A. Ribbon diagram of the ArsD109 dimer showing the extensive interface between monomers. Secondary structural units are N-β1-α1-β2-α2-α3-β3-β4-α4-C. Figures were created with PyMOL (16).

A. The structure of ArsD109 (cyan) was superimposed on the structure of the oxidized form of B. vulgatus 3KTB (red). Cys13 and Cys18 (yellow) in 3KTB are disulfide bonded. 3KTB residue Cys12 (yellow) forms an intersubunit disulfide bond with Cys12 in an adjacent monomer in the unit cell. B. Structure based alignment of ArsD and 3KTB. Secondary structural elements are shown in cartoon form (E = strands, L=loops and H= helices). Residues that form the cysteine-containing loop in 3KTB and the equivalent residues in ArsD109 are highlighted in yellow. Residues that are Identical or similar (+) in ArsD and 3KTB are shown in blue.

The open (nucleotide free) form of ArsA was modeled on the open form of Schizosaccharomyces pombe Get3 (Figure S2B). The three cysteines residues of ArsA that form the As(III) binding site (24, 25) are shown yellow. The structure is shown in two orientations (A and B) that differ by 180° to illustrate the cavity between ArsA1 and ArsA1 into which ArsD is proposed to fit.

The interface in the crystal structure between the two ArsD monomers (green and cyan) is shown with the backbone atoms in ribbon and the side chains of the interfacial residues in ball-and stick. Hydrogen bonds and salt bridges are shown in dotted lines with distances.

Binding of arsenic by apoArsD (A) is proposed to require a minimum of two steps. In the first step, two cysteine thiolates (yellow) bind As(III) (purple sphere) in a transient complex (B). While the order of binding is not known, it is shown here as contributed by Cys13 and Cys18 because those two cysteines from a disulfide bond in the oxidized form of 3KTB. In the second step, the third cysteine thiolate reorients to form the strong three-coordinate binding site (6) (C). Residues 11–22 in ArsD109 were modeled on the corresponding residues in 3KTB. The thiolates of the metalloid binding site (yellow) were placed equidistant from the centrally bound arsenic atom (purple sphere) based on the distances determined by EXAFS (6).

A. An optimized model based on in silico docking analyses is shown in the ribbon diagram. ArsD (cyan) is proposed to fit into the cavity of the open form of ArsA (ArsA1 in green and ArsA2 in orange). The arsenic atom (purple sphere) is bound to the thiolates (yellow ball and sticks) of ArsD residues 12, 13 and 18, which are within 4 Å of the atoms sulfurs of ArsA1 residues Cys113 and Cys172. In a subsequent putative step Cys422 of ArsA2 would reorient to form the third ArsA ligand to As(III). B. The ArsD-ArsA complex is shown vertically rotated 90°, with a surface rendering of ArsA, revealing ArsD residues (black) that are predicted to interact with ArsA residues (white). C. ArsD is shown alone in the same orientation as in B. Residues predicted to interact with ArsA are labeled. Residues on the left side of the dotted line are proposed to interact with residues in ArsA1, and those on the right side of the dotted line with ArsA2. D. ArsA is shown alone in the same orientation as in B. The labeled residues in white are predicted to interact with ArsD.