Transporter Associated With Antigen Processing Complex Research Articles

Natural Spleen Cell Ligandome in Transporter Antigen Processing-Deficient Mice.

Peptides generated by proteases in the cytosol must be translocated to endoplasmic reticulum lumen by the transporter associated with antigen processing (TAP) prior to their assembly with major histocompatibility complex (MHC) class I molecules. Nonfunctional TAP complexes produce a drastic decrease of the MHC class I/peptide complexes presented on the cell surface. Previously, the cellular MHC class I ligandome from TAP-deficient cell lines was determined, but similar analysis from normal tissues remains incomplete. Using high-throughput mass spectrometry to analyze the MHC-bound peptide pools isolated from ex vivo spleen cells of TAP-deficient mice, we identified 210 TAP-independent ligands naturally presented by murine MHC class I molecules. This ligandome showed increased peptide lengths, presence of multiple nested set peptides, and low theoretical MHC binding affinity. The gene ontology enrichment analysis of parental proteins of this TAP-independent subligandome showed almost exclusively enrichment in tissue-specific biological processes related to the immune system as would be expected. Also, cellular components of the extracellular space (namely proteins outside the cell but still within the organism excluding the extracellular matrix) were specifically associated with TAP-independent antigen processing from these ex vivo mice cells. In addition, functional protein association network analysis revealed low protein-protein interactions between parental proteins from the TAP-independent ligandome. Finally, predominant endoproteolytic peptidase specificity for Leu/Phe residues in the P1 position of the scissile bond at both ligand termini was found for the ex vivo TAP-independent ligands. These data indicate that the TAP-independent ligandome from ex vivo cells derives from a more diverse collection of both endoprotease activities and parental proteins and where the cell origin and contribution of the extracellular environment are more relevant than in its equivalent cell lines.

Computational characterization of the peptidome in transporter associated with antigen processing (TAP)-deficient cells.

The transporter associated with antigen processing (TAP) is a key element of the major histocompatibility complex (MHC) class I antigen processing and presentation pathway. Nonfunctional TAP complexes impair the translocation of cytosol-derived proteolytic peptides to the endoplasmic reticulum lumen. This drastic reduction in the available peptide repertoire leads to a significant decrease in MHC class I cell surface expression. Using mass spectrometry, different studies have analyzed the cellular MHC class I ligandome from TAP-deficient cells, but the analysis of the parental proteins, the source of these ligands, still deserves an in-depth analysis. In the present report, several bioinformatics protocols were applied to investigate the nature of parental proteins for the previously identified TAP-independent MHC class I ligands. Antigen processing in TAP-deficient cells mainly focused on small, abundant or highly integral transmembrane proteins of the cellular proteome. This process involved abundant proteins of the central RNA metabolism. In addition, TAP-independent ligands were preferentially cleaved from the N- and C-terminal ends with respect to the central regions of the parental proteins. The abundance of glycine, proline and aromatic residues in the C-terminal sequences from TAP-independently processed proteins allows the accessibility and specificity required for the proteolytic activities that generates the TAP-independent ligandome. This limited proteolytic activity towards a set of preferred proteins in a TAP-negative environment would therefore suffice to promote the survival of TAP-deficient individuals.

Structure and Dynamics of Antigenic Peptides in Complex with TAP.

The transporter associated with antigen processing (TAP) selectively translocates antigenic peptides into the endoplasmic reticulum. Loading onto major histocompatibility complex class I molecules and proofreading of these bound epitopes are orchestrated within the macromolecular peptide-loading complex, which assembles on TAP. This heterodimeric ABC-binding cassette (ABC) transport complex is therefore a major component in the adaptive immune response against virally or malignantly transformed cells. Its pivotal role predestines TAP as a target for infectious diseases and malignant disorders. The development of therapies or drugs therefore requires a detailed comprehension of structure and function of this ABC transporter, but our knowledge about various aspects is still insufficient. This review highlights recent achievements on the structure and dynamics of antigenic peptides in complex with TAP. Understanding the binding mode of antigenic peptides in the TAP complex will crucially impact rational design of inhibitors, drug development, or vaccination strategies.

