Coupled ion binding and structural transitions along the transport cycle of glutamate transporters
- Weill Cornell Medical College, United States
Figures
Figure 1 with 2 supplements
Substrate binding to GltPh-R397A.
(A) Raw binding heat rates measured by isothermal titration calorimetry (top) and binding isotherms (bottom) obtained for GltPh-R397A (left) and wild type GltPh (right) at 25°C in the presence of …
Figure 1—figure supplement 1
Alternating access mechanism in GltPh.
(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 …
Figure 1—figure supplement 2
Structure of GltPh-R397A bound to Na+ and L-asp.
Stereo view of the averaged 2Fo-Fc electron density map contoured at 1σ (grey mesh) around residues in L-asp binding site of GltPh-R397A.
Figure 2
Apo protomer structures.
(A) GltPh protomers in the outward-facing state (left) and a GltPhin protomer (right) viewed from within the plane of the membrane. Shown are superimpositions between apo (colors) and fully bound …
Figure 3 with 2 supplements
Structures of the apo transport domain.
(A) Superimposition of the nearly identical apo transport domains in the outward- and inward-facing states. HP1, HP2, and NMD motif are colored yellow, red, and green, respectively. The remainder of …
Figure 3—figure supplement 1
Apo protomer structures.
Stereo views of the averaged 2Fo-Fc electron density maps contoured at 1σ around HP2 tip of the apo GltPh-R397A (top) and the NMD motif and TM3 of the apo GltPhin (bottom).
Figure 3—figure supplement 2
Structural comparison of the transport domain in various states.
Plotted are root mean square deviations (R.M.S.D.s) of the main chain atoms calculated per residue using VMD for the transport domains in (A) apo outward- and inward-facing states, (B) …
Figure 4 with 1 supplement
Remodeling of L-asp and Na+ binding sites in the apo conformations.
Close-up views of the fully bound (left) and apo (right) transport domains at L-asp binding site (top), Na1 and Na2 sites (middle), and one of the proposed locations for the third Na+ binding site (H…
Figure 4—figure supplement 1
Transport domain remains compact.
Surface representation of the transport domain in fully bound and apo forms.
Figure 5
Met311 is key to the allosteric coupling.
(A) Structural models combining HP2 bound to L-asp and Na+ at Na2 site with apo conformation of the NMD motif (left), and apo conformation of HP2 with the NMD motif bound to Na+ at Na1 site (right). …
Figure 6 with 3 supplements
Structures of ions-only bound transport domain.
(A) Superimposition of the fully bound transport domains (grey) and Tl+-bound GltPhin transport domain in the bound-like conformation (colors), with the averaged anomalous difference Fourier map …
Figure 6—figure supplement 1
Na+ only bound GltPh-R397A.
Stereo view of the averaged 2Fo-Fc electron density map contoured at 1σ around HP1 and HP2.
Figure 6—figure supplement 2
Superimposition of the transport domains bound to Na+ and L-asp (light grey), Na+ and L-TBOA (dark grey) and Na+ only (colors).
Ligands are omitted for clarity, except that L-TBOA is shown as spheres in the right panel. Nostably, the observed additional opening of HP2 is necessary to accommodate L-TBOA.
Figure 6—figure supplement 3
Sequence alignment for the HP2 tip region of GltPh and human EAAT sub-types 1–5.
https://doi.org/10.7554/eLife.02283.019
Figure 7
Modeled Na+-bound early transition intermediate between the outward- and inward-facing states.
(A) Surface representations of the protomer in the fully bound intermediate state (PDB code 3V8G) (left), and the modeled Na+-bound intermediate with an open HP2 tip (right) viewed from the …
Figure 8 with 2 supplements
New cation binding sites.
(A) Superimpositions of GltPhin (left) and outward-facing GltPh-R397A (right) transport domains in the apo form (grey) with Tl+-bound apo-like conformations (colors). Averaged anomalous difference …
Figure 8—figure supplement 1
Transport domain internal cavities.
Internal cavities in the apo GltPhin structure (left). Cavities were calculated using solvent radius of 1.4 Å and colored by local electrostatic potential with red and blue being negative and …
Figure 8—figure supplement 2
Specificity of the new cation binding sites in apo-like GltPhin.
(A) Averaged anomalous difference Fourier maps contoured at 8σ (cyan mesh) for Tl+-soaked (150 mM) GltPhin in the presence of 300 mM K+ (left) and 10 mM Na+ (right) showing a decrease of the Tl+ …
Figure 9 with 2 supplements
Movements of the HP1-TM7a structural module.
