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Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC

  1. Dimitrios Fotiadisa,b,2
  1. aInstitute of Biochemistry and Molecular Medicine, University of Bern, CH-3012 Bern, Switzerland;
  2. bSwiss National Centre of Competence in Research TransCure, University of Bern, CH-3012 Bern, Switzerland;
  3. cComputational Biomolecular Dynamics Group, Max-Planck-Institute for Biophysical Chemistry, D-37077 Goettingen, Germany
  1. Edited by Christopher Miller, Howard Hughes Medical Institute, Brandeis University, Waltham, MA, and approved July 26, 2016 (received for review April 4, 2016)

  1. Fig. 2.

    Cartoon ribbon representations of the Agm-bound and substrate-free AdiC structures. (AC) Superpositions of AgmAdiC-wt (in blue) and apoAdiC-wt (in yellow) dimers. Individual monomers are indicated and transmembrane helices are discerned. The Agm molecule (magenta sticks) bound to AgmAdiC-wt is shown and is located about at the center of the transport path. The location of the substrate-binding pocket is indicated by red ellipses. Three different views are displayed: view from the membrane plane (A), and from the cytoplasmic (B), and periplasmic sides (C). (D) Omit electron density map at 3.0 σ (green) of the Agm molecule (magenta sticks) bound to AgmAdiC-wt.

  2. Fig. 3.

    Comparison of the substrate-binding sites in the Agm-bound and substrate-free AdiC structures. (A) Recognition of the substrate Agm by specific amino acids in AdiC and one protein-associated water molecule (H2O1). Important amino acids are labeled in the one letter code and when interacting with their main-chain carbonyl oxygen atoms additionally labeled with (O). The three Gdm group nitrogen atoms of Agm are labeled Nε, Nη1, and Nη2. The top view from the periplasmic side (Upper) and side view (tilted by 90°) from the membrane plane (Lower) onto the Agm binding site are displayed. For van der Waals interactions between AdiC and the aliphatic portion of Agm (e.g., with the side chain of I205) see Fig. S3A. (B) Same views and labeling as in A for the substrate-free AdiC structure. The AgmAdiC-wt (A) and apoAdiC-wt (B) structures and specific amino acid residues in the substrate-binding pockets are represented as ribbons in blue and yellow, and as sticks in gold and salmon, respectively. Crystallographic water molecules are displayed as red balls, and potential H-bonds and interatomic distances are indicated as dotted lines and in Ångstrom, respectively.

  3. Fig. S1.

    Ligand-binding affinities determined by SPA of AdiC-wt, AdiC-N22A, and AdiC-S26A. Radioligand: l-[3H]arginine ([3H]Arg). Ligands: l-arginine (Arg), agmatine (Agm), l-arginine methyl ester (Arg-OMe) and l-arginine amide (Arg-NH2). Ki: inhibition constant (binding affinity). The determined Ki values are from at least three independent experiments, each in triplicate. Error bars represent SEM. For 95% confidence interval values of the corresponding experiments, see Table 1.

  4. Fig. S2.

    Electron density maps of the AgmAdiC-wt (A) and apoAdiC-wt (B) substrate-binding pockets. For example, electron densities of Agm, water molecules and the different rotamer conformations defining M104 (TM3) are discerned. The 2Fo-Fc electron density maps are contoured at 1.0 σ and colored in magenta (AgmAdiC-wt, A) and blue (apoAdiC-wt, B). The AgmAdiC-wt and apoAdiC-wt structures, and specific amino acid residues in the substrate-binding pockets are represented as ribbons in blue and yellow, and as sticks in gold and salmon, respectively.

  5. Fig. S3.

    Comparison of the van der Waals interactions between protein and aliphatic portion of the substrates in the AgmAdiC-wt and ArgAdiC-N22A structures. (A) Amino acid residues of AgmAdiC-wt involved in van der Waals interactions with the substrate Agm are displayed and labeled in the one letter code. In the Upper part of the figure, a top view from the periplasmic side is shown whereas in the lower part a 90° tilted view is displayed. (B) For the ArgAdiC-N22A structure, same views and labeling as in A are shown. The AgmAdiC-wt (A) and ArgAdiC-N22A (B) structures, and specific amino acids are represented as ribbons in blue and magenta, and as sticks in gold and light-blue, respectively. The substrates are displayed as black (Agm) and yellow (Arg) sticks, and potential interatomic interactions and their distances are indicated as dotted lines and in Å, respectively.

  6. Fig. S4.

    Effect of alanine substitution on substrate binding to AdiC determined by SPA. (A) Relative comparison of l-[3H]arginine binding to AdiC-wt and selected AdiC mutants. (B) Arg and Agm binding affinities of selected AdiC mutants. Radioligand: l-[3H]arginine ([3H]Arg). Ligands: l-arginine (Arg) and agmatine (Agm). Ki: inhibition constant (binding affinity). [3H]Arg binding (A) and the determined Ki values (B) are from at least three independent experiments, each in triplicate. Error bars represent SEM. For 95% confidence interval values of the affinities determined in B, see Table 1.