A dual inhibition mechanism of herpesviral ICP47 arresting a conformationally thermostable TAP complex.

As a centerpiece of antigen processing, the ATP-binding cassette transporter associated with antigen processing (TAP) became a main target for viral immune evasion. The herpesviral ICP47 inhibits TAP function, thereby suppressing an adaptive immune response. Here, we report on a thermostable ICP47-TAP complex, generated by fusion of different ICP47 fragments. These fusion complexes allowed us to determine the direction and positioning in the central cavity of TAP. ICP47-TAP fusion complexes are arrested in a stable conformation, as demonstrated by MHC I surface expression, melting temperature, and the mutual exclusion of herpesviral TAP inhibitors. We unveiled a conserved region next to the active domain of ICP47 as essential for the complete stabilization of the TAP complex. Binding of the active domain of ICP47 arrests TAP in an open inward facing conformation rendering the complex inaccessible for other viral factors. Based on our findings, we propose a dual interaction mechanism for ICP47. A per se destabilizing active domain inhibits the function of TAP, whereas a conserved C-terminal region additionally stabilizes the transporter. These new insights into the ICP47 inhibition mechanism can be applied for future structural analyses of the TAP complex.

The herpesvirus stealth program

Immune evasion represents a critical strategy that many viruses have evolved to ensure survival in individual hosts and entire populations. In vertebrates, particularly mammals that are protected by a highly sophisticated immune system, viruses need to antagonize the many defense line drawn after antigen recognition and the ensuing differentiation and proliferation of virus-specific T and B cells. The Herpesviridae family is characterized by complex viral genomes that contain a number of so-called non-essential genes, which are dispensable for growth in cultured cells but not in vivo. It is no surprise that herpesviruses encode, among many other immune evasion factors, gene products that target antigen presentation by major histocompatibility complex class I (MHC-I) [1]. Due to the drastic reduction of cell surface MHC-I molecules, the signals that activate and recruit cytotoxic T lymphocytes (CTLs) to the infected cell are blocked. As a result, the infected cells cannot be eliminated and thus become a reservoir for the sustained production and release of viruses. The viral inhibitors that cause downregulation of MHC-I on the cell surface can vary widely with respect to mechanistic principles, even among closely related virus species. For instance, some pUL49.5 homologues from the Varicellovirus genus of the Alphaherpesviridae have been shown to interfere with the MHC-I presentation pathway. The viral proteins block the physiological activity of the transporter associated with antigen processing (TAP) that facilitates translocation of peptides produced by the proteasome into the endoplasmic reticulum (ER) and ultimately loading of antigenic peptides onto MHC-I. Regardless of the structural similarities, there are remarkable differences in the molecular functions exerted by pUL49.5 homologues. In the case of bovine herpesvirus 1 (BoHV-1), the TAP complex is tagged for proteasomal degradation by pUL49.5, whereas the pUL49.5 homologues of equine herpesvirus 1 (EHV-1) and EHV-4 impair the function of the TAP complex merely through inhibition of ATP binding (reviewed in [2]). Apart from the pUL49.5 homologues, the EHV-1 pUL56 was shown to be involved in downregulation of cell surface MHC-I [3]. Detailed studies revealed that early in infection the expression of pUL56 correlates with internalization of cell surface MHC-I through dynamin-dependent endocytosis, a process that requires activation of the cellular ubiquitination cascade [4]. Knowing that pUL56 suppresses the MHC-I presentation only during viral infection and not by itself, we speculated that there is at least one viral interactor of pUL56. We, therefore, generated a library of single knockout mutants and attempted to find partner(s) for pUL56. Our investigation resulted in identification of a poorly understood protein equivalent to the HSV-1 (Herpes simplex virus 1) pUL43 homologue that is also involved in the inhibition of the presence of MHC-I molecules on the cell surface. Most importantly, direct interaction of pUL43 and pUL56 was demonstrated, indicating that cell surface MHC-I levels are reduced by cooperation of the two transmembrane proteins [5]. The putative mechanism of MHC-I downregulation induced by the pUL43-pUL56 cooperation is illustrated in Figure ​Figure1.1. Briefly, we presume that ubiquitination of pUL43 is initiated by the interaction between pUL56 and Nedd4 (Neural precursor cell expressed developmentally down-regulated protein 4), which then directs delivery of the internalized tertiary complex MHC-I-pUL43-pUL56 to the early endosome. The cargo eventually traffics to the lysosomal compartments where MHC-I and pUL43 are degraded. We have shown that pUL56 remains stable during infection [3], which indicates that it is excluded from the tertiary complex at the transition from the endosomal to the lysosomal stage. An alternative model would require vesicles derived from the Golgi apparatus to be directed to the early endosome or MVB (Multivesicular body) by the ESCRT machinery (Endosomal sorting complex required for transport) that would then sort MHC-I and pUL43 for lysosomal degradation. During the degradation process, pUL56 late domains (Pro-Pro-x-Tyr motif; x indicates any amino acid) seem critical for bringing the E3 ligase Nedd4 in close proximity with pUL43 and MHC-I. It will be of importance to test if the identified mechanism of early MHC-I degradation is also active in human cells after infection with the EHV-1 cousins HSV and VZV (Varicella-zoster virus). Both viruses encode pUL43 homologues that share structural similarity with that of EHV-1. Figure 1 Model of the presumed cooperation between EHV-1 pUL43 and pUL56 in MHC-I downregulation The identification of a novel potent herpesviral inhibitory mechanism of MHC-I presentation suggests that our knowledge about the arsenal of herpesviruses with respect to escape from host immunity is likely far from complete. The continued adaptive selection of countermeasures to host immune responses is in need of a diversity of viral inhibitors that effectively silence or avoid effector cell populations such as macrophages, natural killer (NK) cells, T and B cells. Therefore, the cooperative action of two viral proteins in escape from CTL responses summarized here is likely not to be the last to be identified.

