Glu-311 in External Loop 4 of the Sodium/Proline Transporter PutP Is Crucial for External Gate Closure

Abstract

The available structural information on LeuT and structurally related transporters suggests that external loop 4 (eL4) and the outer end of transmembrane domain (TM) 10' participate in the reversible occlusion of the outer pathway to the solute binding sites. Here, the functional significance of eL4 and the outer region of TM10' are explored using the sodium/proline symporter PutP as a model. Glu-311 at the tip of eL4, and various amino acids around the outer end of TM10' are identified as particularly crucial for function. Substitutions at these sites inhibit the transport cycle, and affect in part ligand binding. In addition, changes at selected sites induce a global structural alteration in the direction of an outward-open conformation. It is suggested that interactions between the tip of eL4 and the peptide backbone at the end of TM10' participate in coordinating conformational alterations underlying the alternating access mechanism of transport. Together with the structural information on LeuT-like transporters, the results further specify the idea that common design and functional principles are maintained across different transport families.

Keywords: PutP; amino acid transport; electron paramagnetic resonance (EPR); membrane protein; proline; protein chemistry; protein conformation; secondary transport; solute/sodium symport; spin labeling.

FIGURE 1.

Structural model of PutP. A and B, homology and restraint-based model of PutP in an inward-open conformation with eL4 highlighted in orange and TM10′ in turquoise (A, top view; B, side view). The model represents an advancement of earlier models (28, 29), and matches nine DEER-based distances (root mean square deviations for all distance restraints = 0.72 Å). The side chain of Glu-311 is shown in ball-and-stick representation. The indicated putative locations of the binding sites for l-proline and sodium were taken from Olkhova et al. (28). C, sequence alignment of amino acids of eL4 of SSS family members. The alignment was performed with Clustal Omega (53).

FIGURE 2.

Impact of the substitution of Glu-311 in eL4 on sodium-coupled proline uptake and binding. A, normalized initial rates of transport (black) and maximum accumulation of proline inside the cells (gray). Transport of 10 μm l-[¹⁴C]proline into E. coli WG170 was assayed in the presence of 50 mm NaCl and 20 mm d-lactate (sodium salt) at 25 °C under aerobic conditions using a rapid filtration method. Results are shown as percentage of wild-type values, and transport activities are normalized to the amount of PutP based on Western blot analysis. Standard deviations were calculated from triplicate determinations of a representative experiment. B, l-[¹⁴C]proline binding to membrane vesicles containing given PutP variants detected by DRaCALA. E. coli WG170 membrane vesicles were incubated with 50 mm NaCl, 20 μm carbonyl cyanide p-chlorophenylhydrazone, and 5 μm monensin at 25 °C for 30 min before adding 1 μm l-[¹⁴C]proline for 10 min. Aliquots were subsequently pipetted onto dry nitrocellulose in triplicates. nc, negative control with membranes of E. coli WG170 transformed with pTrc99a without putP. All DRaCALA spots are from the same assay.

FIGURE 3.

Accessibility of Cys-311 in PutP(ΔC) to sulfhydryl reagents. A, scheme of the experimental procedure. The efficiency of Cys-311 labeling by MTSEA, MTSES, or MMTS was estimated by analyzing the impact of these compounds on the subsequent labeling with [¹⁴C]N-ethylmaleimide ([¹⁴C]NEM). B, results of the labeling experiment. Right-side out membrane vesicles of E. coli WG170 containing given PutP derivatives were preincubated with 500 μm MTSEA, MTSES, or MMTS at 25 °C for 10 min if indicated. Then, labeling with 500 μm [¹⁴C]NEM was performed at 25 °C for 5 min. Reactions were stopped by addition of 2 mm dithiothreitol. After labeling, PutP was purified and subjected to SDS-PAGE. Radioactivity was detected with a PhosphorImager (i), and total amounts of protein were estimated based on Coomassie Blue staining (ii). The radioactivity was normalized to the amount of protein, and relative labeling yields were determined by arbitrary setting the [¹⁴C]NEM labeling yield in the absence of other sulfhydryl compounds to 1 (iii). Standard deviations were calculated from three independent experiments, whereas a representative PhosphorImager-generated autoradiograph and Coomassie-stained bands are shown.

FIGURE 4.

Role of Arg residues near Glu-311. A, zoom into the PutP model shown in Fig. 1 highlighting eL4 and Arg-150 (TM3′, blue), Arg-312 (eL4, orange), and Arg-396 (loop between TMs 9′ and 10′, turquoise) in the vicinity of Glu-311 (eL4, orange). B, impact of the substitution of the Arg residues on sodium-coupled proline uptake. Transport was analyzed as described in the legend of Fig. 2. Normalized initial rates of transport (black) and maximum accumulation of proline inside the cells (gray) are shown as percentage of wild-type values. Transport activities are normalized to the amount of PutP based on Western blot analysis. Standard deviations were calculated from triplicate determinations of a representative experiment.