(A) Superimposition of GltPhin transport domains when bound to Tl+ in the apo-like conformation (grey) and when prepared in an alkali-free solution (colors). (B) The transport domains of the fully …
Figure 9—figure supplement 1
Alkali-free inward-facing GltPhin.
(A) Stereo view of the 2Fo-Fc (1σ) and Fo-Fc (3σ) omit maps obtained after molecular replacement using Tl+-bound GltPhin and refinement of a model with HP1 and TM7a omitted, in the protomer showing …
Figure 9—figure supplement 2
Surface representation of the alkali-free inward-facing GltPhin transport domain in this protomer after refinement.
A dashed circle indicates a small opening between HP1 and HP2.
Figure 10
Proposed transport cycle for GltPh and EAATs.
Ion binding to the Na1 site of the outward-facing apo transport domain triggers isomerization into bound-like conformation, formation of the L-asp and Na2 binding sites and HP2 opening, impeding …
Tables
Table 1
X-ray crystallographic data and refinement statistics for GltPh-R397A and GltPh-K55C-A364CHg (GltPhin) structures deposited at the PDB
| GltPhin | ||||
|---|---|---|---|---|
| apo | Tl+-bound (apo conf.) | alkali-free | Tl+-bound (bound conf.) | |
| Data collection | ||||
| Space group | C2221 | C2221 | C2221 | C2221 |
| Cell dimensions | ||||
| a, b, c (Å) | 109.93, 201.81, 207.14 | 106.98, 196.56, 206.50 | 106.95, 196.84, 207.48 | 110.83, 200.43, 206.40 |
| α, β, γ (°) | 90.00, 90.00, 90.00 | 90.00, 90.00, 90.00 | 90.00, 90.00, 90.0 | 90.00, 90.00, 90.00 |
| Resolution (Å) | 100.0–3.25 (3.31–3.25) | 100.0–3.75 (3.81–3.75) | 100.0–3.50 (3.56–3.50) | 100.0–4.0 (4.14–4.0) |
| Rsym or Rmerge | 10.9 (88.6) | 14.0 (94.4) | 8.0 (88.1) | 16.3 (75.2) |
| I/σI | 12.3 (1.2) | 8.95 (1.1) | 13.5 (1.2) | 7.9 (1.3) |
| Completeness (%) | 98.7 (88.1) | 99.7 (99.8) | 94.4 (92.7) | 65.2 (6.5) |
| Redundancy | 5.6 (2.8) | 3.8 (3.7) | 3.3 (3.2) | 3.4 (3.5) |
| Refinement | ||||
| Resolution (Å) | 15.0–3.25 | 15.0–3.75 | 15.0–3.5 | 15.0–4.0 |
| No. reflections | 34534 | 21565 | 25446 | 11105 |
| Rwork/Rfree | 22.2/25.8 | 23.0/25.7 | 26.3/27.8 | 25.8/29.6 |
| No. atoms | ||||
| Protein | 9121 | 9114 | 9088 | 8985 |
| Ligand/ion | 3 | 9 | 3 | 9 |
| B-factors | ||||
| Protein | 108.5 | 141.8 | 144.2 | 137.2 |
| Ligand/ion | 135.3 | 170.8 | 214.1 | 102.3 |
| R.m.s. deviations | ||||
| Bond lengths (Å) | 0.010 | 0.013 | 0.005 | 0.012 |
| Bond angles (°) | 1.680 | 1.861 | 1.116 | 1.407 |
| PDB code | 4P19 | 4P1A | 4P3J | 4P6H |
| GltPh-R397A | |||
|---|---|---|---|
| Apo | Na+-bound | Na+/aspartate-bound | |
| Data collection | |||
| Space group | P21 | P31 | P31 |
| Cell dimensions | |||
| a, b, c (Å) | 112.37, 424.42, 113.99 | 110.58, 110.58, 306.92 | 116.96, 116.96, 313.52 |
| α, β, γ (°) | 90.00, 119.40, 90.00 | 90.00, 90.00, 120.00 | 90.00, 90.00, 120.00 |
| Resolution (Å) | 100.0–4.00 (4.14–4.00) | 50.0–3.39 (3.51–3.39) | 100.0–3.50 (3.63–3.50) |
| Rsym or Rmerge | 7.8 (62.2) | 14.0 (>100) | 8.4 (>100) |