An Annular Lipid Belt Is Essential for Allosteric Coupling and Viral Inhibition of the Antigen Translocation Complex TAP (Transporter Associated with Antigen Processing)

The transporter associated with antigen processing (TAP) constitutes a focal element in the adaptive immune response against infected or malignantly transformed cells. TAP shuttles proteasomal degradation products into the lumen of the endoplasmic reticulum for loading of major histocompatibility complex (MHC) class I molecules. Here, the heterodimeric TAP complex was purified and reconstituted in nanodiscs in defined stoichiometry. We demonstrate that a single heterodimeric core-TAP complex is active in peptide binding, which is tightly coupled to ATP hydrolysis. Notably, with increasing peptide length, the ATP turnover was gradually decreased, revealing that ATP hydrolysis is coupled to the movement of peptide through the ATP-binding cassette transporter. In addition, all-atom molecular dynamics simulations show that the observed 22 lipids are sufficient to form an annular belt surrounding the TAP complex. This lipid belt is essential for high affinity inhibition by the herpesvirus immune evasin ICP47. In conclusion, nanodiscs are a powerful approach to study the important role of lipids as well as the function, interaction, and modulation of the antigen translocation machinery.

Analyses of Conformational States of the Transporter Associated with Antigen Processing (TAP) Protein in a Native Cellular Membrane Environment

The transporter associated with antigen processing (TAP) plays a critical role in the MHC class I antigen presentation pathway. TAP translocates cellular peptides across the endoplasmic reticulum membrane in an ATP hydrolysis-dependent manner. We used FRET spectroscopy in permeabilized cells to delineate different conformational states of TAP in a native subcellular membrane environment. For these studies, we tagged the TAP1 and TAP2 subunits with enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, respectively, C-terminally to their nucleotide binding domains (NBDs), and measured FRET efficiencies under different conditions. Our data indicate that both ATP and ADP enhance the FRET efficiencies but that neither induces a maximally closed NBD conformation. Additionally, peptide binding induces a large and significant increase in NBD proximity with a concentration dependence that is reflective of individual peptide affinities for TAP, revealing the underlying mechanism of peptide-stimulated ATPase activity of TAP. Maximal NBD closure is induced by the combination of peptide and non-hydrolysable ATP analogs. Thus, TAP1-TAP2 NBD dimers are not fully stabilized by nucleotides alone, and substrate binding plays a key role in inducing the transition state conformations of the NBD. Taken together, these findings show that at least three steps are involved in the transport of peptides across the endoplasmic reticulum membrane for antigen presentation, corresponding to three dynamically and structurally distinct conformational states of TAP. Our studies elucidate structural changes in the TAP NBD in response to nucleotides and substrate, providing new insights into the mechanism of ATP-binding cassette transporter function.

