The small multidrug resistance (SMR) family of membrane proteins is prominent because of its rare dual topology architecture, simplicity, and small size. Its best studied member, EmrE, is an important model system in several fields related to membrane protein biology, from evolution to mechanism. But despite decades of work on these multidrug transporters, the native function of the SMR family has remained a mystery, and many highly similar SMR homologs do not transport drugs at all. Here we establish that representative SMR proteins, selected from each of the major clades in the phylogeny, function as guanidinium ion exporters. Drug-exporting SMRs are all clustered in a single minority clade. Using membrane transport experiments, we show that these guanidinium exporters, which we term Gdx, are very selective for guanidinium and strictly and stoichiometrically couple its export with the import of two protons. These findings draw important mechanistic distinctions with the notably promiscuous and weakly coupled drug exporters like EmrE.

By providing broad resistance to environmental biocides, transporters from the small multidrug resistance (SMR) family drive the spread of multidrug resistance cassettes among bacterial populations. A fundamental understanding of substrate selectivity by SMR transporters is needed to identify the types of selective pressures that contribute to this process. Using solid-supported membrane electrophysiology, we find that promiscuous transport of hydrophobic substituted cations is a general feature of SMR transporters. To understand the molecular basis for promiscuity, we solved X-ray crystal structures of a SMR transporter Gdx-Clo in complex with substrates to a maximum resolution of 2.3 Å. These structures confirm the family’s extremely rare dual topology architecture and reveal a cleft between two helices that provides accommodation in the membrane for the hydrophobic substituents of transported drug-like cations.

Recent evidence established that the cell envelope of Mycobacterium tuberculosis, the bacillus causing tuberculosis (TB), is coated by an α-glucan-containing capsule that has been implicated in persistence in a mouse infection model. As one of three known metabolic routes to α-glucan in mycobacteria, the cytoplasmic GlgE-pathway converts trehalose to α(1 → 4),α(1 → 6)-linked glucan in 4 steps. Whether individual reaction steps, catalyzed by trehalose synthase TreS, maltokinase Pep2, and glycosyltransferases GlgE and GlgB, occur independently or in a coordinated fashion is not known. Here, we report the crystal structure of M. tuberculosis TreS, and show by small-angle X-ray scattering and analytical ultracentrifugation that TreS forms tetramers in solution. Together with Pep2, TreS forms a hetero-octameric complex, and we demonstrate that complex formation markedly accelerates maltokinase activity of Pep2. Thus, complex formation may act as part of a regulatory mechanism of the GlgE pathway, which overall must avoid accumulation of toxic pathway intermediates, such as maltose-1-phosphate, and optimize the use of scarce nutrients.

G protein-coupled receptors (GPCRs) are physiologically abundant signaling hubs routing hundreds of extracellular signal substances and drugs into intracellular pathways. The GPCR database, GPCRdb supports >5000 interdisciplinary researchers every month with reference data, analysis, visualization, experiment design and dissemination. Here, we present our fifth major GPCRdb release setting out with an overview of the many resources for receptor sequences, structures, and ligands. This includes recently published additions of class D generic residue numbers, a comparative structure analysis tool to identify functional determinants, trees clustering GPCR structures by 3D conformation, and mutations stabilizing inactive/active states. We provide new state-specific structure models of all human non-olfactory GPCRs built using AlphaFold2-MultiState. We also provide a new resource of endogenous ligands along with a larger number of surrogate ligands with bioactivity, vendor, and physiochemical descriptor data. The one-stop-shop ligand resources integrate ligands/data from the ChEMBL, Guide to Pharmacology, PDSP Ki and PubChem database. The GPCRdb is available at https://gpcrdb.org.

