...

Archaeal glutamate transporter homologs catalyze the coupled uptake of aspartate and three sodium ions. After the delivery of the substrate and sodium ions to the cytoplasm, the empty binding site must reorient to the outward-facing conformation to reset the transporter. Here, we report a crystal structure of the substrate-free transporter GltTk fr...

Energy-coupling factor (ECF)-type transporters mediate the uptake of micro-nutrients in many bacteria. They consist of a substrate-translocating subunit (S-component) and an ATP-hydrolysing motor (ECF module) Previous data indicate that the S-component topples within the membrane to alternately expose the binding site to either side of the membrane...

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 majorit...

The construction from scratch of synthetic cells by assembling molecular building blocks is unquestionably an ambitious goal from a scientific and technological point of view. To realize functional life-like systems, minimal enzymatic modules are required to sustain the processes underlying the out-of-equilibrium thermodynamic status hallmarking li...

Life-like systems need to maintain a basal metabolism, which includes importing a variety of building blocks required for macromolecule synthesis, exporting dead-end products, and recycling cofactors and metabolic intermediates, while maintaining steady internal physical and chemical conditions (physicochemical homeostasis). A compartment, such as...

Episodic ataxias (EAs) are rare neurological conditions affecting the nervous system and typically leading to motor impairment. EA6 is linked to the mutation of a highly conserved proline into an arginine in the glutamate transporter EAAT1. In vitro studies showed that this mutation leads to a reduction in the substrates transport and an increase i...

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...

The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS-CoV-2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo-EM confirms that Sb#15...

A wide variety of organisms encode cobalamin-dependent enzymes catalyzing essential metabolic reactions, but the cofactor cobalamin (vitamin B12) is only synthesized by a subset of bacteria and archaea. The biosynthesis of cobalamin is complex and energetically costly, making cobalamin variants and precursors metabolically valuable. Auxotrophs for...

Significance As cellular membranes are impermeable to most molecules, transporter proteins are typically present in the membrane to transport small molecules in or out of the cell. Due to the small, nanometer size of these transporters, it is challenging to study their transport mechanism. Here, we use advanced microscopy approaches to study in rea...

The energy-coupling factor (ECF) transporters are a family of transmembrane proteins involved in the uptake of vitamins in a wide range of bacteria. Inhibition of the activity of these proteins could reduce the viability of pathogens that depend on vitamin uptake. Their central role in the metabolism of bacteria and absence in humans make the ECF t...

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...

Significance The glutamine transporter ASCT2 is an emerging therapeutic target for various cancer types. Here, we use an integrated computational and experimental approach to develop unique ASCT2 inhibitors targeting a conformational state useful for rational drug design. We apply computational chemistry tools such as molecular docking and molecula...

Significance Gram-positive bacteria that lack biosynthesis pathways for micronutrients such as vitamins use a class of membrane proteins to acquire them from the environment. These membrane proteins are called energy-coupling factor (ECF) transporters and function in an unusual way. A membrane-embedded substrate-binding protein rotates within the m...

Supplementary material

It is well-established that the secondary active transporters Glt Tk and Glt Ph catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. Here, we systematically measured aspartate uptake rates in proteoliposomes containing purified Glt Tk , and derived the rate equation for a mech...

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 cons...

Photopharmacology addresses the challenge of drug selectivity and side effects through creation of photoresponsive molecules activated with light with high spatiotemporal precision. This is achieved through incorporation of molecular photoswitches and photocages into the pharmacophore. However, the structural basis for the light-induced modulation...

Energy-coupling factor (ECF) transporters mediate import of micronutrients in prokaryotes. They consist of an integral membrane S-component (that binds substrate) and ECF module (that powers transport by ATP hydrolysis). It has been proposed that different S-components compete for docking onto the same ECF module, but a minimal liposome-reconstitut...

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 expedien...

The COVID-19 pandemic has resulted in a global crisis. Here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. We identified a sybody pair (Sb#15 and Sb#68) that can bind simultaneously to the RBD, and block ACE2 binding, thereby neutralizing pseudotyped and l...

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesti...

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 ABC transporters, whereas other members are truncated homologues that lack the nucleotide‐bindin...