Diversity of Natural Self-Derived Ligands Presented by Different HLA Class I Molecules in Transporter Antigen Processing-Deficient Cells

The transporter associated with antigen processing (TAP) translocates the cytosol-derived proteolytic peptides to the endoplasmic reticulum lumen where they complex with nascent human leukocyte antigen (HLA) class I molecules. Non-functional TAP complexes and viral or tumoral blocking of these transporters leads to reduced HLA class I surface expression and a drastic change in the available peptide repertoire. Using mass spectrometry to analyze complex human leukocyte antigen HLA-bound peptide pools isolated from large numbers of TAP-deficient cells, we identified 334 TAP-independent ligands naturally presented by four different HLA-A, -B, and -C class I molecules with very different TAP dependency from the same cell line. The repertoire of TAP-independent peptides examined favored increased peptide lengths and a lack of strict binding motifs for all four HLA class I molecules studied. The TAP-independent peptidome arose from 182 parental proteins, the majority of which yielded one HLA ligand. In contrast, TAP-independent antigen processing of very few cellular proteins generated multiple HLA ligands. Comparison between TAP-independent peptidome and proteome of several subcellular locations suggests that the secretory vesicle-like organelles could be a relevant source of parental proteins for TAP-independent HLA ligands. Finally, a predominant endoproteolytic peptidase specificity for Arg/Lys or Leu/Phe residues in the P1 position of the scissile bond was found for the TAP-independent ligands. These data draw a new and intricate picture of TAP-independent pathways.

A Viral, Transporter Associated with Antigen Processing (TAP)-independent, High Affinity Ligand with Alternative Interactions Endogenously Presented by the Nonclassical Human Leukocyte Antigen E Class I Molecule

The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.

Molecular architecture of the MHC I peptide‐loading complex: one tapasin molecule is essential and sufficient for antigen processing

The loading of antigen-derived peptides onto MHC class I molecules for presentation to cytotoxic T cells is a key process in adaptive immune defense. Loading of MHC I is achieved by a sophisticated machinery, the peptide-loading complex (PLC), which is organized around the transporter associated with antigen processing (TAP) with the help of several auxiliary proteins. As an essential adapter protein recruiting MHC I molecules to TAP, tapasin catalyzes peptide loading of MHC I. However, the exact stoichiometry and basic molecular architecture of TAP and tapasin within the PLC remains elusive. Here, we demonstrate that two tapasin molecules are assembled in the PLC, with one tapasin bound to each TAP subunit. However, one tapasin molecule bound either to TAP1 or TAP2 is sufficient for efficient MHC I antigen presentation. By specifically blocking the interaction between tapasin-MHC I complexes and the translocation complex TAP, the MHC I surface expression is impaired to the same extent as with soluble tapasin. Thus, the proximity of the peptide supplier TAP to the acceptor MHC I is crucial for antigen processing. In summary, the human PLC consists maximally of 2× tapasin-ERp57/MHC I per TAP complex, but one tapasin-ERp57/MHC I in the PLC is essential and sufficient for antigen processing.

