Additional file 1 of High-resolution structure of the amino acid transporter AdiC reveals insights into the role of water molecules and networks in oligomerization and substrate binding
Creators: name: Ilgü, Hüseyin
name: Jeckelmann, Jean-Marc
name: Kalbermatter, David
name: Ucurum, Zöhre
name: Lemmin, Thomas
name: Fotiadis, Dimitrios
Publisher: name: figshare
Year: 2021
Subjects: subject: Biophysics
subject: Biochemistry
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: 69999 Biological Sciences not elsewhere classified
subject: FOS: Biological sciences
subject: 80699 Information Systems not elsewhere classified
subject: FOS: Computer and information sciences
subject: Hematology
subject: Computational Biology

description: 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