Energy-coupling factor type (ECF-transporters) represent trace nutrient acquisition systems. Substrate binding components of ECF-transporters are membrane proteins with extraordinary affinity, allowing them to scavenge trace amounts of ligand. A number of molecules have been described as substrates of ECF-transporters, but an involvement in iron-ac...

Energy-coupling factor type transporters (ECF) represent trace nutrient acquisition systems. Substrate binding components of ECF-transporters are membrane proteins with extraordinary affinity, allowing them to scavenge trace amounts of ligand. A number of molecules have been described as substrates of ECF-transporters, but an involvement in iron-ac...

Energy-coupling factor type (ECF-transporters) represent trace nutrient acquisition systems. Substrate binding components of ECF-transporters are membrane proteins with extraordinary affinity, allowing them to scavenge trace amounts of ligand. A number of molecules have been described as substrates of ECF-transporters, but an involvement in iron-ac...

ASCT2 (SLC1A5) is a sodium-dependent neutral amino acid transporter that controls amino acid homeostasis in peripheral tissues. ASCT2 is upregulated in cancer, where it modulates intracellular glutamine levels, fueling cell proliferation. Nutrient deprivation via ASCT2 inhibition provides an emerging strategy for cancer therapy. Here, guided by a h...

Energy coupling factor (ECF) transporters are responsible for the uptake of micronutrients in bacteria and archaea. They consist of an integral membrane unit, the S-component, and a tripartite ECF module. It has been proposed that the S-component mediates the substrate transport by toppling over in the membrane when docking onto an ECF module. Here...

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 tra...

NadR is a bifunctional enzyme that converts nicotinamide riboside (NR) into nicotinamide mononucleotide (NMN), which is then converted into nicotinamide adenine dinucleotide (NAD). Although a crystal structure of the enzyme from the Gram-negative bacterium Haemophilus influenzae is known, structural understanding of its catalytic mechanism remains...

Mycobacterium tuberculosis (Mtb) is an obligate human pathogen and the causative agent of tuberculosis1,2,3. Although Mtb can synthesize vitamin B12 (cobalamin) de novo, uptake of cobalamin has been linked to pathogenesis of tuberculosis². Mtb does not encode any characterized cobalamin transporter4,5,6; however, the gene rv1819c was found to be es...

Glutamate transporters are cation-coupled secondary active membrane transporters that clear the neurotransmitter L-glutamate from the synaptic cleft. These transporters are homotrimers, with each protomer functioning independently by an elevator-type mechanism, in which a mobile transport domain alternates between inward-and outward-oriented states...

Here, we report on the virtual screening, design, synthesis and structure–activity relationships (SARs) of the first class of selective, antibacterial agents against the energy-coupling factor (ECF) transporters. The ECF transporters are a family of transmembrane proteins involved in the uptake of vitamins in a wide range of bacteria. Inhibition of...

Efficient capture of glycans, the prime metabolic resources in the human gut, confers a key competitive advantage for gut microbiota members equipped with extracellular glycoside hydrolases (GHs) to target these substrates. The association of glycans to the bacterial cell surface is typically mediated by carbohydrate binding modules (CBMs). Here, w...

The human Alanine Serine Cysteine Transporter 2 (ASCT2) is a neutral amino acid exchanger that belongs to the solute carrier family 1 (SLC1A). SLC1A structures have revealed an elevator-type mechanism, in which the substrate is translocated across the cell membrane by a large displacement of the transport domain, whereas a small movement of hairpin...

Energy-coupling factor (ECF)–type ATP-binding cassette (ABC) transporters catalyze membrane transport of micronutrients in prokaryotes. Crystal structures and biochemical characterization have revealed that ECF transporters are mechanistically distinct from other ABC transport systems. Notably, ECF transporters make use of small integral membrane s...

Human gut bifidobacteria rely on ATP‐binding cassette (ABC) transporters for oligosaccharide uptake. Multiple oligosaccharide‐specific solute binding protein (SBP) genes are occasionally associated with a single ABC transporter, but the significance of this multiplicity remains unclear. Here, we characterize BlMnBP1 and BlMnBP2, the two SBPs associ...

Mammalian glutamate transporters are crucial players in neuronal communication as they perform neurotransmitter reuptake from the synaptic cleft. Besides L-glutamate and L-aspartate, they also recognize D-aspartate, which might participate in mammalian neurotransmission and/or neuromodulation. Much of the mechanistic insight in glutamate transport...