Role of Metalloproteases in Vaccinia Virus Epitope Processing for Transporter Associated with Antigen Processing (TAP)-independent Human Leukocyte Antigen (HLA)-B7 Class I Antigen Presentation

The transporter associated with antigen processing (TAP) translocates the viral proteolytic peptides generated by the proteasome and other proteases in the cytosol to the endoplasmic reticulum lumen. There, they complex with nascent human leukocyte antigen (HLA) class I molecules, which are subsequently recognized by the CD8(+) lymphocyte cellular response. However, individuals with nonfunctional TAP complexes or tumor or infected cells with blocked TAP molecules are able to present HLA class I ligands generated by TAP-independent processing pathways. Herein, using a TAP-independent polyclonal vaccinia virus-polyspecific CD8(+) T cell line, two conserved vaccinia-derived TAP-independent HLA-B*0702 epitopes were identified. The presentation of these epitopes in normal cells occurs via complex antigen-processing pathways involving the proteasome and/or different subsets of metalloproteinases (amino-, carboxy-, and endoproteases), which were blocked in infected cells with specific chemical inhibitors. These data support the hypothesis that the abundant cellular proteolytic systems contribute to the supply of peptides recognized by the antiviral cellular immune response, thereby facilitating immunosurveillance. These data may explain why TAP-deficient individuals live normal life spans without any increased susceptibility to viral infections.

Epstein-Barr Viral BNLF2a Protein Hijacks the Tail-anchored Protein Insertion Machinery to Block Antigen Processing by the Transport Complex TAP

Virus-infected cells are eliminated by cytotoxic T lymphocytes, which recognize viral epitopes displayed on major histocompatibility complex class I molecules at the cell surface. Herpesviruses have evolved sophisticated strategies to escape this immune surveillance. During the lytic phase of EBV infection, the viral factor BNLF2a interferes with antigen processing by preventing peptide loading of major histocompatibility complex class I molecules. Here we reveal details of the inhibition mechanism of this EBV protein. We demonstrate that BNLF2a acts as a tail-anchored protein, exploiting the mammalian Asna-1/WRB (Get3/Get1) machinery for posttranslational insertion into the endoplasmic reticulum membrane, where it subsequently blocks antigen translocation by the transporter associated with antigen processing (TAP). BNLF2a binds directly to the core TAP complex arresting the ATP-binding cassette transporter in a transport-incompetent conformation. The inhibition mechanism of EBV BNLF2a is distinct and mutually exclusive of other viral TAP inhibitors.

Structural and functional analysis of the TAP-inhibiting UL49.5 proteins of varicelloviruses

Viral infections are counteracted by virus-specific cytotoxic T cells that recognize the infected cell via MHC class I (MHC I) molecules presenting virus-derived peptides. The loading of the peptides onto MHC I molecules occurs in the endoplasmic reticulum (ER) and is facilitated by the peptide loading complex. A key player in this complex is the transporter associated with antigen processing (TAP), which translocates the viral peptides from the cytosol into the ER. Herpesviruses have developed many strategies to evade cytotoxic T cells. Several members of the genus Varicellovirus encode a UL49.5 protein that prevents peptide transport through TAP. These include bovine herpesvirus (BoHV) 1, BoHV-5, bubaline herpesvirus 1, cervid herpesvirus 1, pseudorabies virus, felid herpesvirus 1, and equine herpesvirus 1 and 4. BoHV-1 UL49.5 inhibits TAP by preventing conformational changes essential for peptide transport and by inducing degradation of the TAP complex. UL49.5 consists of an ER luminal N-terminal domain, a transmembrane domain and a cytosolic C-terminal tail domain.In this study, the following features of UL49.5 were deciphered: (1) chimeric constructs of BoHV-1 and VZV UL49.5 attribute the lack of TAP inhibition by VZV UL49.5 to its ER-luminal domain, (2) the ER-luminal and TM domains of UL49.5 are required for efficient interaction with and inhibition of TAP, (3) the C-terminal RXRX sequence is essential for TAP degradation by BoHV-1 UL49.5, and (4) in addition to the RXRX sequence, the cytoplasmic tail of BoHV-1 UL49.5 carries a motif that is required for efficient TAP inhibition by the protein. A model is presented depicting how the different domains of UL49.5 may block the translocation of peptides by TAP and target TAP for proteasomal degradation.

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

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.

