Engineering the glutamate transporter homologue GltPh using protein semisynthesis

Abstract

Glutamate transporters catalyze the concentrative uptake of glutamate from synapses and are essential for normal synaptic function. Despite extensive investigations of glutamate transporters, the mechanisms underlying substrate recognition, ion selectivity, and the coupling of substrate and ion transport are not well-understood. Deciphering these mechanisms requires the ability to precisely engineer the transporter. In this study, we describe the semisynthesis of GltPh, an archaeal homologue of glutamate transporters. Semisynthesis allows the precise engineering of GltPh through the incorporation of unnatural amino acids and peptide backbone modifications. In the semisynthesis, the GltPh polypeptide is initially assembled from a recombinantly expressed thioester peptide and a chemically synthesized peptide using the native chemical ligation reaction followed by in vitro folding to the native state. We have developed a robust procedure for the in vitro folding of GltPh. Biochemical characterization of the semisynthetic GltPh indicates that it is similar to the native transporter. We used semisynthesis to substitute Arg397, a highly conserved residue in the substrate binding site, with the unnatural analogue, citrulline. Our studies demonstrate that Arg397 is required for high-affinity substrate binding, and on the basis of our results, we propose that Arg397 is involved in a Na+-dependent remodeling of the substrate binding site required for high-affinity Asp binding. We anticipate that the semisynthetic approach developed in this study will be extremely useful in investigating functional mechanisms in GltPh. Further, the approach developed in this study should also be applicable to other membrane transport proteins.

Figure 1. Structure of Glt_Ph and semisynthesis using native chemical ligation

A) Top view of the trimeric Glt_Ph transporter (pdb: 2nwx) shows the central trimerization domain (colored wheat) and the peripheral transport domain (colored green and purple). B) Topology and structure of a single subunit of Glt_Ph. The regions of the subunit that contribute to the trimerization and the transport domain are colored as in panel A. Asp (space fill) and Na⁺ ions (yellow spheres) bound to the transport domain are shown. C) Close-up view of the Asp and Na⁺ binding sites. Asp is shown in stick representation and Na⁺ ions are shown as yellow spheres. Interactions between the bound ligands and Glt_Ph are indicated by dashed lines. D) Semisynthesis of Glt_Ph using native chemical ligation. The Glt_Ph polypeptide is assembled by the ligation reaction of a recombinantly expressed thioester peptide (N-peptide: residues 1-384, blue) and a synthetic peptide with an N-terminal Cys (C-peptide: residues 385-418, red). The ligation product is folded in vitro to the native state. The Cys residue at the ligation site, M385C is represented as a yellow sphere.

Figure 2. In vitro folding of Glt_Ph

A) SDS PAGE gel showing the native (N) and unfolded (U) Glt_Ph with (+) and without (−) glutaraldehyde cross-linking. Native Glt_Ph cross-links to a trimer while the unfolded protein is monomeric. B) Size exclusion chromatography showing a similar elution profile for the native (black) and the refolded Glt_Ph (red). Inset shows glutaraldehyde cross-linking of the peak fraction for native and refolded Glt_Ph before (−) and after (+) treatment with 1 % SDS. In panels A and B, the oligomeric nature of the cross-linked band (1X, 2X, 3X) is indicated. C) Asp uptake by the refolded Glt_Ph. Time course of ¹⁴C-Asp uptake into vesicles containing the refolded Glt_Ph in the presence of a Na⁺ gradient (circles, n= 3). ¹⁴C-Asp uptake is not observed in the absence of a Na⁺ gradient (100 mM K⁺ on both sides of the membrane, triangles). D) Asp binding by native and refolded Glt_Ph. The fraction of the protein bound (F_bound) was determined by dividing the fluorescence change upon addition of Asp to the total change at the end of the titration. Solid lines are fits to the data using the equation described in methods with a K_d value of 97 ± 4.8 μM (n = 7) for the native (black) and 154 ± 19 μM (n = 6) for refolded (red) Glt_Ph. The binding assays were carried out in 1 mM Na⁺. Error bars correspond to SEM.

