1. Volume 2, Issue 1, Pages 75-84 (July 1998)
The Structure of VanX Reveals a Novel Amino-Dipeptidase Involved in Mediating Transposon-Based Vancomycin Resistance 
Dirksen E. Bussiere, Steven D. Pratt, Leonard Katz, Jean M. Severin, Thomas Holzman, Chang H. Park 
Molecular Cell 
Volume 2, Issue 1, Pages (July 1998)
DOI: /S (00)80115-X

2. Figure 1 Mechanism and Inhibitors of VanX
(A) The proposed mechanism of VanX. (i) An incoming dipeptide displaces a zinc-bound water molecule toward Glu-181; (ii) the water molecule is activated by the influence of the catalytic zinc and Glu-181 and attacks the polarized carbonyl to form a tetrahedral intermediate; (iii) the tetrahedral intermediate forms a bidentate complex with the zinc and is stablized further by interactions with Arg-71; Glu-181 subsequently donates the proton to the scissile nitrogen, completing the reaction; (iv) D-alanine is released.
(B) The phosphinate transition-state analog. This compound has a reported Ki of 0.3 μM (Wu et al. 1995).
(C) The phosphonate transition-state analog. This compound has a reported Ki of 0.3 mM (Wu et al. 1995).
Molecular Cell 1998 2, 75-84DOI: ( /S (00)80115-X)

3. Figure 2 VanX Electron Density Maps Contoured at 1.2 σ
(A) Electron density map calculated from the MIR/solvent-flattened/noncrystallographic symmetry averaged phases at 2.8 Å resolution. A portion of one of the β strands is shown.
(B) 2Fo − Fc electron density map calculated from the refined atomic model of VanX at 2.25 Å resolution. The equivalent portion of the β strand of (A) is shown.
Molecular Cell 1998 2, 75-84DOI: ( /S (00)80115-X)

4. Figure 3 Stereo Diagram of the VanX Monomer
Key portions of secondary structure are labeled, and the N and C termini are also identified. β strands are shown in red, turns/coil in orange, 310 helices in light blue, and α helices in green. The zinc ion is shown as a yellow sphere. The illustration was prepared using RIBBONS (Carson 1987).
Molecular Cell 1998 2, 75-84DOI: ( /S (00)80115-X)

5. Figure 4
Zinc, Substrate, and Transition-State Analog Binding in the VanX Active Site Cavity
Key protein–substrate/product or –inhibitor interactions are shown. The catalytic zinc is shown as a yellow sphere. Distances between interacting groups are described within the figure text. In the case of (B) and (C), the struc-tures of the substrate/product complexes and the analog complexes have been superimposed for the purpose of comparison.
(A) The active site of VanX. The catalytic zinc coordinates to His-116, Asp-123, His-184, and the structural water (shown as a red sphere) at a distance of 2.45 Å, 3.01 Å, 2.50 Å, and 3.14 Å, respectively. Glu-181, which is postulated to aid in activation of the catalytic base, is shown, as is Arg-71, which stabilizes the transition state. The β strands are shown as arrows.
(B) The binding of D-ala-D-ala (substrate) and D-ala (product) within the active site. The D-ala-D-ala dipeptide is shown with its carbon atoms colored white. Its amino terminus labeled with a red “N” and the carboxyl terminus labeled “O.” The D-ala amino acid’s carbon atoms are colored magenta. The yellow arrow indicates the entrance of the active site cavity. Ligand–side chain interactions are identified by dashed lines (white for the dipeptide, magenta for the amino acid). The amino group donates hydrogen bonds to Tyr-21, Asp-123, and Asp-142, with distances of 3.16 Å, 3.51 Å, and 2.90 Å; the carbonyl coordinates directly to the catalytic zinc at a distance of 2.48 Å. The bridging amide (within the green circle) donates a hydrogen to the backbone carbonyl of Tyr-109; the carboxylate accepts hydrogen bonds from Ser-115 at a distance of 3.16 Å. The D-ala amino acid shows a more narrow range of interaction. The amino group donates hydrogen bonds to Tyr-21, Asp-123, and Asp-142, with distances of 3.84 Å, 3.23 Å, and 3.32 Å. One oxygen of the carboxylate coordinates to the catalytic zinc at a distance of 2.86 Å; the other oxygen coordinates to the backbone amide of Ala-111, which is brought into position by a rearrangement of residues 108–113 (shown by the magenta arrow) in those molecules that bound D-ala.
(C) The binding of the phosphinate and phosphonate transition-state analogs within the active site. The amino terminus of the analogs is labeled with a red “N,” the phosphorous portion with a red “P,” and the carboxyl terminus with a red “O.” The phosphonate analog is shown with white carbons and the phosphinate analog is shown with magenta carbons. Analog–side chain interactions are shown in dashed lines (white for the phosphonate and magenta for the phosphinate). In the phosphinate, the amino group donates hydrogen bonds to Tyr-21, Asp-123, and Asp-142, with distances of 2.96 Å, 3.03 Å, and 2.82 Å; both oxygens of the phosphinate moiety coordinate directly to the catalytic zinc at a distance of approximately 2.5 Å; the carboxylate accepts hydrogen bonds from Ser-114 at a distance of 2.78 Å. In the phosphonate complex, the interactions are equivalent, except for the amino group that is rotated 180° about the carbon–phosphorous bond. The amino group donates hydrogen bonds to Asp-142 and Glu-181 with distances of 3.59 Å and 3.88 Å; both oxygens of the phosphinate moiety coordinate directly to the catalytic zinc at a distance of approximately 2.5 Å; the carboxylate accepts hydrogen bonds from Ser-114 at a distance of 2.64 Å.
(D) Surface electrostatic potential of the VanX protein. The electrostatic potential is superimposed on the solvent-accessible surface of VanX calculated using a water probe radius of 1.4 Å. The calculations were performed using GRASP (Nicholls et al. 1991). The ranges of red (negative) and blue (positive) represent electrostatic potentials of <−15 to >+15 kbT, where kb is the Boltzmann constant and T is the temperature. White represents nonpolar residues. In the calculations, the pH is 7.0, and the dielectric constants for the solvent and protein are 80 and 2, respectively. The active site cavity is identified by a green box, and the phosphinate analog is shown within the cavity.
Molecular Cell 1998 2, 75-84DOI: ( /S (00)80115-X)

6. Figure 5
Multiple Sequence Alignment of Identified VanX Proteins from Gram-Positive Bacteria
Residues shaded in black at a particular position indicate a 100% identity over all sequences. Residues shaded in gray at a particular position indicate amino acid homology between three sequences. Residues that form the active site are labeled with an “A.” Alignment was performed using CLUSTAL W (Thompson et al. 1994) and illustrated using the program BOXSHADE.
Molecular Cell 1998 2, 75-84DOI: ( /S (00)80115-X)

7. Figure 6 Structural Homologies of the VanX Protein
(A) Structural alignments of the VanX protein (red) with the N-terminal fragment of murine Sonic hedgehog (green), and D-ala-D-ala carboxypeptidase (gold). The active site position of all of the enzymes is labelled. All structural alignments were done using the program SARF (Alexandrov 1996).
(B) Structural homology within the VanX (red), N-terminal murine Sonic hedgehog (green), and D-ala-D-ala carboxypeptidase of S. albus G (gold) active sites. Homologous side chains and main chains are shown and are labeled in equivalent colors to their respective molecules.
Molecular Cell 1998 2, 75-84DOI: ( /S (00)80115-X)