Purification and Reconstitution of the Antigen Transport Complex TAP

The transporter associated with antigen processing (TAP) is an essential machine of the adaptive immune system that translocates antigenic peptides from the cytosol into the endoplasmic reticulum lumen for loading of major histocompatibility class I molecules. To examine this ABC transport complex in mechanistic detail, we have established, after extensive screening and optimization, the solubilization, purification, and reconstitution for TAP to preserve its function in each step. This allowed us to determine the substrate-binding stoichiometry of the TAP complex by fluorescence cross-correlation spectroscopy. In addition, the TAP complex shows strict coupling between peptide binding and ATP hydrolysis, revealing no basal ATPase activity in the absence of peptides. These results represent an optimal starting point for detailed mechanistic studies of the transport cycle of TAP by single molecule experiments to analyze single steps of peptide translocation and the stoichiometry between peptide transport and ATP hydrolysis.

Structural arrangement of the transmission interface in the antigen ABC transport complex TAP

The transporter associated with antigen processing (TAP) represents a focal point in the immune recognition of virally or malignantly transformed cells by translocating proteasomal degradation products into the endoplasmic reticulum-lumen for loading of MHC class I molecules. Based on a number of experimental data and the homology to the bacterial ABC exporter Sav1866, we constructed a 3D structural model of the core TAP complex and used it to examine the interface between the transmembrane and nucleotide-binding domains (NBD) by cysteine-scanning and cross-linking approaches. Herein, we demonstrate the functional importance of the newly identified X-loop in the NBD in coupling substrate binding to downstream events in the transport cycle. We further verified domain swapping in a heterodimeric ABC half-transporter complex by cysteine cross-linking. Strikingly, either substrate binding or translocation can be blocked by cross-linking the X-loop to coupling helix 2 or 1, respectively. These results resolve the structural arrangement of the transmission interface and point to different functions of the cytosolic loops and coupling helices in substrate binding, signaling, and transport.

A CD8+ T cell immune evasion protein specific to Epstein-Barr virus and its close relatives in Old World primates

γ1-Herpesviruses such as Epstein-Barr virus (EBV) have a unique ability to amplify virus loads in vivo through latent growth-transforming infection. Whether they, like α- and β-herpesviruses, have been driven to actively evade immune detection of replicative (lytic) infection remains a moot point. We were prompted to readdress this question by recent work (Pudney, V.A., A.M. Leese, A.B. Rickinson, and A.D. Hislop. 2005. J. Exp. Med. 201:349–360; Ressing, M.E., S.E. Keating, D. van Leeuwen, D. Koppers-Lalic, I.Y. Pappworth, E.J.H.J. Wiertz, and M. Rowe. 2005. J. Immunol. 174:6829–6838) showing that, as EBV-infected cells move through the lytic cycle, their susceptibility to EBV-specific CD8+ T cell recognition falls dramatically, concomitant with a reductions in transporter associated with antigen processing (TAP) function and surface human histocompatibility leukocyte antigen (HLA) class I expression. Screening of genes that are unique to EBV and closely related γ1-herpesviruses of Old World primates identified an early EBV lytic cycle gene, BNLF2a, which efficiently blocks antigen-specific CD8+ T cell recognition through HLA-A–, HLA-B–, and HLA-C–restricting alleles when expressed in target cells in vitro. The small (60–amino acid) BNLF2a protein mediated its effects through interacting with the TAP complex and inhibiting both its peptide- and ATP-binding functions. Furthermore, this targeting of the major histocompatibility complex class I pathway appears to be conserved among the BNLF2a homologues of Old World primate γ1-herpesviruses. Thus, even the acquisition of latent cycle genes endowing unique growth-transforming ability has not liberated these agents from evolutionary pressure to evade CD8+ T cell control over virus replicative foci.