Figure 3. Semisynthesis of Glt_Ph

A) Strategy for the semisynthesis of Glt_Ph. Glt_Ph residues 1-384 are sandwiched between glutathione-S-transferase (GST) and the gyrA intein (I). A thrombin cleavage site and His₆ tag are present between GST and the Glt_Ph sequence. Proteolysis with thrombin releases the Glt_Ph-intein fusion (IF), which is purified using the His₆ tag and cleaved with MESNA to provide the N-peptide thioester (N). The N-peptide thioester is ligated to a synthetic C-peptide (residues 385- 418) with an N-terminal Cys (yellow sphere) and a C-terminal Strep tag. The ligation reaction yields the full length Glt_Ph polypeptide (L), which is purified using the Strep tag and then folded in vitro to the native state. B) SDS-PAGE gel detailing the assembly and the purification of the semisynthetic Glt_Ph polypeptide. Lane 1: Treatment of the Glt_Ph-intein fusion (IF) with MESNA cleaves the N-peptide thioester (N) from the intein (I); Lane 2: NCL of the N-peptide thioester with the synthetic C-peptide yields the semisynthetic Glt_Ph polypeptide (L); Lane 3: The semisynthetic Glt_Ph polypeptide following purification using the C-terminal Strep tag. C) Size exclusion chromatography of semsiysnthetic Glt_Ph. Inset: SDS-PAGE gel of the semisynthetic Glt_Ph with (+) and without (−) glutaraldehdye cross-linking. D) Asp uptake by the semisynthetic Glt_Ph. Time course of ¹⁴C-Asp uptake into vesicles containing the semisynthetic Glt_Ph in the presence (open circles, n = 3) and in the absence of a Na⁺ gradient (filled circles, n = 3). E) Asp binding by semisynthetic Glt_Ph. Asp binding assays as described in Fig. 2D for the semisynthetic (red circles, K_d = 229 ± 28 μM, n=3) and the native control, M385C-Glt_Ph (black squares, K_d = 210 ± 21 μM, n=3).

Figure 4. Arg397 in the substrate binding site of Glt_Ph

A) Close up view of the Asp binding site highlighting the interaction of Arg397 with the β-carboxyl group of the bound Asp. B) Structures of the side chains of Arg and the unnatural amino acid Citrulline (Cit). C) Time course for ¹⁴C-Asp uptake into vesicles containing the R397Cit Glt_Ph in the presence 0.1 μM ¹⁴C-Asp (open diamonds, n = 2) and 1.0 μM ¹⁴C-Asp (filled diamonds, n = 3). For comparison, the time course for ¹⁴C-Asp uptake into vesicles containing the semisynthetic WT Glt_Ph in the presence of 0.1 μM ¹⁴C-Asp (open circles) is also shown (data from Fig. 3D). For clarity, data presented has been corrected for background uptake determined in the absence of a Na⁺ gradient (100 mM K⁺ on both sides of the membrane). D and E) Asp binding to R397Cit (D) and the WT (E) at 1 mM (filled symbols) and 100 mM Na⁺ (open symbols) carried out as described in Fig. 2D. A shift in Na⁺ from 1 mM to 100 mM Na⁺ decreases the K_d for Asp binding to the native control (M385C-Glt_Ph) from 210 ± 21 μM (n= 3) to 3.01 ± 0.6 nM (n= 4) while only a modest decrease [961 ± 102 μM (n = 3) to 204 ± 15 μM (n =4)] is observed for the R397Cit-Glt_Ph. F) Logarithmic plots of Asp K_d (μM) values against log [Na⁺] (mM) are shown for the native control (black squares) and R397Cit Glt_Ph (blue diamonds).

Figure 5. Ion-induced conformational change in substrate binding site of Glt_Ph

A and B) Crystal structures of the inward-facing conformation of Glt_Ph in the apo-state (A, pdb: 4p19) and in the presence of Tl⁺ (B, pdb: 4p6h). Close-up view of the substrate binding site showing HP1 and TM7 (colored grey) and TM8 (blue). Arg397 is shown in stick representation. The cation-binding sites, Na1 and Na2 are represented as yellow spheres. C) Model depicting a proposed role for Arg397. Binding of a Na⁺ ion causes a conformational change in the substrate binding site that reorients Arg397 to create a high affinity Asp binding site. Binding of Asp is followed by the binding of another Na⁺ ion that is concomitant with the closure of HP2 leading to transport. Only the sodium sites visualized in the Glt_Ph crystal structures are shown in the model.