  7. Fig. S5.

    MD simulations of apoAdiC-wt. (A) Water densities observed in the MD simulations of both monomers of the apoAdiC-wt dimer are depicted. The density profiles are consistent for both monomers and are represented at a contour level of 1.0 σ. The crystallographically resolved water positions H2O1-H2O4 are displayed as red balls. (B) Analysis of hydrogen bonds (H-Bonds) formed by the A96 (TM3) and S357 (TM10) amino acids with water molecules averaged over ten molecular dynamics simulations of 100 ns each of apoAdiC-wt: number of H-bonds (Left), H-bond energies (Right). Error bars represent SEM.

  8. Fig. S6.

    Comparison of the substrate-binding sites of the Arg-bound AdiC-N22A and the Agm-bound AdiC-wt structures. (A) Recognition of the substrate Arg by specific amino acids of AdiC-N22A. Amino acids interacting with the substrate are labeled in the one letter code and if the main-chain carbonyl oxygen atom or amide nitrogen are involved additionally labeled with (O) or (N). In the Upper part of the panel a top view from the periplasmic side is shown, whereas in the Lower part a 90° tilted view is displayed. (B) For the AgmAdiC-wt structure same views and labeling as in A are shown. The ArgAdiC-N22A (A) and AgmAdiC-wt (B) structures, and specific amino acids in the substrate-binding pockets, are represented as ribbons in magenta and blue, and as sticks in light-blue and gold, respectively. The substrates are displayed as yellow (Arg) and black (Agm) sticks and the crystallographic water molecule (H2O1) found in the AgmAdiC-wt substrate-binding pocket is represented as red ball. Potential H-bonds and interatomic distances are indicated as dotted lines and in Ångstroms, respectively.

  9. Fig. S7.

    Superposition and stereoview representation of the substrate-binding sites of the ArgAdiC-N22A and the AgmAdiC-wt structures. Amino acids interacting with the substrates are labeled in the one letter code and if the main-chain carbonyl oxygen atom or amide nitrogen are involved additionally labeled with (O) or (N). Views from the periplasmic side (A) and membrane plane (B). The ArgAdiC-N22A and AgmAdiC-wt structures, and specific amino acids in the substrate-binding pockets, are represented as ribbons in magenta and blue, and as sticks in light-blue and gold, respectively. The substrates are displayed as yellow (Arg) and black (Agm) sticks and the crystallographic water molecule (H2O1) found in the AgmAdiC-wt substrate-binding pocket is represented as red ball.

  10. Fig. S8.

    Comparison of selected amino acids located at the substrate-binding site and the intracellular (distal) gate of Agm-bound AdiC in the outward-facing occluded (A; AgmAdiC-N22A) and outward-open conformation (B; AgmAdiC-wt). The former was generated from the published ArgAdiC-N22A structure (PDB ID code 3L1L) (10) by removing in silico the carboxylate group from the substrate Arg to obtain Agm. Specific amino acids are labeled in the one letter code. In the Upper part of the panel a top view from the periplasmic side is shown whereas in the Lower part a 90° tilted view is displayed (view from the membrane plane). (B) For the AgmAdiC-wt structure the same views and labeling as in A are shown. The AgmAdiC-N22A (A) and AgmAdiC-wt (B) structures, specific amino acids directly interacting with Agm, and amino acids belonging to the intracellular gate are represented as ribbons in transparent magenta and transparent blue, as sticks in light-blue and gold, and as sticks in magenta and blue, respectively. Agm is displayed as black sticks and the crystallographic water molecule (H2O1) found in the AgmAdiC-wt substrate-binding pocket is represented as red ball. Potential H-bonds and interatomic distances are indicated as dotted lines and in Ångstroms, respectively.

  11. Fig. S9.

    AdiC, CadB, and PotE are virtual proton pump exchangers from E. coli and members from APC superfamily. Amino acid residues discussed in the text are indicated in bold and on yellow background. Amino acid sequence alignment was performed with Clustal Omega (www.clustal.org/omega/). The UniProt ID codes of AdiC, CadB, and PotE are P60061, P0AAE8, and P0AAF1, respectively. The three characters (asterisk, colon, dot) indicate positions that have a single, fully conserved residue (*), and conservation between groups of strongly (:), and weakly similar properties (·). The strong and weak groups are defined as strong score >0.5 and weak score ≤0.5 occurring in the Gonnet PAM 250 matrix. Color coding of amino acid residues is according to their physicochemical properties: that is, small and hydrophobic (including aromatic except tyrosine) (red), acidic (blue), basic (magenta), and others (green) amino acid residues.

  12. Fig. S10.

    Potential conformational changes of AdiC involved in Agm release into the periplasmic space. To illustrate the differences between outward-facing occluded (magenta; ArgAdiC-N22A structure, PDB ID code: 3L1L) (10) and outward-open conformations (blue; AgmAdiC-wt structure) of AdiC, both structures were superpositioned and are represented as cartoon ribbon (helices shown as solid cylinders). Most pronounced movements in this conformational change (indicated by yellow arrows) are within transmembrane regions, i.e., transmembrane α-helices (TMs) 2, 6 and 10. Minor differences found on the periplasmic side (top) are the result of crystal contacts; for example, the region marked by an asterisk.

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