Modulation of the Antigenic Peptide Transporter TAP by Recombinant Antibodies Binding to the Last Five Residues of TAP1

The transporter associated with antigen processing (TAP) plays a pivotal role in the major histocompatibility complex (MHC) class I mediated immune response against infected or malignantly transformed cells. It belongs to the ATP-binding cassette (ABC) superfamily and consists of TAP1 (ABCB2) and TAP2 (ABCB3), each of which possesses a transmembrane and a nucleotide-binding domain (NBD). Here we describe the generation of recombinant Fv and Fab antibody fragments to human TAP from a hybridoma cell line expressing the TAP1-specific monoclonal antibody mAb148.3. The epitope of the antibody was mapped to the very last five C-terminal amino acid residues of TAP1 on solid-supported peptide arrays. The recombinant antibody fragments were heterologously expressed in Escherichia coli and purified to homogeneity from periplasmic extracts by affinity chromatography. The monoclonal and recombinant antibodies bind with nanomolar affinity to the last five C-terminal amino acid residues of TAP1 as demonstrated by ELISA and surface plasmon resonance. Strikingly, the recombinant antibody fragments confer thermal stability to the heterodimeric TAP complex. At the same time TAP is arrested in a peptide transport incompetent conformation, although ATP and peptide binding to TAP are not affected. Based on our results we suggest that the C terminus of TAP1 modulates TAP function presumably as part of the dimer interface of the NBDs.

Catalytic Site Modifications of TAP1 and TAP2 and Their Functional Consequences

The transporter associated with antigen processing (TAP), a member of the ATP binding cassette (ABC) family of transmembrane transporters, transports peptides across the endoplasmic reticulum membrane for assembly of major histocompatibility complex class I molecules. Two subunits, TAP1 and TAP2, are required for peptide transport, and ATP hydrolysis by TAP1.TAP2 complexes is important for transport activity. Two nucleotide binding sites are present in TAP1.TAP2 complexes. Compared with other ABC transporters, the first nucleotide binding site contains non-consensus catalytic site residues, including Asp(668) in the Walker B region of TAP1 (in place of a highly conserved glutamic acid), and Gln(701) in the switch region of TAP1 (in place of a highly conserved histidine). At the second nucleotide binding site, a glutamic acid (TAP2 Glu(632)) follows the Walker B motif, and the switch region contains a histidine (TAP2 His(661)). We found that alterations at Glu(632) and His(661) of TAP2 significantly reduced peptide translocation and/or TAP-induced major histocompatibility complex class I surface expression. Alterations of TAP1 Asp(668) alone or in combination with TAP1 Gln(701) had only small effects on TAP activity. Thus, the naturally occurring Asp(668) and Gln(701) alterations of TAP1 are likely to contribute to attenuated catalytic activity at the first nucleotide binding site (the TAP1 site) of TAP complexes. Due to its enhanced catalytic activity, the second nucleotide binding site (the TAP2 site) appears to be the main site driving peptide transport. A mechanistic model involving one main active site is likely to apply to other ABC transporters that have an asymmetric distribution of catalytic site residues within the two nucleotide binding sites.

Structure and Dynamics of Membrane-associated ICP47, a Viral Inhibitor of the MHC I Antigen-processing Machinery

To evade the host's immune response, herpes simplex virus employs the immediate early gene product ICP47 (IE12) to suppress antigen presentation to cytotoxic T-lymphocytes by inhibition of the ATP-binding cassette transporter associated with antigen processing (TAP). ICP47 is a membrane-associated protein adopting an alpha-helical conformation. Its active domain was mapped to residues 3-34 and shown to encode all functional properties of the full-length protein. The active domain of ICP47 was reconstituted into oriented phospholipid bilayers and studied by proton-decoupled 15N and 2H solid-state NMR spectroscopy. In phospholipid bilayers, the protein adopts a helix-loop-helix structure, where the average tilt angle of the helices relative to the membrane surface is approximately 15 degrees (+/- 7 degrees ). The alignment of both structured domains exhibits a mosaic spread of approximately 10 degrees . A flexible dynamic loop encompassing residues 17 and 18 separates the two helices. Refinement of the experimental data indicates that helix 1 inserts more deeply into the membrane. These novel insights into the structure of ICP47 represent an important step toward a molecular understanding of the immune evasion mechanism of herpes simplex virus and are instrumental for the design of new therapeutics.