Figure 1. Substrate binding to Glt_Ph-R397A.

(A) Raw binding heat rates measured by isothermal titration calorimetry (top) and binding isotherms (bottom) obtained for Glt_Ph-R397A (left) and wild type Glt_Ph (right) at 25°C in the presence of 100 mM NaCl. The solid lines through the data are fits to the independent binding sites model with the following parameters for Glt_Ph-R397A and wild type Glt_Ph, respectively: enthalpy change (ΔH) of −3.2 and −14.3 kcal/mol; the apparent number of binding sites (n) of 0.8 and 0.7 per monomer; dissociation constant (K_d) of 6.6 µM and 27 nM. Note that L-asp binding to the wild type transporter is too tight at 100 mM NaCl to be accurately measured in this experiment. The binding K_d has been estimated to be ∼1 nM (Boudker et al., 2007). (B) L-asp binding site in Glt_Ph-R397A (left) and wild type Glt_Ph (right). L-asp and residues coordinating the side chain carboxylate are shown as sticks with carbon atoms colored light brown and blue, respectively. Potential hydrogen bonds (distances less than 3.5 Å) between the L-asp side chain carboxylate and transporter residues are shown as dashed lines. Note that Y317, which forms cation-π interactions with guanidium group of R397 in wild type Glt_Ph, interacts directly with L-asp in Glt_Ph-R397A.

DOI: http://dx.doi.org/10.7554/eLife.02283.008

Figure 1—figure supplement 1. Alternating access mechanism in Glt_Ph.

(A) The transporter consists of a rigid trimerization domain (light brown) and a dynamic transport domain (blue, with HP1 yellow and HP2 red). The apo outward-facing transporter binds the substrate (blue square) and Na⁺ ions (pink circles) within the transport domain under the tip of HP2. The transport domain moves into the inward-facing state, in which the ligands are occluded from the cytoplasm by the tips of HP1 and HP2, and then released. (B) Glt_Ph trimer viewed from the extracellular medium. (C) Single protomers of the outward-facing Glt_Ph (PDB code 2NWX) (left), and Glt_Phⁱⁿ (PDB code 3KBC) (right). Cα atoms of the cross-linked cysteine residues and Hg²⁺ ion are shown as spheres. The bar next to the structures indicates approximately the thickness of the membrane, separating the extracellular (Ext) and intracellular (Int) solutions.

DOI: http://dx.doi.org/10.7554/eLife.02283.009

Figure 1—figure supplement 2. Structure of Glt_Ph-R397A bound to Na⁺ and L-asp.

Stereo view of the averaged 2F_o-F_c electron density map contoured at 1σ (grey mesh) around residues in L-asp binding site of Glt_Ph-R397A.

DOI: http://dx.doi.org/10.7554/eLife.02283.010