Nucleotide-dependent conformational changes direct peptide export by the transporter associated with antigen processing

Abstract

The transporter associated with antigen processing (TAP) delivers peptide antigens from the cytoplasm into the endoplasmic reticulum (ER) for loading onto major histocompatibility complex class I (MHC-I) molecules. To examine the mechanisms of peptide transport and release into the ER, we determined cryo-electron microscopy structures of the human TAP heterodimer in multiple functional states along the transport cycle. In the inward-facing conformation, when the peptide translocation cavity within the TAP heterodimer is exposed to the cytosol, ATP binding strengthened intradomain assembly. Transition to the outward-facing conformation, when the transporter opens to the ER lumen, led to a complete reconfiguration of the peptide-binding site, facilitating peptide release. ATP hydrolysis opened the catalytically active nucleotide-binding consensus site, and the subsequent separation of the nucleotide-binding domains reset the transport cycle. These findings establish a comprehensive structural framework for understanding unilateral peptide transport, vanadate trapping, and trans-inhibition-an internal feedback mechanism that prevents excessive peptide accumulation and activation of the ER stress response.

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

Figure 1.. ATP binding to wild-type TAP stabilizes nucleotide-binding domain 1

(A) Schematic of the TAP transport cycle. ATP binding stabilizes the inward-facing state. (B) The coupling helices (CHs) connect the soluble nucleotide-binding domains (NBDs) to the transmembrane domains (TMDs). TAP is represented as a white ribbon. The TAP CHs are represented as colored tubes. The boundaries of the membrane are shown as silver lines. (C) Cryo-EM density of TAP bound to a 9-mer peptide has a flexible NBD1 (EMDB: EMD-41029). Densities corresponding to the TAP TMDs, NBD1, and NBD2 are colored as silver, sky blue, and gold, respectively, and contoured to 0.125 standard deviations (SDs). (D) Cryo-EM density of TAP bound to a 9-mer peptide and ATP has a rigid NBD1. Density is contoured to 0.8 SDs. (E) TAP1 CH1 and TAP2 CH3 interact with NBD1 near the ATP-binding site at the interface between TMD and NBD1. TAP is shown as cartoon tubes, and ATP is shown as sticks. (F and G) Zoomed-in view of the TMD/NBD1 interface as boxed in (E) in the absence (PDB: 8T4F) (F) or presence (G) of ATP. The dotted line represents the unresolved CH1. (H) ATP binding is associated with a rotation of NBD1 that brings the two NBDs to face each other. Superposition of TAP in the absence (white) and presence (colored) of ATP. The arrow indicates the movement of NBD1 upon ATP binding. See also Figure S1.

Figure 2.. ATP binding to ATP hydrolysis-deficient TAP(EQ) adopts the outward-facing state

(A) NBD dimerization stabilizes the outward-facing state. (B) Molecular model of TAP(EQ) in the NBD-dimerized outward-facing ATP-bound state. TAP1 and TAP2 are shown as ribbons and colored in sky blue and gold, respectively. ATP is shown as spheres. The boundaries of the membrane are shown as silver lines. (C) Molecular surface models of TAP(EQ) in the NBD-dimerized outward-facing ATP-bound state. The closed cytosolic and open ER luminal gates are boxed. (D) The translocation pathway is open to the ER lumen. (E and F) TAP2 TM3 is flexible and adopts two conformations in the outward-facing state: an outward-facing open (E) and kinked (F) state. The molecular models of TAP1 and TM3 are represented as cartoons with the side chains represented as sticks. Cryo-EM densities corresponding to TAP1 and TAP2 TM3 are colored in transparent sky blue and gold, respectively, and contoured to 0.12 and 0.196 SDs, respectively. The N- and C-pockets of the peptide-binding sites are labeled. See also Figures S2–S4.

Figure 3.. Conformational changes upon NBD dimerization in outward-facing TAP(EQ) enable substrate release

(A) Global conformational changes upon NBD dimerization as viewed from the cytoplasm. Superposition of NBD1 in the NBD-separated (white) and NBD-dimerized (colored) conformations. The arrow indicates the movement of NBD2. (B) Local conformational changes in the NBDs upon NBD dimerization. Superposition of NBD1 and NBD2 individually in the NBD-separated (white) and NBD-dimerized (colored) conformations. The D-loop is highlighted, and the arrow indicates movement of the D-loop to interact with ATP in the degenerate site. (C) Zoom-in view of the degenerate nucleotide-binding site in NBD1. Hydrogen bonds are represented as dashed lines. (D and E) The TAP translocation pathway in the 9-mer peptide-bound inward-facing (D) or outward-facing (E) conformation as viewed from the ER lumen. The helices of TAP are numbered and shown as colored cartoon tubes. The main-chain backbone of the peptide substrate is shown as silver sticks. The N-pocket and C-pocket are boxed as indicated, and residues that comprise each pocket are shown as sticks. (F) Superposition of the TAP translocation pathway in the inward-facing peptide-bound (silver) and outward-facing (colored) conformations. Arrows indicate movement of the TAP helices. (G and H) Conformation of TAP TM3 in the 9-mer peptide-bound inward-facing (G) or outward-facing (H) conformation as viewed from the membrane. (I) Superposition of TAP TM3 in the inward-facing peptide-bound (silver) and outward-facing (colored) conformations. (J and K) Zoom-in of the TAP N- (J) and C- (K) pockets in the 9-mer peptide-bound inward-facing (left) or outward-facing (right) conformation. Hydrogen bonds are shown as dotted lines. The alpha-carbons of the peptide substrate are numbered. The peptide from the inward-facing state is superimposed upon the outward-facing state and is represented as transparent sticks (right). Arrows indicate movement of the TAP TM3. See also Figures S2–S4.

Figure 4.. WT TAP incubated with ATP at 37° C captures a post-hydrolytic outward-facing state with asymmetrically separated NBDs

(A) The post-hydrolytic state can adopt an outward-facing state. (B) Molecular model of WT TAP in the NBD-dimerized outward-facing post-hydrolytic state. The degenerate and consensus ATP-binding sites are labeled. (C) ATP is bound in the NBD1 degenerate site, and ADP is bound in the NBD2 consensus site. ATP is shown as sticks, and the magnesium atom is shown as spheres. Density corresponding to bound nucleotide is shown as a gray surface and contoured to 0.22 SDs. (D) Superposition of NBD1 before (white) and after (colored) ATP hydrolysis as viewed from the cytoplasm. Arrows indicate conformational changes in NBD2 after ATP hydrolysis. (E) Zoom-in view of the NBD2 consensus site as boxed in (D). Arrows indicate local conformational changes in the D-loop of NBD1 and the H-loop of NBD2 after ATP hydrolysis. See also Figure S5.

Figure 5.. WT TAP incubated with ATP at 37° C also adopts the NBD-separated post-hydrolytic conformation

(A) NBD separation resets the transport cycle. (B) Molecular model of WT TAP in the NBD-separated outward-facing post-hydrolytic state. Bound nucleotide is shown as sticks, and the magnesium atom is shown as spheres. The degenerate and consensus ATP-binding sites are labeled. (C) ATP is bound in the NBD1 degenerate site (top), while ADP is bound in the NBD2 consensus site (bottom). Density corresponding to bound nucleotide is shown as a gray surface and contoured to 0.33 SDs. See also Figure S5.