Principles of peptide selection by the transporter associated with antigen processing

Abstract

Our ability to fight pathogens relies on major histocompatibility complex class I (MHC-I) molecules presenting diverse antigens on the surface of diseased cells. The transporter associated with antigen processing (TAP) transports nearly the entire repertoire of antigenic peptides into the endoplasmic reticulum for MHC-I loading. How TAP transports peptides specific for MHC-I is unclear. In this study, we used cryo-EM to determine a series of structures of human TAP, both in the absence and presence of peptides with various sequences and lengths. The structures revealed that peptides of eight or nine residues in length bind in a similarly extended conformation, despite having little sequence overlap. We also identified two peptide-anchoring pockets on either side of the transmembrane cavity, each engaging one end of a peptide with primarily main chain atoms. Occupation of both pockets results in a global conformational change in TAP, bringing the two halves of the transporter closer together to prime it for isomerization and ATP hydrolysis. Shorter peptides are able to bind to each pocket separately but are not long enough to bridge the cavity to bind to both simultaneously. Mutations that disrupt hydrogen bonds with the N and C termini of peptides almost abolish MHC-I surface expression. Our findings reveal that TAP functions as a molecular caliper that selects peptides according to length rather than sequence, providing antigen diversity for MHC-I presentation.

Keywords: ABC transporter; MHC-I; adaptive immunity; antigen presentation; transporter associated with antigen processing.

Fig. 1.

Peptide-binding brings the nucleotide-binding domains together. (A) Topology diagram of the TAP complex. TAP1 and TAP2 are colored in cyan and yellow, respectively. (B) ATPase activity as a function of b27 concentration. Data represent measurements from three technical replicates (n = 3) collected at 28 °C using 0.5 µM TAP and 3 mM ATP. (C) ATPase activity in the presence of b27 variants. Data represent means and SEs from three technical replicates of three biological replicates for −ATP, +ATP, and +ATP/b27 conditions (n = 9) and three technical replicates of two biological replicates for termini-modified peptides (n = 6). Concentrations: TAP, 0.5 µM; peptides, 20 µM; ATP, 3 mM. Statistical significance tested by one-way ANOVA. ns, not significant; ****P < 0.0001. (D) Peptides used in this study. (E) cryo-EM densities (Upper) and ribbon representations (Lower) of TAP in the presence and absence of peptide substrates. Densities are sliced perpendicular to the membrane to show the translocation pathway. TAP1, TAP2, and peptides are colored in skyblue, gold, and magenta, respectively. Unassigned densities are colored white. Membrane boundaries are marked by gray lines and the distance between TAP1 D668 and TAP2 E632 are indicated. TAP reconstructions corresponding to apo, b27″, b27′, b27, a2, c4, and b35 are contoured to 0.22, 0.55, 0.20, 0.10, 0.23, 0.20, and 0.14 SDs, respectively.

Fig. 2.

9-mer peptides bind in extended conformations parallel to the membrane. (A) Cryo-EM densities of the TAP translocation pathway with bound a2, b27, and c4. Reconstructions are contoured to the same values as in Fig. 1E. (B) Cross-sectional surface representation of the peptide-binding site viewed from the ER lumen. (C) Peptide-binding residues on TAP1 (Left) and TAP2 (Right). Only transmembrane helices within 4 Å of the peptide are shown. Hydrogen bonds are marked as black dashed lines. (D) Schematic of the residues in TAP involved in peptide binding. Residues that interact with all three 9-mer peptides are indicated as filled ovals and those that interact with only 1 or 2 peptides are indicated as open ovals. (E) Superposition of the three 9-mer peptides and adjacent transmembrane helices (side chains have been omitted for clarity). Insets display conserved interactions at the N and C termini. (F) Schematic showing TAP–peptide interactions that were observed for all three 9-mers. (G) Flow cytometry analysis of MHC-I cell surface levels in TAP KO cells expressing GFP-tagged TAP variants. Data represent median fluorescence intensity of three technical replicates of three biological replicates (n = 9). Statistical significance tested by one-way ANOVA. ns, not significant; ****P < 0.0001.

Fig. 3.

The structure and electrostatics of each binding pocket confer sequence preferences. Electrostatic surface representations of the peptide-binding site. Center, binding of the entire b27 peptide. Left, insertion of the first residue of b27 (Top), a2 (Middle), and c4 (Bottom) into the N-pocket. Right, binding of the last residue of each peptide to the C-pocket.

Fig. 4.

At least eight residues are required to engage N- and C-pockets concurrently. (A) The 8-mer (b27′) but not the 7-mer [b27″ and b27″(Q)] peptides stimulated ATP hydrolysis. Data represent means and SEs from three technical replicates of three biological replicates (n = 9). Statistical significance tested by one-way ANOVA. ns, not significant; ****P < 0.0001. (B and C) EM density for bound 8-mer b27′ (B) and 7-mer b27″ (C) peptides, shown as transparent magenta surfaces. Density corresponding to b27′ and b27″ are contoured to 0.11 and 0.45 SDs, respectively. (D) Overlay of peptide backbones of the 9-mer (gray) and two partially built 7-mer peptides (colored).

Fig. 5.

TAP binds longer peptides by allowing flexibility in their central residues. (A) Densities corresponding to the 14-mer b35 peptide are shown as transparent magenta surfaces and contoured to 0.14 SDs. (B) b35 makes fewer interactions with the N-pocket than the 9-mer b27. The side chains for each peptide have been removed for clarity. (C) b35 makes an additional hydrogen bond with the C-pocket compared to b27. Only the last two residues of each peptide are shown. View is from the same perspective as Fig. 3 (Right).