Title: Three-dimensional Structure of Transporter Associated with Antigen Processing (TAP) Obtained by Single Particle Image Analysis
Abstract: The transporter associated with antigen processing (TAP) is an ATP binding cassette transporter responsible for peptide translocation into the lumen of the endoplasmic reticulum for assembly with major histocompatibility complex class I molecules. Immunoaffinity-purified TAP particles comprising TAP1 and TAP2 polypeptides, and TAP2 particles alone were characterized after detergent solubilization and studied by electron microscopy. Projection structures of TAP1+2 particles reveal a molecule ∼10 nm across with a deeply staining central region, whereas TAP2 molecules are smaller in projection. A three-dimensional structure of TAP reveals it is isolated as a single heterodimeric complex, with the TAP1 and TAP2 subunits combining to create a central 3-nm-diameter pocket on the predicted endoplasmic reticulum-lumenal side. Its structural similarity to other ABC transporters demonstrates a common tertiary structure for this diverse family of membrane proteins. The transporter associated with antigen processing (TAP) is an ATP binding cassette transporter responsible for peptide translocation into the lumen of the endoplasmic reticulum for assembly with major histocompatibility complex class I molecules. Immunoaffinity-purified TAP particles comprising TAP1 and TAP2 polypeptides, and TAP2 particles alone were characterized after detergent solubilization and studied by electron microscopy. Projection structures of TAP1+2 particles reveal a molecule ∼10 nm across with a deeply staining central region, whereas TAP2 molecules are smaller in projection. A three-dimensional structure of TAP reveals it is isolated as a single heterodimeric complex, with the TAP1 and TAP2 subunits combining to create a central 3-nm-diameter pocket on the predicted endoplasmic reticulum-lumenal side. Its structural similarity to other ABC transporters demonstrates a common tertiary structure for this diverse family of membrane proteins. ATP binding cassette contrast transfer function endoplasmic reticulum major histocompatibility complex nucleotide binding domain transporter associated with antigen presentation transmembrane domain azidoadenosine 5′-triphosphate polyacrylamide gel electrophoresis ethylene glycol bis(succininmidylsuccinate) hydroxysulfosuccinimidyl-4-azidobenzoate ATP binding cassette (ABC)1 transporters are ubiquitous and can form a significant component of an organism's genome, for example 2% in Escherichia coli (1Blattner F.R. Plunkett III, G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (5987) Google Scholar). They are defined by the ABC domain, a nucleotide hydrolysis domain whose activity powers substrate transport (2Higgins C.F. Annu. Rev. 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In this report we have studied the structure of TAP by electron microscopy of detergent-solubilized immunopurified particles. Single particle analysis was used to compare TAP from cells expressing the TAP1-TAP2 heterodimer with particles from cells expressing TAP2 alone. Three-dimensional reconstruction of the TAP heterodimer was also carried out that allowed direct comparison with the structure of P-glycoprotein. The resulting assignment of features in the molecule to previously characterized domains provides the first direct structural evidence supporting models of TAP that have until now been predominantly predictive in nature. T2, T2-TAP2 (expressing rat TAP2 only), T2-TAP1 (expressing rat TAP1 only), and T2 TAP1+2 (expressing rat TAP1 and TAP2) cells (39Momburg F. Ortiz-Navarrete V. Neefjes J. Goulmy E. van de Wal Y. Spits H. Powis S.J. Butcher G.W. Howard J.C. Walden P. Hammerling G.J. Nature. 1992; 360: 174-177Crossref PubMed Scopus (232) Google Scholar, 40Powis S.J. Eur. J. Immunol. 1997; 27: 2744-2747Crossref PubMed Scopus (33) Google Scholar) were maintained in RPMI 1640 medium with 5% fetal calf serum and 1.0 mg/ml G418 (Life Technologies, Inc.). Cells were washed into PFN medium (phosphate-buffered saline, 2% fetal calf serum, 0.1% azide) and incubated with monoclonal antibody MEI (anti-HLA B) tissue culture supernatant (a gift from J. Taurog, University of Texas) followed by fluorescein isothiocyanate-conjugated anti-mouse IgG (Sigma). Analysis was performed on a Becton Dickinson Facsort using CellQuest software. Immunoisolation utilized sheep anti-TAP2 antisera raised against the C-terminal 14 residues of rat TAP2 (KVYAHLVQQRLEA) (40Powis S.J. Eur. J. Immunol. 