FIGURE 5.

Interactions of eL4 with TM10′ in transporters with a LeuT-fold. A, detail of the PutP model shown in Fig. 1 highlighting eL4 with Glu-311 and TM10′ with Ala-404. B, detail of the crystal structure of vSGLT (Protein Data Bank 3DH4 (17)) highlighting the interaction between Asp-336 in eL4 and Ala-423 in TM10′. C, detail of the crystal structure of LeuT in an inward-open conformation (Protein Data Bank 3TT3 (13)) highlighting the interaction between Ala-319 in eL4 and Asp-401 in TM10. Distances highlighted by dotted lines are in Å.

FIGURE 6.

Functional significance and accessibility of the region around the end of TM10′. A, relative amounts of PutP with given amino acid replacementsin E. coli WG170 membranes as determined by Western blot analysis. B, normalized initial rates of transport (black) and maximum accumulation of proline in E. coli WG170 (gray). Transport was analyzed as described in the legend of Fig. 2. Transport activities are normalized to the amount of PutP based on Western blot analysis. C, l-proline binding to membrane vesicles containing PutP variants detected by DRaCALA. Binding was analyzed as described in the legend of Fig. 2. All DRaCALA spots are from the same assay. D, accessibility of TM10′ residues to FM. Membrane vesicles of E. coli WG170 transformed with pTrc99a/putP(ΔC) containing a single Cys residue were incubated with 200 μm FM at 25 °C for 10 min. Labeling reactions were stopped by addition of 10 mm β-mercaptoethanol, PutP was purified as described (41), and equal amounts of protein were subjected to SDS-PAGE. (i) fluorescent bands of PutP; (ii) total amounts of PutP based on Coomassie staining; (iii) FM fluorescence normalized to the total amount of PutP. The yield of Cys labeling with the native Glu at position 311 was arbitrarily set to 1 in each experiment. Standard deviations were calculated from three independent experiments, whereas representative fluorescent and Coomassie-stained bands are shown. All bands of an individual experiment come from the same gel.

FIGURE 7.

Impact of the substitution of Ala-404 on PutP function. A, normalized initial rates of transport (black) and maximum accumulation of proline in E. coli WG170 (gray). Transport was analyzed as described in the legend of Fig. 2. Transport activities are normalized to the amount of PutP based on Western blot analysis. B, relative amounts of PutP with given amino acid replacements in E. coli WG170 membranes as determined by Western blot analysis. C, l-proline binding to membrane vesicles containing PutP variants detected by DRaCALA. Binding was analyzed as described in the legend of Fig. 2.

FIGURE 8.

Impact of the E311A and A404E substitutions on the putative inner and outer pathways to the substrate binding sites. A, schematic presentation of the experiment. Cys residues (green circles) were placed into the putative pathways, and modification by FM was analyzed in PutP(ΔC), PutP(ΔC)-E311A, and -A404E containing the given Cys substitutions. B, impact of the substitution of E311A or A404E on the accessibility of Cys-309 and Cys-315 in eL4. C, impact of the substitution of E311A or A404E on the accessibility of Cys-341 and Cys-344 lining the putative inner pathway. Membrane vesicles of E. coli WG170 transformed with pTrc99a/putP(ΔC) containing the given substitutions were incubated with 200 μm FM at 25 °C for 1 min (Cys-309 and Cys-315) or 0.5 min (Cys-341 and Cys-344). Sample preparation for SDS-PAGE was performed as described in the legend of Fig. 6. (i) Fluorescent bands of PutP; (ii) total amounts of PutP based on Coomassie staining; (iii) FM fluorescence normalized to the total amount of PutP. The yield of Cys labeling with the native Glu at position 311 and the native Ala at position 404 was arbitrary set to 1 in each experiment. Standard deviations were calculated from three independent experiments, whereas representative fluorescent and Coomassie-stained bands are shown. All bands of an individual experiment come from the same gel.

FIGURE 9.

DEER analysis of the impact of Glu-311 and Ala-404 substitutions on (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)-methanethiosulfonate (MTSSL)-labeled PutP(ΔC)-R1₂₉₈/R1₃₂₆. A, schematic presentation of the site-directed spin labeling experiment highlighting the sites of labeling. B, DEER analysis of PutP(ΔC)-R1₂₉₈/R1₃₂₆ in detergent solution. C, DEER analysis of PutP(ΔC)-R1₂₉₈/R1₃₂₆ reconstituted into liposomes. Shown are the echo amplitude in the DEER experiment as a function of dipolar evolution time and normalized to the value at zero time (left column) with background fits (dashed black lines), the background-corrected and renormalized echo amplitude (form factor) scaled to the same depth of the dipolar modulation (middle column), and distance distributions computed by Tikhonov regularization with regularization factor of 10 and normalized to the maximum amplitude of probability density P(r) of finding distance r (right column). In the left column, data for E311A and A404E are vertically shifted for better visibility. Data were analyzed with DeerAnalysis (54).