| I/σI | 9.3 (1.3) | 13.8 (1.4) | 10.6 (0.4) |
| Completeness (%) | 67.9 (13.0) | 87.3 (12.0) | 98.1 (96.6) |
| Redundancy | 1.8 (2.0) | 11.8 (8.6) | 4.5 (4.2) |
| Refinement | |||
| Resolution (Å) | 20.0–4.0 | 12.0–3.41 | 15.0–3.50 |
| No. reflections | 52068 | 48366 | 55613 |
| Rwork/Rfree | 24.9/26.6 | 28.4/29.3 | 24.3/26.8 |
| No. atoms | |||
| Protein | 35277 | 17580 | 18192 |
| Ligand/ion | N/A | 6 | 54/12 |
| Water | N/A | 6 | 6 |
| B-factors | |||
| Protein | 139.5 | 152.0 | 97.1 |
| Ligand/ion | N/A | 145.1 | 84.7/86.9 |
| Water | N/A | 102.6 | 144.6 |
| R.m.s. deviations | |||
| Bond lengths (Å) | 0.010 | 0.010 | 0.015 |
| Bond angles (°) | 1.393 | 1.468 | 1.735 |
| PDB code | 4OYE | 4OYF | 4OYG |
Table 2
Completeness of datasets corrected for anisotropy
| Tl+-bound GltPhin (bound conformation) | Na+-bound GltPh-R397A | ||
|---|---|---|---|
| Resolution range (Å) | Completeness (%) | Resolution range (Å) | Completeness (%) |
| 100.0–8.62 | 99.3 | 50.00–7.30 | 99.6 |
| 8.62–6.84 | 99.9 | 7.30–5.79 | 100.0 |
| 6.84–5.97 | 100.0 | 5.79–5.06 | 100.0 |
| 5.97–5.43 | 99.9 | 5.06–4.60 | 100.0 |
| 5.43–5.04 | 99.9 | 4.60–4.27 | 100.0 |
| 5.04–4.74 | 69.6 | 4.27–4.02 | 100.0 |
| 4.74–4.50 | 39.2 | 4.02–3.82 | 100.0 |
| 4.50–4.31 | 23.6 | 3.82–3.65 | 98.6 |
| 4.31–4.14 | 14.4 | 3.65–3.51 | 63.0 |
| 4.14–4.00 | 6.5 | 3.51–3.39 | 12.0 |
| Apo GltPh-R397A | |
|---|---|
| Resolution range (Å) | Completeness (%) |
| 100.0–8.62 | 85.0 |
| 8.62–6.84 | 75.6 |
| 6.84–5.97 | 75.5 |
| 5.97–5.43 | 75.3 |
| 5.43–5.04 | 75.2 |
| 5.04–4.74 | 75.8 |
| 4.74–4.50 | 75.3 |
| 4.50–4.31 | 75.4 |
| 4.31–4.14 | 51.7 |
| 4.14–4.00 | 13.0 |
Table 3
X-ray crystallographic data and refinement statistics for GltPh-R397A and GltPh-K55C-A364CHg structures not deposited at the PDB
| GltPh-R397A | GltPhin | ||
|---|---|---|---|
| Tl+-bound (apo conf.) | Tl+/Na+ (apo conf.) | Tl+/k+ (apo conf.) | |
| Data collection | |||
| Space group | P21 | C2221 | C2221 |
| Cell dimensions | |||
| a, b, c (Å) | 115.18, 428.53, 116.61 | 108.11, 198.86, 206.34 | 106.59, 198.48, 205.82 |
| α, β, γ (°) | 90.00, 119.49, 90.00 | 90.00, 90.00, 90.00 | 90.00, 90.00, 90.00 |
| Resolution (Å) | 30.0–5.0 (5.18–5.00) | 100.0–4.0 (4.07–4.00) | 100.0–4.15 (4.22–4.15) |
| Rsym or Rmerge | 10.9 (>100) | 15.0 (92.2) | 13.9 (94.1) |
| I/σI | 13.8 (1.9) | 8.9 (1.5) | 9.2 (1.5) |
| Completeness (%) | 86.4 (75.1) | 99.9 (100) | 94.5 (90.2) |
| Redundancy | 5.5 (5.8) | 3.9 (3.9) | 4.0 (3.9) |
| Refinement | |||
| Resolution (Å) | 20.0–5.0 | 15.0–4.0 | 15.0–4.15 |
| No. reflections | 34747 | 18184 | 15419 |
| Rwork/Rfree | 22.0/26.5 | 28.2/31.7 | 28.3/31.2 |
| No. atoms | |||
| Protein | 35107 | 9135 | 9135 |
| Ligand/ion | N/A | N/A | N/A |
| Water | N/A | N/A | N/A |
| B-factors | |||
| Protein | 223.00 | 183.6 | 194.4 |
| Ligand/ion | N/A | N/A | N/A |
| Water | N/A | N/A | N/A |
| R.m.s. deviations | |||
| Bond lengths (Å) | 0.008 | 0.006 | 0.008 |
| Bond angles (°) | 1.186 | 1.266 | 1.440 |