1997; 27: 2744-2747Crossref PubMed Scopus (33) Google Scholar). 2 × 108 cells were lysed in detergent buffer (1% Triton X-100, 150 mm NaCl, 10 mm Tris, pH 7.6, 5 mm MgCl2, 1 mm phenymethylsulfonyl fluoride). Immunoaffinity beads were prepared by equilibrating 5 μg of purified anti-TAP2 antisera with 50 μl of protein G-Sepharose. Pre-cleared lysates (50 μl of protein G-Sepharose, 1 h) were incubated sequentially in 1.5-ml aliquots with the same immunoaffinity beads for 30 min each. Beads were washed extensively before eluting with 50 μm above C-terminal TAP2 peptide. Eluted samples were cleared of any residual immunoglobulin by two further clearing steps with protein G-Sepharose with 20,000 × g-centrifuge spins in between. Samples were adjusted to 20% glycerol as cryoprotectant and stored at −80 °C. Western blotting of detergent-solubilized cells was carried out as previously described (40Powis S.J. Eur. J. Immunol. 1997; 27: 2744-2747Crossref PubMed Scopus (33) Google Scholar). Antibody immobilized using sheep anti-TAP1 or -TAP2 antisera (40Powis S.J. Eur. J. Immunol. 1997; 27: 2744-2747Crossref PubMed Scopus (33) Google Scholar) or peptide-eluted TAP polypeptides was incubated in a volume of 30 μl of lysis buffer with 1 μCi of 8-azidoadenosine 5′- [α-32P]triphosphate (Affinity Labeling Technology, Lexington, KY) for 15 min on ice, then exposed to UV irradiation at 365 nm for 10 min on ice. In some experiments unlabeled ATP was added at a concentration of 1 mm. For antibody-immobilized TAP polypeptides, free 8N3ATP was removed by washes in lysis buffer. Particles in solution were diluted in 500 μl of lysis buffer and re-immunoprecipitated with monoclonal antibody MAC394 (41Knittler M.R. Gulow K. Seelig A. Howard J.C. J. Immunol. 1998; 161: 5967-5977PubMed Google Scholar), recognizing rat TAP2 (at an epitope different from the C-terminal sheep sera), which was a gift from M. Knittler and J. Howard (University of Cologne). For SDS denaturation, samples were adjusted to contain 1% w/v SDS, heated to 80 °C for 5 min, cooled on ice, diluted as above in lysis buffer, and immunoprecipitated with sheep anti-rat TAP1 antisera or monoclonal antibody MAC394. Samples were heated in reducing sample buffer and analyzed by 6% SDS-PAGE. Ethylene glycol bis(succininmidylsuccinate) (EGS, Sigma) labeling was performed on cell lysates prepared at a concentration of 20 × 108 in phosphate-buffered saline containing 1% Triton X-100 and 1 mm phenylmethylsulfonyl fluoride. Peptides were added to a final concentration of 20 μm for 30 min on ice followed by EGS at a concentration of 1 mm for a further 30 min. Samples were heated in reducing sample buffer and analyzed by 6% SDS-PAGE and Western blotting with rabbit anti-TAP2 sera 116 (41Knittler M.R. Gulow K. Seelig A. Howard J.C. J. Immunol. 1998; 161: 5967-5977PubMed Google Scholar). Reporter peptide TNKTVARYV was coupled to hydroxysulfosuccinimidyl-4-azidobenzoate (HSAB; Pierce) in phosphate-buffered saline for 1 h at room temperature and iodinated with iodogen. Coupling of the peptide to HSAB was confirmed by mass spectrometry. Immunoisolated or peptide-eluted TAP particles were incubated with ∼5 μm reporter peptide in the presence or absence of 10 μm inhibitor peptides for 10 min on ice, and cross-linking was induced at 365 nm as described above. Immunoimmobilized TAP polypeptides were washed in lysis buffer. Eluted TAP particles were re-immunoprecipitated with MAC394 and analyzed by SDS-PAGE as described above. Densitometry was performed using NIH Image software. Samples were added to freshly glow-discharged (rendered hydrophilic) copper/carbon mesh grids for 30 s, and the sides were blotted with Whatman grade 50 filter paper and negatively stained in 4% uranyl acetate. Micrographs of the grids were taken with a Philips CM10 (University of Leeds) and a Philips Technai 10 (UMIST) electron microscope operating at 100 kV. The micrographs were scanned using a Leaf Microdensitometer (University of Leeds) and a Zeiss scanner (University of Sheffield). Image files were initially contrast transfer function (CTF)-corrected using the CRISP image processing package (Calidris Software, Stockholm). Particles were then selected using SPIDER (42Frank J. Radermacher M. Penczek P. Zhu J. Li Y. Ladjadj M. Leith A. J. Struct. Biol. 1996; 116: 190-199Crossref PubMed Scopus (1804) Google