Single residue within the antigen translocation complex TAP controls the epitope repertoire by stabilizing a receptive conformation

Abstract

The recognition of virus infected or malignantly transformed cells by cytotoxic T lymphocytes critically depends on the transporter associated with antigen processing (TAP), which delivers proteasomal degradation products into the endoplasmic reticulum lumen for subsequent loading of major histocompatibility complex class I molecules. Here we have identified a single cysteinyl residue in the TAP complex that modulates peptide binding and translocation, thereby restricting the epitope repertoire. Cysteine 213 in human TAP2 was found to be part of a newly uncovered substrate-binding site crucial for peptide recognition. This residue contacts the peptide in the binding pocket in an orientated manner. The translocation complex can be reversibly inactivated by thiol modification of this cysteinyl residue. As part of an unexpected mechanism, this residue is crucial in complementing the binding pocket for a given subset of epitopes as well as in maintaining a substrate-receptive conformation of the translocation complex.

Fig. 1.

Function of cysteines in the human TAP complex. (A) Wild-type and Cys-less TAP subunits have a different impact on peptide binding and translocation. ATP-dependent transport assays were performed with microsomes (normalized to TAP2 expression) for 3 min at 32 °C using 1 μM of R9L-F (RRYQNSTC^(F)L, black bars) and R10T-A (RYWANATRC^(A)T, gray bars). After lysis, N-core glycosylated peptides were bound to ConA beads, eluted with methyl-D-mannopyranoside, and quantified by fluorescence detection. Transport of R9L-F by wild-type TAP was set to 100%. TAP-containing microsomes (20 μg protein per lane) were analyzed by SDS-PAGE (10%) followed by immunoblotting against TAP1 and TAP2 (mAb148.3 and mAb435.3, respectively). Peptide-binding studies were performed with microsomes (normalized to TAP2 expression) and 1 μM radiolabeled RR(¹²⁵I)YQKSTEL for 20 min on ice and corrected for background binding. Peptide binding of wild-type TAP was set to 100%. All experiments were performed as triplicates. (B) C213 in TAP2 is critical for substrate binding and transport. Transport of R9L-F in the presence (black bar) or absence of MgATP (3 mM, open bar) was carried as described in A. Expression of and peptide binding to TAP mutants were analyzed as described in A. R9L-F binding of wild-type TAP was set to 100%. All experiments were performed as triplicates.

Fig. 2.

Functional importance of the C213 in TAP2. (A) Oxidative cross-linking of CL/CL(C213) in microsomes (0.5 mg of total protein) with radiolabeled peptides containing a single cysteine (1.25 μM, ). Cross-linking was induced by adding 1 mM of copper phenanthroline in the presence or absence of the competitor RRYQKSTEL (0.25 mM). After metal affinity purification of the TAP complex, cross-linked products were analyzed by nonreducing SDS-PAGE (10%) and autoradiography. The TAP expression was confirmed by SDS-PAGE and immunoblotting against TAP1 and TAP2 (mAb148.3 and mAb435.3, respectively). The asterisk indicates a TAP1 degradation product. (B) Modification of C213 blocks peptide binding of TAP. CL/CL(C213) containing microsomes (20 μg of total protein) were incubated with various thiol-specific reagents [0.5 mM NEM; 10 mM MTSES; 1 mM [2-{trimethylammonium)ethyl]methanethiosulfonate; 2.5 mM (2-aminoethyl)methanethiosulfonate; 0.25 mM fluorescein-5-maleimide (F-Mal); 0.25 mM 5-iodoacetamidofluorescein (5-IAF)] for 15 min on ice. Subsequently, peptide-binding assays were performed using radiolabeled RR(¹²⁵I)YQKSTEL (1 μM) as a reporter. (C) Inhibition of peptide binding by MTSES. To determine the half-maximum inhibition value (IC₅₀) of MTSES, CL/CL(C213) containing microsomes were incubated with an increasing concentration of MTSES followed by peptide-binding studies under conditions as described in Fig. 2A. The IC₅₀ was determined to be 52 ± 7 μM (Eq. S2). (D) Reversible TAP inhibition by thiol-specific modification. CL/CL(C213) containing microsomes (20 μg of total protein) were incubated with 10 mM of MTSES for 15 min on ice and subsequently incubated with or without β-ME (100 mM) for 30 min on ice. Peptide binding was assayed with 1 μM of radiolabeled RR(¹²⁵I)YQKSTEL. (E) Efficiency of MTS labeling was determined by means of accessibility for alkylation by 5-IAF. After MTS labeling, samples were incubated with 5-IAF (250 μM) for 15 min on ice and the relative amounts of 5-IAF-modified TAP were determined by in-gel fluorescence. To determine the maximal labeling capacity by 5-IAF (shown as 100%), TAP was denatured by SDS (2%) for 20 min at room temperature and then labeled with 250 μM of 5-IAF for 3 min before SDS-PAGE (10%) and immunoblotting. Asterisks indicate nonspecific labeling.

Fig. 3.

C213 in TAP2 controls the substrate specificity. (A) Peptide length specificity of the Cys-less (▾) and wild-type TAP (▪) complex. Competition assays were performed with TAP-containing microsomes (20 μg of total protein), 1 μM of radiolabeled R(¹²⁵I)YWANATRST (R10T), and increasing concentrations of the peptide library X₁₀. The IC₅₀ of each library was determined by Eq. S2. (B) Peptide length specificity of the Cys-less (black) and wild-type TAP complex (open). The IC₅₀/IC_50,ref for competition of radiolabeled R10T was determined as described before. Competition of wild-type TAP by the X₉ library served as reference (IC_50,ref). (C–F) Cys-less (black) and wild-type TAP (open) display a different binding motif. Competition assays were performed with TAP-containing microsomes (20 μg of total protein) using 1 μM of R(¹²⁵I)YWANATRST as reporter peptide. The concentration of the sublibraries was set to the IC₅₀ value of the X₉ library (10 μM). For comparison, the competition values of human TAP (WT/WT) were taken from Ref. . All experiments were performed in triplicates.

Fig. 4.

Dual-color translocation assays reveal restrictions in a shared TAP1/2 interface. (A) ATP-dependent transport of RRYQNSTC^(F)L (green bars) and RYWANATRC^(A)T (orange bars) by CL/CL and WT/WT were performed as described in Fig. 1A. The competitor peptides RRYKQNSTEL (NST) or EPGYTNSTD (E9D) were applied in equimolar amount. The transport of R9L-F by wild-type TAP was normalized to 100%. A model of the altered binding and translocation modes of wild-type and Cys-less TAP is given below. (B) 3D homology model of the core TAP complex composed of TAP1 (cyan) and TAP2 (green) based on the X-ray structure of Sav1866 (PDB 2HYD) (25, 29). The extra N-terminal domains (TMD0) of each subunit are illustrated schematically. The location of the 10 and 9 cysteines in TAP1 and TAP2, respectively, are indicated in red. (C) Key residues (red) lining the putative substrate-binding pocket and translocation pathway viewed from the ER membrane toward the TMD–NBD interface. The transmembrane helices of core TAP1 and TAP2 are numbered.