Advancing drug discovery through assay development: a survey of tool compounds within the human solute carrier superfamily.

With over 450 genes, solute carriers (SLCs) constitute the largest transporter superfamily responsible for the uptake and efflux of nutrients, metabolites, and xenobiotics in human cells. SLCs are associated with a wide variety of human diseases, including cancer, diabetes, and metabolic and neurological disorders. They represent an important therapeutic target class that remains only partly exploited as therapeutics that target SLCs are scarce.

Pho pictures provide powerful perspectives of phosphate importing proteins.

In this issue of Structure, Schneider et al. report multiple structures of the low-affinity inorganic-phosphate transporter Pho90 from Saccharomyces cerevisiae. With remarkable resolution of the Divalent Anion Sodium Symporter family member, their cryo-EM studies of this fungal protein reveal new modes of sodium, substrate, and lipid binding.

Structure and function of the SIT1 proline transporter in complex with the COVID-19 receptor ACE2.

Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy.

Protein Binder Toolbox for Studies of Solute Carrier Transporters.

Transporters of the solute carrier superfamily (SLCs) are responsible for the transmembrane traffic of the majority of chemical substances in cells and tissues and are therefore of fundamental biological importance. As is often the case with membrane proteins that can be heavily glycosylated, a lack of reliable high-affinity binders hinders their functional analysis. Purifying and reconstituting transmembrane proteins in their lipidic environments remains challenging and standard approaches to generate binders for multi-transmembrane proteins, such as SLCs, channels or G protein-coupled receptors (GPCRs) are lacking.

The binding and mechanism of a positive allosteric modulator of Kv3 channels.

Small-molecule modulators of diverse voltage-gated K (Kv) channels may help treat a wide range of neurological disorders. However, developing effective modulators requires understanding of their mechanism of action. We apply an orthogonal approach to elucidate the mechanism of action of an imidazolidinedione derivative (AUT5), a highly selective positive allosteric modulator of Kv3.

Still in Search for an EAAT Activator: GT949 Does Not Activate EAAT2, nor EAAT3 in Impedance and Radioligand Uptake Assays.

Excitatory amino acid transporters (EAATs) are important regulators of amino acid transport and in particular glutamate. Recently, more interest has arisen in these transporters in the context of neurodegenerative diseases. This calls for ways to modulate these targets to drive glutamate transport, EAAT2 and EAAT3 in particular.

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies.

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins.

The solute carrier SPNS2 recruits PI(4,5)P to synergistically regulate transport of sphingosine-1-phosphate.

Solute carrier spinster homolog 2 (SPNS2), one of only four known major facilitator superfamily (MFS) lysolipid transporters in humans, exports sphingosine-1-phosphate (S1P) across cell membranes. Here, we explore the synergistic effects of lipid binding and conformational dynamics on SPNS2's transport mechanism. Using mass spectrometry, we discovered that SPNS2 interacts preferentially with PI(4,5)P.

Structural basis of ion - substrate coupling in the Na-dependent dicarboxylate transporter VcINDY.

The Na-dependent dicarboxylate transporter from Vibrio cholerae (VcINDY) is a prototype for the divalent anion sodium symporter (DASS) family. While the utilization of an electrochemical Na gradient to power substrate transport is well established for VcINDY, the structural basis of this coupling between sodium and substrate binding is not currently understood. Here, using a combination of cryo-EM structure determination, succinate binding and site-directed cysteine alkylation assays, we demonstrate that the VcINDY protein couples sodium- and substrate-binding via a previously unseen cooperative mechanism by conformational selection.

Structural basis for inhibition of the drug efflux pump NorA from Staphylococcus aureus.

Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet, there are no clinically approved drugs to inhibit efflux pumps, which would potentiate the efficacy of existing antibiotics rendered ineffective by drug efflux. Here we identified synthetic antigen-binding fragments (Fabs) that inhibit the quinolone transporter NorA from methicillin-resistant Staphylococcus aureus (MRSA).

The ups and downs of elevator-type di-/tricarboxylate membrane transporters.

The divalent anion sodium symporter (DASS) family contains both sodium-driven anion cotransporters and anion/anion exchangers. The family belongs to a broader ion transporter superfamily (ITS), which comprises 24 families of transporters, including those of AbgT antibiotic efflux transporters. The human proteins in the DASS family play major physiological roles and are drug targets.

Structure and inhibition mechanism of the human citrate transporter NaCT.

Citrate is best known as an intermediate in the tricarboxylic acid cycle of the cell. In addition to this essential role in energy metabolism, the tricarboxylate anion also acts as both a precursor and a regulator of fatty acid synthesis. Thus, the rate of fatty acid synthesis correlates directly with the cytosolic concentration of citrate.

