Structure of the ABC ATPase domain of human TAP1, the transporter associated with antigen processing

Abstract

The transporter associated with antigen processing (TAP) is an ABC transporter formed of two subunits, TAP1 and TAP2, each of which has an N-terminal membrane-spanning domain and a C-terminal ABC ATPase domain. We report the structure of the C-terminal ABC ATPase domain of TAP1 (cTAP1) bound to ADP. cTAP1 forms an L-shaped molecule with two domains, a RecA-like domain and a small alpha-helical domain. The diphosphate group of ADP interacts with the P-loop as expected. Residues thought to be involved in gamma-phosphate binding and hydrolysis show flexibility in the ADP-bound state as evidenced by their high B-factors. Comparisons of cTAP1 with other ABC ATPases from the ABC transporter family as well as ABC ATPases involved in DNA maintenance and repair reveal key regions and residues specific to each family. Three ATPase subfamilies are identified which have distinct adenosine recognition motifs, as well as distinct subdomains that may be specific to the different functions of each subfamily. Differences between TAP1 and TAP2 in the nucleotide-binding site may be related to the observed asymmetry during peptide transport.

Fig. 1. Structure of cTAP1. (A) Topology diagram of cTAP1 color-coded as in (B). (B) Ribbon diagrams of cTAP1, highlighting the helical domain in blue, the RecA-like domain in red and the conserved sequence motifs in yellow. The top shows a ‘side’ view of cTAP1, whereas the bottom shows cTAP1 viewed from the bottom (90° rotation from the top view). The β-strands and α-helices are labeled according to the HisP topology. The ADP (green) and Mg²⁺ ion (cyan) are shown as ball-and-stick models.

Fig. 2. Domain movements. (A) The two MalK monomers forming a dimer in the asymmetric unit (Diederichs et al., 2000) are superimposed using their RecA-like domain to show domain motion of the helical domain. cTAP1 is similarly superimposed onto the MalK A monomer (B) and the MalK B monomer (C) revealing that cTAP1 more closely resembles the MalK B monomer. The MalK A monomer is cyan (RecA-like domain, 1–88 and 158–244) and blue (helical domain, 89–157), the MalK B monomer is light pink (RecA-like domain, 1–88 and 158–244) and magenta (helical domain, 89–157), and cTAP1 is yellow (RecA-like domain, 492–586 and 661–742) and orange (helical domain, 587–660). The ions and nucleotides (ADP–Mg²⁺ for cTAP1, pyrophosphate for MalK A, and pyrophosphate–Mg²⁺ for MalK B) are represented in ball-and-stick in the same color as the corresponding helical domain.

Fig. 3. Structure-based sequence alignment of all ABC ATPases for which structures have been published: human cTAP1, Salmonella typhimurium HisP crystallized with ATPγS (1b0u; Hung et al., 1998), Thermococcus litoralis MalK showing pyrophosphate in the active site (1g29; Diederichs et al., 2000), Pyrococcus furiosus RAD50 bound to ATP (1f2t and 1f2up; Hopfner et al., 2000), Thermotoga maritima SMC with no nucleotide bound (1e69; Lowe et al., 2001), E.coli MutS with ADP (1e3m; Lamers et al., 2000) and T.aquaticus MutS with ADP (1ewq and 1ewr; Obmolova et al., 2000). The sequence of human TAP2 was also aligned, based on homology to human TAP1. β-strands and α-helices are boxed green and blue, respectively, and numbered according to the cTAP1 terminology (derived from HisP) for simplicity. Nucleotide-interacting residues are highlighted (adenosine interactions in yellow, phosphate interactions in pink, adenosine interactions from the opposing monomer in the dimer in orange, and cross-dimer phosphate interactions in cyan). Subfamily-specific sequences are colored as in Figure 4. Conserved sequence motifs are labeled with side-to-side arrows: WA (Walker A), Q (Q-loop), C (signature or C motif), WB (Walker B), D (D-loop) and switch (switch region). Down-arrows indicate the beginning and end of the cTAP1 structure. The lines below the alignment identify regions that are structurally overlapping in all structures. The small dots above the alignment indicate increments of 10 in the TAP1 sequence. ‘cc’ in the RAD50 and SMC sequences (and * above it the alignment) shows the location of the coiled-coil domain inserted in RAD50 (150–734) and SMC (147–1022).

Fig. 4. (A) Ribbon diagram of cTAP1 highlighting in red the regions specific to the transporter subfamily of ABC ATPases, conserved in cTAP1, HisP (1b0u; Hung et al., 1998) and MalK (1g29; Diederichs et al., 2000). (B) Ribbon diagram of RAD50 (1f2u; Hopfner et al., 2000) highlighting in blue the regions structurally specific to the RAD50/SMC type ABC ATPase DNA repair proteins. (C) Ribbon diagram of E.coli MutS (1e3m; Lamers et al., 2000) with the region specific to the MSH subfamily of ABC ATPases colored green.

Fig. 5. The nucleotide-binding site. (A) Stereo representation of the σA-weighted 2F_o–F_c electron density map contoured at 1.2σ in the vicinity of the nucleotide-binding site showing clear density corresponding to ADP. Residues contacting the ADP, as well as other conserved residues in the vicinity of the active site are labeled. Hydrogen-bonding and salt-bridge interactions listed in Table II are marked by red dotted lines. (B) Stereo view of the three different adenosine conformations seen in the ABC ATPase structures. For reference, the cTAP1 ADP is shown in an orientation similar to that in (A). The cTAP1 ADP is in green, the RAD50 ATP in blue and the MutS ADP in pink.