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The transporter associated with antigen processing (TAP) is a key component of the cellular immune system. As a member of the ATP-binding cassette (ABC) superfamily, TAP hydrolyzes ATP to energize the transport of peptides from the cytosol into the lumen of the endoplasmic reticulum. TAP is composed of TAP1 and TAP2, each containing a transmembrane domain and a nucleotide-binding domain (NBD). Here we investigated the role of the ABC signature motif (C-loop) on the functional non-equivalence of the NBDs, which contain a canonical C-loop (LSGGQ) for TAP1 and a degenerate C-loop (LAAGQ) for TAP2. Mutation of the leucine or glycine (LSGGQ) in TAP1 fully abolished peptide transport. However, TAP complexes with equivalent mutations in TAP2 still showed residual peptide transport activity. To elucidate the origin of the asymmetry of the NBDs of TAP, we further examined TAP complexes with exchanged C-loops. Strikingly, the chimera with two canonical C-loops showed the highest transport rate whereas the chimera with two degenerate C-loops had the lowest transport rate, demonstrating that the ABC signature motifs control peptide transport efficiency. All single site mutants and chimeras showed similar activities in peptide or ATP binding, implying that these mutations affect the ATPase activity of TAP. In addition, these results prove that the serine of the C-loop is not essential for TAP function but rather coordinates, together with other residues of the C-loop, the ATP hydrolysis in both nucleotide-binding sites.
The family of scaffold attachment factor B (SAFB) proteins comprises three members and was first identified as binders of the nuclear matrix/scaffold. Over the past two decades, SAFBs were shown to act in DNA repair, mRNA/(l)ncRNA processing, and as part of protein complexes with chromatin-modifying enzymes. SAFB proteins are approximately-100-kDa-sized dual nucleic acid-binding proteins with dedicated domains in an otherwise largely unstructured context, but whether and how they discriminate DNA- and RNA-binding has remained enigmatic. We here provide the SAFB2 DNA- and RNA-binding SAP and RRM domains in their functional boundaries and use solution NMR spectroscopy to ascribe DNA- and RNA-binding functions. We give insight into their target nucleic acid preferences and map the interfaces with respective nucleic acids on sparse data-derived SAP and RRM domain structures. Further, we provide evidence that the SAP domain exhibits intra-domain dynamics and a potential tendency to dimerise, which may expand its specifically targeted DNA sequence range. Our data provide a first molecular basis of and a starting point towards deciphering DNA- and RNA-binding functions of SAFB2 on the molecular level and serve a basis for understanding its localization to specific regions of chromatin and its involvement in the processing of specific RNA species.
Identification of a lysosomal peptide transport system induced during dendritic cell development
(2007)
The delivery of protein fragments to major histocompatibility complex (MHC)-loading compartments of professional antigen-presenting cells is essential in the adaptive immune response against pathogens. Apart from the crucial role of the transporter associated with antigen processing (TAP) for peptide loading of MHC class I molecules in the endoplasmic reticulum, TAP-independent translocation pathways have been proposed but not identified so far. Based on its overlapping substrate specificity with TAP, we herein investigated the ABC transporter ABCB9, also named TAP-like (TAPL). Remarkably, TAPL expression is strongly induced during differentiation of monocytes to dendritic cells and to macrophages. TAPL does not, however, restore MHC class I surface expression in TAP-deficient cells, demonstrating that TAPL alone or in combination with single TAP subunits does not form a functional transport complex required for peptide loading of MHC I in the endoplasmic reticulum. In fact, by using quantitative immunofluorescence and subcellular fractionation, TAPL was detected in the lysosomal compartment co-localizing with the lysosome-associated membrane protein LAMP-2. By in vitro assays, we demonstrate a TAPL-specific translocation of peptides into isolated lysosomes, which strictly requires ATP hydrolysis. These results suggest a mechanism by which antigenic peptides have access to the lysosomal compartment in professional antigen-presenting cells.
The purification and functional reconstitution of a five-component oligopeptide ATP-binding cassette transporter with a remarkably wide substrate specificity are described. High-affinity peptide uptake was dependent on liganded substrate-binding protein OppA, which interacts with the translocator OppBCDF with higher affinity than unliganded OppA. Transport screening with combinatorial peptide libraries revealed that (i) the Opp transporter is not selective with respect to amino acid side chains of the transported peptides; (ii) any peptide that can bind to OppA is transported via Opp, including very long peptides up to 35 residues long; and (iii) the binding specificity of OppA largely determines the overall transport selectivity.