Scholar), and the densities were normalized between several micrographs. Reference-free alignment was carried out, and the results were statistically analyzed. Particles were sorted into groups for averaging using hierarchical ascendant classification with complete linkage. For each average, Fourier ring correlation between two sub-averages was used to assess its resolution. A cut-off point at which the Fourier ring correlation values dropped below 0.5 was chosen and was determined by fitting the data with a four-parameter Boltzman function. A threshold for each class that gave the best averages in terms of resolution was thus determined. The random conical reconstruction methods we used were part of a suite of procedures written for SPIDER (www.wadsworth.org/spider_doc/spider/docs/spider.html), adapted to include CTF correction in SPIDER. The change in the CTF across the micrographs of the tilted specimen was determined using the program ctfind2.com (43Crowther R.A. Henderson R. Smith J.M. J. Struct. Biol. 1996; 116: 9-16Crossref PubMed Scopus (665) Google Scholar). Particles from untilted micrographs were selected with their equivalent tilted partners (40°) from the underfocused side of the tilted one. An individual CTF correction was applied to each of the tilted particles selected based on its positional coordinates. CTF correction was not needed to align the untilted data set, but a Fermi low pass filter was used to improve the fidelity of the alignment. Reference-free alignment was carried out on the untilted data set (1013 particles), and hierarchical clustering was used to sort these into the major groups. Three classes (215, 215, and 251 particles) were chosen as before for reconstruction of volumes by back projection from the tilted particles (44Radermacher M. J. Electron Microsc. Technol. 1988; 9: 359-394Crossref PubMed Scopus (416) Google Scholar). Each volume was put through an angular refinement step (see Pawel's random conical reconstruction methods on the above-cited SPIDER web site) by generating projections from the initial three-dimensional structure and translationally realigning the original tilted particles to these newly generated projections. The realigned particles were then used to recreate a new volume. This refinement step was carried out six times, and the final volume was used for merging the data. The refined volumes from the three separate groups were then compared and rotationally aligned using a three-dimensional correlation search before combining to give a merged reconstruction. This reconstruction was also subject to a similar refinement procedure. This volume represented 67% (683 particles) of the initial data set (1013 particles). Resolution was assessed by splitting its tilted projection data set into two, constructing two “sub-volumes,” and calculating the Fourier shell correlation between them using a cut-off of 2/N 1/2 (where N is the number of pixels in each Fourier shell). We utilized the TAP-deficient T2 human lymphoblastoid cell line, which had been transfected with both rat TAP1 and TAP2 or with rat TAP2 alone. Peptide specificity and transport activity, and MHC class I-chaperone interactions with TAP polypeptides have been well documented in these transfectant lines (39Momburg F. Ortiz-Navarrete V. Neefjes J. Goulmy E. van de Wal Y. Spits H. Powis S.J. Butcher G.W. Howard J.C. Walden P. Hammerling G.J. Nature. 1992; 360: 174-177Crossref PubMed Scopus (232) Google Scholar, 40Powis S.J. Eur. J. Immunol. 1997; 27: 2744-2747Crossref PubMed Scopus (33) Google Scholar, 45Momburg F. Roelse J. Hammerling G.J. Neefjes J.J. J. Exp. Med. 1994; 179: 1613-1623Crossref PubMed Scopus (186) Google Scholar, 46Momburg F. Roelse J. Howard J.C. Butcher G.W. Hammerling G.J. Neefjes J.J. Nature. 1994; 367: 648-651Crossref PubMed Scopus (304) Google Scholar, 47Neefjes J. Gottfried E. Roelse J. Gromme M. Obst R. Hammerling G.J. Momburg F. Eur. J. Immunol. 