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Additional file 1: Supplementary Figures and Tables. Fig. S1. Schematic representation of the alternating access mechanism. The major conformational changes and states of the transporter are shown, which are necessary to allow alternating substrate access from either side of the membrane to the substrate-binding site. Transporter, substrate and lipid molecules are colored in blue, orange and light brown, respectively. The different conformational states shown are a: outward-open, substrate-free; b: outward-open, substrate-bound; c: outward-facing, substrate occluded; d: substrate-bound, fully occluded; e: inward-facing, substrate occluded; f: inward-open, substrate-bound; g: inward-open, substrate-free; h: substrate-free, fully occluded. Fig. S2. Quality of the electron density of AdiC. All TMs are displayed as viewed from the membrane plane and boxed into TM-groups belonging to inverted repeats (left) and the dimerization interface (right). Starting and ending amino acid residues of corresponding TMs are labeled. The TMs are displayed as sticks (cyan) and the corresponding 2Fo-Fc electron density map is contoured at 1.0 σ and shown as blue colored mesh. Fig. S3. Structural details of AdiC. (A) TM1 and TM6 are discontinuous and their loop regions connecting the α-helical segments are in close proximity and involved in substrate-binding. TM1 and TM6 are represented as light blue ribbons and the side chains as lines. The α-helical segments of TM1 (TM1a and TM1b) and TM6 (TM6a and TM6b) are highlighted as cylinders and labeled accordingly. The respective N- and C-terminal residues of these segments are labeled as well. (B) The largest interface contribution for AdiC homodimer formation is the interaction between TM11 and TM12. TM12 of monomer A (blue) and TM11 of monomer B (yellow) are displayed as ribbons. Interdigitating, non-polar amino acid residues involved in dimer formation are shown as sticks and labeled. Fig. S4. Comparison of the number of water molecules found in the substrate-binding sites of AdiC structures solved at different resolutions. (A) In the 2.2 Å AdiC structure [16], seven water molecules could be found in the substrate-binding sites of each monomer. (B) In the 1.7 Å AdiC structure of the present study, twenty-one and fifteen water molecules were detected in monomers A and B, respectively. The substrate-binding site cavity was defined as the solvent accessible volume between the indole nitrogen atom of W293 and the side chain oxygen atom of the S26 (borders are indicated with dashed lines and respective amino acids are shown as gray sticks). The two AdiC monomers are represented as ribbons and are colored in blue and yellow color hues. Water molecules are displayed as red spheres. Fig. S5. Water induced linkage of TM domains. (A-D) Interactions of water molecules (orange) with amino acid main chains (blue sticks) and side chains (cyan sticks). Respective interactions between atoms are shown with dashed lines (distances are given in Å). The corresponding 2Fo-Fc electron density map of the interaction regions is contoured at 1.0 σ and is displayed with a light blue mesh representation. Involved TMs are indicated, and amino acids are labeled in the one letter code and when interacting with their main-chain carbonyl oxygen or nitrogen atom additionally labeled with (O) or (N). Fig. S6. Side chain torsional angles sampled during molecular dynamics simulations. Density plots representing the χ1 and χ2 conformations of W202 (A) and W293 (B). The marginal distributions are obtained using a kernel density estimate. The centroids from a k-means clustering are shown with orange triangles. Fig. S7. Multiple sequence alignment of AdiC from E. coli K12 and the pathogenic E. coli strains O104:H4 and O157:H7, AdiC/CadB homologs (hAdiC/hCadB) from other pathogenic enterobacteria and the amino acid-diamine transporters PotE and CadB from E. coli K12. Amino acid sequence alignment was performed with Clustal Omega ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ). The UniProt ID or NCBI reference sequence codes are as follows: AdiC E. coli K12 (P60061), AdiC E. coli O104:H4 (A0A0E0Y6U0), AdiC E. coli O157:H7 (P60063), PotE E. coli K12 (P0AAF1), CadB E. coli K12 (P0AAE8), hAdiC Salmonella typhi (P60065), hAdiC Shigella flexneri (P60064), hCadB Vibrio cholerae (WP_000097425.1) and hAdiC Yersinia enterocolitica (WP_174848373.1). Positions with a single, fully conserved residue are indicated by (*), conservation between groups of strongly and weakly similar properties are indicated by (:) and (·), respectively. Color-coding of amino acid residues is based on their physicochemical properties, i.e., small and hydrophobic (red), acidic (blue), basic (magenta) and other (green) amino acid residues. The highly conserved gating residues W202 and W293 are highlighted in bold and yellow, and the N- and C-terminal numbers of the corresponding amino acid sequence stretches are indicated. Fig. S8. Cavity at the dimer interface. Two different views into the heart-shaped inter-dimeric cavity are shown, i.e., as viewed from the membrane plane (A) and as viewed from the periplasmic side (B). The top images represent overviews of the overall AdiC structure displayed as transparent surfaces and ribbons. The panels in the middle illustrate a vertical cut through the structure revealing the presence of a buried cavity (highlighted in red). The boxed structural regions are displayed enlarged in the bottom images. In all panels, side chains shaping the cavity are shown as sticks. All structural elements and labels are colored according to the individual monomeric color code, i.e., in blue and yellow. Fig. S9. Water inside the interdimeric cavity. (A) Positive residual Fo-Fc electron density map after the first refinement run. (B) After positioning eight waters (a-c, e-h and j), the second refinement run revealed additional positive residual Fo-Fc electron density. (C) 2Fo-Fc density after the third refinement run with all waters positioned (a-k). (D) Distances between water molecules are shown as black dashed lines and are given in Å. (E) Contour map representing the density probability of the water molecules in the cavity calculated from molecular dynamics simulations. The positions of crystallographic water molecules a-k are indicated with yellow crosses. (F) Corresponding wire-frame rendering of the isosurface contoured at 10% probability. In panels (A-D) and (F), the AdiC structure (transparent ribbons), important amino acid side chains (sticks) and the labels are colored according to the individual monomeric color code, i.e., in blue and yellow. Water molecules are represented as red spheres and are labeled with small case letters. Positive Fo-Fc and 2Fo-Fc electron density maps are represented by meshes, colored in green and blue, and contoured at 3.0 and 1.0 σ, respectively. Fig. S10. Different possibilities of water binding at the AdiC dimer interface cavity, i.e., binding of one (A-D) or two (E-G) water molecules. In all panels, distances are in Å and shown as black dashed lines. The AdiC structure (transparent ribbons), important amino acid side chains (sticks) and the labels are colored according to the individual monomeric color code, i.e., in blue and yellow. Water molecules are represented as red spheres and are labeled with the small case letters a-d. Corresponding frequencies (%) according to molecular dynamics simulations of water configurations are indicated in the lower, left corners. Fig. S11. Size exclusion chromatography (SEC) of AdiC variants. Wild-type AdiC (AdiC-wt), and AdiC mutants Q88E (AdiC-Q88E), Y367F (AdiC-Y367F) and T421V (AdiC-T421V) eluted at similar elution volumes indicating preservation of the dimeric state. SEC analysis was performed as described in Methods for thermostability analysis of AdiC variants. Table S1. Data collection, processing and refinement statistics. Table S2. Selected interactions in the substrate-binding site involving water molecules

The bacterial phosphoenolpyruvate: sugar phosphotransferase system serves the combined uptake and phosphorylation of carbohydrates. This structurally and functionally complex system is composed of several conserved functional units that, through a cascade of phosphorylated intermediates, catalyze the transfer of the phosphate moiety from phosphoenolpyruvate to the substrate, which is bound to the integral membrane domain IIC. The wild-type glucose-specific IIC domain (wt-IIC(glc)) of Escherichia coli was cloned, overexpressed and purified for biochemical and functional characterization. Size-exclusion chromatography and scintillation-proximity binding assays showed that purified wt-IIC(glc) was homogenous and able to bind glucose. Crystallization was pursued following two different approaches: (i) reconstitution of wt-IIC(glc) into a lipid bilayer by detergent removal through dialysis, which yielded tubular 2D crystals, and (ii) vapor-diffusion crystallization of detergent-solubilized wt-IIC(glc), which yielded rhombohedral 3D crystals. Analysis of the 2D crystals by cryo-electron microscopy and the 3D crystals by X-ray diffraction indicated resolutions of better than 6Å and 4Å, respectively. Furthermore, a complete X-ray diffraction data set could be collected and processed to 3.93Å resolution. These 2D and 3D crystals of wt-IIC(glc) lay the foundation for the determination of the first structure of a bacterial glucose-specific IIC domain.

Multiple enveloped RNA viruses of the family Paramyxoviridae and Pneumoviridae, like measles virus (MeV), Nipah virus (NiV), canine distemper virus (CDV), or respiratory syncytial virus (RSV), are of high clinical relevance. Each year a huge number of lives are lost as a result of these viral infections. Worldwide, MeV infection alone is responsible for over a hundred thousand deaths each year despite available vaccine. Therefore, there is an urgent need for treatment options to counteract these viral infections. The development of antiviral drugs in general stands as a huge challenge due to the rapid emergence of viral escape mutants. Here, we disclose the discovery of a small-molecule antiviral, compound 1 (ZHAWOC9045), active against several pneumo-/paramyxoviruses, including MeV, NiV, CDV, RSV, and parainfluenza virus type 5 (PIV-5). A series of mechanistic characterizations revealed that compound 1 targets a host factor which is indispensable for viral genome replication. Drug resistance profiling against a paramyxovirus model (CDV) demonstrated no detectable adaptation despite prolonged time of investigation, thereby mitigating the rapid emergence of escape variants. Furthermore, a thorough structure-activity relationship analysis of compound 1 led to the invention of 100-times-more potent-derivatives, e.g., compound 2 (ZHAWOC21026). Collectively, we present in this study an attractive host-directed pneumoviral/paramyxoviral replication inhibitor with potential therapeutic application. IMPORTANCE Measles virus, respiratory syncytial virus, canine distemper virus, and Nipah virus are some of the clinically significant RNA viruses that threaten substantial number of lives each year. Limited to no availability of treatment options for these viral infections makes it arduous to handle the outbreaks. This highlights the major importance of developing antivirals to fight not only ongoing infections but also potential future epidemics. Most of the discovered antivirals, in clinical trials currently, are virus targeted, which consequently poses the challenge of rapid emergence of escape variants. Here, we present compound 1 (ZHAWOC9045), discovered to target viral replication in a host-dependent manner, thereby exhibiting broad-spectrum activity against several members of the family Pneumo-/Paramyxoviridae. The inability of viruses to mutate against the inhibitor mitigated the critical issue of generation of escape variants. Importantly, compound 1 was successfully optimized to a highly potent variant, compound 2 (ZHAWOC21026), with a promising profile for pharmacological intervention.