The transporter associated with antigen processing (TAP) plays a key role in the adaptive immune response by pumping antigenic peptides into the endoplasmic reticulum for subsequent loading of major histocompatibility complex class I molecules. TAP is a heterodimer consisting of TAP1 and TAP2. Each subunit is composed of a transmembrane domain and a nucleotide-binding domain, which energizes the peptide transport. To analyze ATP hydrolysis of each subunit we developed a method of trapping 8-azido-nucleotides to TAP in the presence of phosphate transition state analogs followed by photocross-linking, immunoprecipitation, and high resolution SDS-PAGE. Strikingly, trapping of both TAP subunits by beryllium fluoride is peptide-specific. The peptide concentration required for half-maximal trapping is identical for TAP1 and TAP2 and directly correlates with the peptide binding affinity. Only a background level of trapping was observed for low affinity peptides or in the presence of the herpes simplex viral protein ICP47, which specifically blocks peptide binding to TAP. Importantly, the peptide-induced trapped state is reached after ATP hydrolysis and not in a backward reaction of ADP binding and trapping. In the trapped state, TAP can neither bind nor exchange nucleotides, whereas peptide binding is not affected. In summary, these data support the model that peptide binding induces a conformation that triggers ATP hydrolysis in both subunits of the TAP complex within the catalytic cycle.
The lysosomal ABC transporter associated with antigen processing-like (TAPL, ABCB9) acts as an ATP-dependent polypeptide transporter with broad length selectivity. To characterize in detail its substrate specificity, a procedure for functional reconstitution of human TAPL was developed. By intensive screening of detergents, ideal solubilization conditions were evolved with respect to efficiency, long term stability, and functionality of TAPL. TAPL was isolated in a two-step procedure with high purity and, subsequently, reconstituted into proteoliposomes. The peptide transport activity of reconstituted TAPL strongly depends on the lipid composition. With the help of combinatorial peptide libraries, the key positions of the peptides were localized to the N- and C-terminal residues with respect to peptide transport. At both ends, TAPL favors positively charged, aromatic, or hydrophobic residues and disfavors negatively charged residues as well as asparagine and methionine. Besides specific interactions of both terminal residues, electrostatic interactions are important, since peptides with positive net charge are more efficiently transported than negatively charged ones.
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.
1H, 13C, and 15N backbone chemical shift assignments of coronavirus-2 non-structural protein Nsp10
(2020)
The international Covid19-NMR consortium aims at the comprehensive spectroscopic characterization of SARS-CoV-2 RNA elements and proteins and will provide NMR chemical shift assignments of the molecular components of this virus. The SARS-CoV-2 genome encodes approximately 30 different proteins. Four of these proteins are involved in forming the viral envelope or in the packaging of the RNA genome and are therefore called structural proteins. The other proteins fulfill a variety of functions during the viral life cycle and comprise the so-called non-structural proteins (nsps). Here, we report the near-complete NMR resonance assignment for the backbone chemical shifts of the non-structural protein 10 (nsp10). Nsp10 is part of the viral replication-transcription complex (RTC). It aids in synthesizing and modifying the genomic and subgenomic RNAs. Via its interaction with nsp14, it ensures transcriptional fidelity of the RNA-dependent RNA polymerase, and through its stimulation of the methyltransferase activity of nsp16, it aids in synthesizing the RNA cap structures which protect the viral RNAs from being recognized by the innate immune system. Both of these functions can be potentially targeted by drugs. Our data will aid in performing additional NMR-based characterizations, and provide a basis for the identification of possible small molecule ligands interfering with nsp10 exerting its essential role in viral replication.
By translocating proteasomal degradation products into the endoplasmic reticulum for loading of major histocompatibility complex I molecules, the ABC transporter TAP plays a focal role in the adaptive immunity against infected or malignantly transformed cells. A key question regarding the transport mechanism is how the quality of the incoming peptide is detected and how this information is transmitted to the ATPase domains. To identify residues involved in this process, we evolved a Trojan horse strategy in which a small artificial protease is inserted into antigenic epitopes. After binding, the TAP backbone in contact is cleaved, allowing the peptide sensor site to be mapped by mass spectrometry. Within this sensor site, we identified residues that are essential for tight coupling of peptide binding and transport. This sensor and transmission interface is restructured during the ATP hydrolysis cycle, emphasizing its important function in the cross-talk between the transmembrane and the nucleotide-binding domains. This allocrite sensor may be similarly positioned in other members of the ABC exporter family.
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.