1995; 25: 1133-1136Crossref PubMed Scopus (87) Google Scholar). Western blot analysis of detergent-solubilized cell lysates confirmed the expression of both TAP polypeptides in the T2-TAP1+2 transfectant and of TAP2 only in T2-TAP2 cells (Fig. 1 b). Flow cytometric analysis of the cell surface levels of MHC class I confirmed no increase in MHC class I on T2-TAP2 cells compared with T2 cells, whereas T2-TAP1+2 displayed at least 10-fold more MHC class I (Fig.1 a). This confirmed that before immunoprecipitation, the TAP heterodimer is functional in peptide transport, whereas TAP2 on its own is not. We chose to purify TAP by a rapid immunoaffinity-based methodology to avoid lengthy chromatography-based isolation, thus limiting the time-dependent inactivation of TAP after detergent solubilization (48Uebel S. Plantinga T. Weber P.J. Beck-Sickinger A.G. Tampe R. FEBS Lett. 1997; 416: 359-363Crossref PubMed Scopus (11) Google Scholar). Triton X-100 was chosen to isolate the core subunits of TAP1 and -2 in the absence of associated class I MHC-chaperone complexes. Immunopurification was performed utilizing anti-rat TAP2 antisera immobilized on protein G-Sepharose. After extensive washing of the immune complexes, TAP was specifically eluted by the addition of the peptide, against which the antisera was generated. The purity of the TAP preparations was determined by analyzing a portion of the eluate by SDS-PAGE and silver staining (Fig.1 c). Similar attempts to isolate TAP1 using the relevant peptide resulted in very inefficient recovery of TAP1 and was not pursued further in this study. On average purifications using TAP2 reagents yielded between 50 and 200 ng of TAP polypeptides from ∼2 × 108 cells. T2-TAP2 cells produced a predominantly single species of approximate molecular mass 75,000 Da, characteristic of TAP2, whereas a doublet of similar mass representing TAP2 and TAP1 was isolated from T2-TAP1+2 cells. Western blotting verified the identity of the components (not shown). The co-isolation of TAP1 using anti-TAP2 antisera confirmed the functional interaction of these polypeptides, suggesting also that they have been purified in an unaltered conformation. Control purifications from untransfected T2 cell lysates produced no detectable TAP species (Fig. 1 c). Some higher molecular mass contaminants were visible in all preparations including the control T2 lysate; however, the relatively high purity of the TAP preparations was confirmed. Once solubilized in detergent, it is difficult to determine the peptide transport activity of TAP; we therefore studied the integrity of the ATP binding domains of TAP, which are known to intimately control the ability of TAP to bind and transport peptides (36Alberts P. Daumke O. Deverson E.V. Howard J.C. Knittler M.R. Curr. Biol. 2001; 11: 242-251Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 37Gorbulev S. Abele R. Tampe R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3732-3737Crossref PubMed Scopus (109) Google Scholar, 49Knittler M.R. Alberts P. Deverson E.V. Howard J.C. Curr. Biol. 1999; 9: 999-1008Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 50Karttunen J.T. Lehner P.J. Gupta S.S. Hewitt E.W. Cresswell P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7431-7436Crossref PubMed Scopus (64) Google Scholar). The presence of an ATP-binding site was probed by incubation of immunoisolated TAPs with 8N3ATP, which can be cross-linked once bound by exposure to UV irradiation. We first tested the ability of the TAP heterodimer, TAP2 alone, and also TAP1 alone (in T2-TAP1 cells) to be labeled by 8N3ATP while still attached to antibody in immune complexes. As shown in Fig.2 a, both TAP2 and TAP1 labeled strongly when expressed singly. In the case of the heterodimer, whether immunoisolated by anti-TAP2 or -TAP1 reagents, a band was obtained that in comparison to single TAP chains, suggested that both ATP binding sites were equally accessible to 8N3ATP. A strikingly different pattern of labeling was obtained when we examined TAP particles free in solution after peptide-specific elution. In this instance poor labeling was observed with TAP2 alone (Fig.2 b), whereas in the TAP heterodimer, the majority of the labeling appeared to be on material of lower molecular mass than TAP2, suggesting almost exclusive labeling of TAP1. In both cases the presence of nonradioactive ATP inhibited the labeling. To confirm this observation we performed the same experiment but denatured a portion of the TAP heterodimer sample in SDS after labeling with 8N3ATP and re-immunoprecipitated with reagents specific for TAP1 or TAP2. The majority of the labeling was cross-linked to TAP1 (Fig. 2 c). Thus the ATP binding sites in the TAP heterodimer appear to differ in their ability to bind ATP when immobilized with antibody. Possibly the presence of antibody directed at the C terminus of either TAP1 or TAP2 may alter the conformation of the NBDs. However, the preferential labeling of TAP1 of TAP particles free in solution correlates with recent data that indicates that the ATP-binding site on TAP1 is more readily labeled than TAP2 (36Alberts P. Daumke O. Deverson E.V. Howard J.C. Knittler M.R. Curr. Biol. 2001; 11: 242-251Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 51Hewitt E.W. Gupta S.S. Lehner P.J. EMBO J. 2001; 20: 387-396Crossref PubMed Scopus (150) Google Scholar). We also studied the ability of purified TAP particles to bind peptides. An iodinated model peptide TNKTVARYV (termed pep1 in Fig. 2) coupled to the photoreactive cross-linker HSAB (52Androlewicz M.J. Cresswell P. Immunity. 