Membrane proteins play critical physiological roles in all organisms, from ion transport and signal transduction to multidrug resistance. Elucidating their 3D structures is essential for understanding their functions, and this information can also be exploited for structure‐aided drug discovery efforts. In this regard, X‐ray crystallography has been the most widely used technique for determining the high‐resolution 3D structures of membrane proteins. However, the success of this technique is dependent on efficient protein extraction, solubilization, stabilization, and generating diffracting crystals. Each of these steps can impose great challenges for membrane protein crystallographers. In this review, the process of generating membrane protein crystals from protein extraction and solubilization to structure determination is discussed. In addition, the current methods for precrystallization screening and a few strategies to increase the chance of crystallizing challenging membrane proteins are introduced.

Proteins from the bacterial small multidrug resistance (SMR) family are proton-coupled exporters of diverse antiseptics and antimicrobials, including polyaromatic cations and quaternary ammonium compounds. The transport mechanism of the Escherichia coli transporter, EmrE, has been studied extensively, but a lack of high-resolution structural information has impeded a structural description of its molecular mechanism. Here we apply a novel approach, multipurpose crystallization chaperones, to solve several structures of EmrE, including a 2.9 Å structure at low pH without substrate. We report five additional structures in complex with structurally diverse transported substrates, including quaternary phosphonium, quaternary ammonium, and planar polyaromatic compounds. These structures show that binding site tryptophan and glutamate residues adopt different rotamers to conform to disparate structures without requiring major rearrangements of the backbone structure. Structural and functional comparison to Gdx-Clo, an SMR protein that transports a much narrower spectrum of substrates, suggests that in EmrE, a relatively sparse hydrogen bond network among binding site residues permits increased sidechain flexibility.

Proteins from the bacterial small multidrug resistance (SMR) family are proton-coupled exporters of diverse antiseptics and antimicrobials, including polyaromatic cations and quaternary ammonium compounds. The transport mechanism of the Escherichia coli transporter, EmrE, has been studied extensively, but a lack of high-resolution structural information has impeded a structural description of its molecular mechanism. Here we apply a novel approach, multipurpose crystallization chaperones, to solve several structures of EmrE, including a 2.9 Å structure at low pH without substrate. We report five additional structures in complex with structurally diverse transported substrates, including quaternary phosphonium, quaternary ammonium, and planar polyaromatic compounds. These structures show that binding site tryptophan and glutamate residues adopt different rotamers to conform to disparate structures without requiring major rearrangements of the backbone structure. Structural and functional comparison to Gdx-Clo, an SMR protein that transports a much narrower spectrum of substrates, suggests that in EmrE, a relatively sparse hydrogen bond network among binding site residues permits increased sidechain flexibility.

A growing body of evidence implicates the mycobacterial capsule, the outermost layer of the mycobacterial cell envelope, in modulation of the host immune response and virulence of mycobacteria. Mycobacteria synthesize the dominant capsule component, α(1→4)-linked glucan, via three interconnected and potentially redundant metabolic pathways. Here, we report the crystal structure of the Mycobacterium smegmatis TreS:Pep2 complex, containing trehalose synthase (TreS) and maltokinase (Pep2), which converts trehalose to maltose 1-phosphate as part of the TreS:Pep2–GlgE pathway. The structure, at 3.6 Å resolution, revealed that a diamond-shaped TreS tetramer forms the core of the complex and that pairs of Pep2 monomers bind to opposite apices of the tetramer in a 4 + 4 configuration. However, for the M. smegmatis orthologues, results from isothermal titration calorimetry and analytical ultracentrifugation experiments indicated that the prevalent stoichiometry in solution is 4 TreS + 2 Pep2 protomers. The observed discrepancy between the crystallized complex and the behavior in the solution state may be explained by the relatively weak affinity of Pep2 for TreS ( K d 3.5 μ m at mildly acidic pH) and crystal packing favoring the 4 + 4 complex. Proximity of the ATP-binding site in Pep2 to the complex interface provides a rational basis for rate enhancement of Pep2 upon binding to TreS, but the complex structure appears to rule out substrate channeling between the active sites of TreS and Pep2. Our findings provide a structural model for the trehalose synthase:maltokinase complex in M. smegmatis that offers critical insights into capsule assembly.