With high-resolution structures available for many ion-coupled (secondary active) transporters, a major challenge for the field is to determine how coupling is accomplished. Knowledge of the kinetic mechanism of the transport reaction, which defines the binding order of substrate and co-ions, together with the sequence with which all relevant state...

In an attempt to find new antibiotics, novel ways of interfering with important biological functions should be explored, especially with those which are necessary or even irreplaceable for bacterial survival, growth, and virulence. The purpose of this review is to highlight B‐type vitamin transporters from the energy‐coupling factor (ECF) family, w...

Uptake of vitamin B12 is essential for many prokaryotes, but in most cases the membrane proteins involved are yet to be identified. We present the biochemical characterization and high-resolution crystal structure of BtuM, a predicted bacterial vitamin B12 uptake system. BtuM binds vitamin B12 in its base-off conformation, with a cysteine residue a...

Human ASCT2 belongs to the SLC1 family of secondary transporters and is specific for the transport of small neutral amino acids. ASCT2 is upregulated in cancer cells and serves as the receptor for many retroviruses; hence, it has importance as a potential drug target. Here we used single-particle cryo-EM to determine a structure of the functional a...

Vitamin B12 (cobalamin) is the most complex B-type vitamin and is synthetized exclusively in a limited number of prokaryotes. Its biologically active variants contain rare organometallic bonds, which are used by enzymes in a variety of central metabolic pathways such as L-methionine synthesis and ribonucleotide reduction. Although its biosynthesis...

Data collection, phasing and refinement statistics.

Primer list used in this study.

Glutamate is an important signaling molecule in the nervous system and its extracellular levels are regulated by amino acid transporters. Studies on the role of glutamate transport have benefitted from the development of small molecule inhibitors. Most inhibitors, however, cannot be remotely controlled with respect to the time and place of their ac...

Membrane transporters of the bacterial pyridine nucleotide uptake (Pnu) family mediate the uptake of various B-type vitamins. For example, the PnuT transporters have specificity for vitamin B1 (thiamine). It has been hypothesized that Pnu transporters are facilitators that allow passive transport of the vitamin substrate across the membrane. Metabo...

Background: To optimize the production of membrane and secretory proteins in Escherichia coli, it is critical to harmonize the expression rates of the genes encoding these proteins with the capacity of their biogenesis machineries. Therefore, we engineered the Lemo21(DE3) strain, which is derived from the T7 RNA polymerase-based BL21(DE3) protein...

ATP-binding cassette (ABC) transporters form the largest class of active membrane transport proteins. Binding and hydrolysis of ATP by their highly conserved nucleotide-binding domains drive conformational changes of the complex that mediate transport of substrate across the membrane. The vitamin B12 importer BtuCD-F in Escherichia coli is an exten...

Zinc is an essential microelement to sustain all forms of life. However, excess of zinc is toxic, therefore dedicated import, export and storage proteins for tight regulation of the zinc concentration have evolved. In Enterobacteriaceae, several membrane transporters are involved in zinc homeostasis and linked to virulence. ZntB has been proposed t...

Crystal structures provide visual models of biological macromolecules, which are widely used to interpret data from functional studies and generate new mechanistic hypotheses. Because the quality of the collected x-ray diffraction data directly affects the reliability of the structural model, it is essential that the limitations of the models are c...

We applied dynamic combinatorial chemistry (DCC) to identify ligands of ThiT, the S-component of the energy-coupling factor (ECF) transporter for thiamine in Lactococcus lactis. We used a pre-equilibrated dynamic combinatorial library (DCL) and saturation-transfer difference (STD)-NMR to identify ligands of ThiT. This is the first report in which D...

The recently determined crystal structure of the bacterial Na⁺-citrate symporter CitS provides unexpected structural and mechanistic insights. The protein has a fold that has not been seen in other proteins, but the oligomeric state, domain organization and proposed transport mechanism strongly resemble those of the sodium-dicarboxylate symporter v...

Cells are delineated by a lipid bilayer that physically separates the inside from the outer environment. Most polar, charged, or large molecules require proteins to reduce the energetic barrier for passage across the membrane and to achieve transport rates that are relevant for life. Here, we describe techniques to visualize the functioning of memb...