1994; 1: 7-14Abstract Full Text PDF PubMed Scopus (153) Google Scholar) labeled the immunoimmobilized TAP heterodimer but not TAP2 alone (Fig.2 d). However, initial attempts to label TAP particles in solution with pep1 were unsuccessful. We reasoned that the presence of the C-terminal TAP2 peptide KVYAHLVQQRLEA (termed pep2) used to competitively elute TAP particles at 50 μm may inhibit the binding of the photoreactive reporter peptide. To test whether pep2 was capable of binding TAP, we utilized the ability of the chemical cross-linker EGS to cross-link TAP polypeptides into a large molecular mass complex, the formation of which is enhanced by TAP-binding peptides (50Karttunen J.T. Lehner P.J. Gupta S.S. Hewitt E.W. Cresswell P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7431-7436Crossref PubMed Scopus (64) Google Scholar, 53Lacaille V.G. Androlewicz M.J. J. Biol. Chem. 1998; 273: 17386-17390Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Detergent lysates of T2 TAP1+2 cells were incubated with pep1 or pep2, and cross-linking was induced by the addition of 1 mm EGS. Western blot analysis of the lysates indicated that both peptides increased the formation of a cross-linked TAP product of relative molecular mass 220 kDa (Fig. 2 e). Thus the C-terminal TAP2 peptide binds to TAP and is likely to compete with the photoreactive reporter peptide. We therefore eluted TAP particles with a 10-fold reduced concentration of pep2 and retested the binding ability of iodinated HSAB-pep1. Under these conditions iodinated HSAB-pep1 labeled TAP particles, and the addition of 10 μm unlabeled pep1 or pep2 resulted in inhibition of 78 and 43% binding, respectively (Fig. 2 f). Thus eluted TAP particles in solution retain the ability to bind peptides. Taken together with the ATP binding data, this suggests that the TAP particles in solution maintain good overall structural integrity. Transmission electron micrographs of the TAP heterodimer, TAP2, and untransfected T2 control preparations are shown in Fig. 3. Untransfected T2 cell preparations contained only very small debris, whereas the other two samples contained much larger particles. Thus the immunoisolation reaction per se does not appear to contribute particulate matter of similar size to the TAP immunoisolates. Particles in the TAP2 micrographs were on average 7–9 nm in diameter, whereas the TAP heterodimer particles were 9–12 nm in diameter. This information assisted in the TAP heterodimer and TAP2 particle selection process to help improve the data set. Images of 686 TAP2 and 1436 TAP1+2 particles were selected (Fig. 3). Each image represents a projection (a two-dimensional view) of the protein structure. After reference-free alignment, particles were sorted into groups, and averages of different orientations of each molecule were generated (Fig.4 A). The choice of threshold to apply in the clustering was selected to maximize resolution (Fig.4 B). For each molecule, five of the highest resolution averages are shown magnified (Fig. 4 C). TAP1+2 averages (Fig. 4 C, top row) are ∼11–12 nm along the longest axis and 8–9 nm perpendicular to the longest axis. These averages have a spatial resolution of 38–42 Å, as determined by Fourier ring correlation (Fig. 4 B). There is pseudo-2-fold symmetry in the orientations. Averages B andC are examples of projections that show a central 3-nm-diameter-stain-accumulating region. Five of the most significant averages of TAP2 are also shown at the same scale as the TAP1+2 averages (Fig. 4 C, bottom row). These averages have a spatial resolution of 26–35 Å, determined by Fourier ring correlation (Fig. 4 B). Some orientations have a diamond shape (Fig. 4 C, bottom row, d ande), and others have a trapezoid shape (Fig. 4 C,bottom row, a–c). Along one axis, the length of the molecule changes from one side, 6–7 nm, to the