Structural basis for the reaction cycle of DASS dicarboxylate transporters.

Citrate, α-ketoglutarate and succinate are TCA cycle intermediates that also play essential roles in metabolic signaling and cellular regulation. These di- and tricarboxylates are imported into the cell by the divalent anion sodium symporter (DASS) family of plasma membrane transporters, which contains both cotransporters and exchangers. While DASS proteins transport substrates via an elevator mechanism, to date structures are only available for a single DASS cotransporter protein in a substrate-bound, inward-facing state.

Predicting the optimal growth temperatures of prokaryotes using only genome derived features.

Motivation: Optimal growth temperature is a fundamental characteristic of all living organisms. Knowledge of this temperature is central to the study of a prokaryote, the thermal stability and temperature dependent activity of its genes, and the bioprospecting of its genome for thermally adapted proteins. While high throughput sequencing methods have dramatically increased the availability of genomic information, the growth temperatures of the source organisms are often unknown.

Rapid Bioinformatic Identification of Thermostabilizing Mutations.

Ex vivo stability is a valuable protein characteristic but is laborious to improve experimentally. In addition to biopharmaceutical and industrial applications, stable protein is important for biochemical and structural studies. Taking advantage of the large number of available genomic sequences and growth temperature data, we present two bioinformatic methods to identify a limited set of amino acids or positions that likely underlie thermostability.

The conserved potassium channel filter can have distinct ion binding profiles: structural analysis of rubidium, cesium, and barium binding in NaK2K.

Potassium channels are highly selective for K(+) over the smaller Na(+). Intriguingly, they are permeable to larger monovalent cations such as Rb(+) and Cs(+) but are specifically blocked by the similarly sized Ba(2+). In this study, we used structural analysis to determine the binding profiles for these permeant and blocking ions in the selectivity filter of the potassium-selective NaK channel mutant NaK2K and also performed permeation experiments using single-channel recordings.

Sodium and potassium competition in potassium-selective and non-selective channels.

Potassium channels selectively conduct K(+), primarily to the exclusion of Na(+), despite the fact that both ions can bind within the selectivity filter. Here we perform crystallographic titration and single-channel electrophysiology to examine the competition of Na(+) and K(+) binding within the filter of two NaK channel mutants; one is the potassium-selective NaK2K mutant and the other is the non-selective NaK2CNG, a CNG channel pore mimic. With high-resolution structures of these engineered NaK channel constructs, we explicitly describe the changes in K(+) occupancy within the filter upon Na(+) competition by anomalous diffraction.

Distinct gating mechanisms revealed by the structures of a multi-ligand gated K(+) channel.

The gating ring-forming RCK domain regulates channel gating in response to various cellular chemical stimuli in eukaryotic Slo channel families and the majority of ligand-gated prokaryotic K(+) channels and transporters. Here we present structural and functional studies of a dual RCK-containing, multi-ligand gated K(+) channel from Geobacter sulfurreducens, named GsuK. We demonstrate that ADP and NAD(+) activate the GsuK channel, whereas Ca(2+) serves as an allosteric inhibitor.

Structural insight into the ion-exchange mechanism of the sodium/calcium exchanger.

Sodium/calcium (Na(+)/Ca(2+)) exchangers (NCX) are membrane transporters that play an essential role in maintaining the homeostasis of cytosolic Ca(2+) for cell signaling. We demonstrated the Na(+)/Ca(2+)-exchange function of an NCX from Methanococcus jannaschii (NCX_Mj) and report its 1.9 angstrom crystal structure in an outward-facing conformation.

Protein interactions central to stabilizing the K+ channel selectivity filter in a four-sited configuration for selective K+ permeation.

The structural and functional conversion of the nonselective NaK channel to a K(+) selective channel (NaK2K) allows us to identify two key residues, Tyr and Asp in the filter sequence of TVGYGD, that participate in interactions central to stabilizing the K(+) channel selectivity filter. By using protein crystallography and channel electrophysiology, we demonstrate that the K(+) channel filter exists as an energetically strained structure and requires these key protein interactions working in concert to hold the filter in the precisely defined four-sited configuration that is essential for selective K(+) permeation. Disruption of either interaction, as tested on both the NaK2K and eukaryotic K(v)1.

Tuning the ion selectivity of tetrameric cation channels by changing the number of ion binding sites.

Selective ion conduction across ion channel pores is central to cellular physiology. To understand the underlying principles of ion selectivity in tetrameric cation channels, we engineered a set of cation channel pores based on the nonselective NaK channel and determined their structures to high resolution. These structures showcase an ensemble of selectivity filters with a various number of contiguous ion binding sites ranging from 2 to 4, with each individual site maintaining a geometry and ligand environment virtually identical to that of equivalent sites in K(+) channel selectivity filters.