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Membrane receptors are central to cell-cell communication. Receptor clustering at the plasma membrane modulates physiological responses, and mesoscale receptor organization is critical for downstream signaling. Spatially restricted cluster formation of the neuropeptide Y2 hormone receptor (Y2R) was observed in vivo; however, the relevance of this confinement is not fully understood. Here, we controlled Y2R clustering in situ by a chelator nanotool. Due to the multivalent interaction, we observed a dynamic exchange in the microscale confined regions. Fast Y2R enrichment in clustered areas triggered a ligand-independent downstream signaling determined by an increase in cytosolic calcium, cell spreading, and migration. We revealed that the cell response to ligand-induced activation was amplified when cells were pre-clustered by the nanotool. Ligand-independent signaling by clustering differed from ligand-induced activation in the binding of arrestin-3 as downstream effector, which was recruited to the confined regions only in the presence of the ligand. This approach enables in situ clustering of membrane receptors and raises the possibility to explore different modalities of receptor activation.
Many interesting and important membrane proteins are hetero-oligomeric. However, besides naturally abundant examples, the structures of relatively few such complexes are known. Partly, this is due to difficulties in expression, stoichiometric assembly, and in the evaluation of their stability prior to crystallization trials. Here we describe a new approach, which allows rapid assessment of protein complex quality, assembly and stoichiometry, simplifying the search for conditions conducive to long-term stability and crystallization. Multicolour fluorescence size-exclusion chromatography (MC-FSEC) is used to enable tracking of individual subunits through expression, solubilization and purification steps. We show how the method has been applied to the heterodimeric transporter associated with antigen processing (TAP) and demonstrate how it may be extended in order to analyse membrane multisubunit assemblies.
ATP-binding cassette (ABC) systems translocate a wide range of solutes across cellular membranes. The thermophilic Gram-negative eubacterium Thermus thermophilus, a model organism for structural genomics and systems biology, discloses ∼46 ABC proteins, which are largely uncharacterized. Here, we functionally analyzed the first two and only ABC half-transporters of the hyperthermophilic bacterium, TmrA and TmrB. The ABC system mediates uptake of the drug Hoechst 33342 in inside-out oriented vesicles that is inhibited by verapamil. TmrA and TmrB form a stable heterodimeric complex hydrolyzing ATP with a Km of 0.9 mm and kcat of 9 s−1 at 68 °C. Two nucleotides can be trapped in the heterodimeric ABC complex either by vanadate or by mutation inhibiting ATP hydrolysis. Nucleotide trapping requires permissive temperatures, at which a conformational ATP switch is possible. We further demonstrate that the canonic glutamate 523 of TmrA is essential for rapid conversion of the ATP/ATP-bound complex into its ADP/ATP state, whereas the corresponding aspartate in TmrB (Asp-500) has only a regulatory role. Notably, exchange of this single noncanonic residue into a catalytic glutamate cannot rescue the function of the E523Q/D500E complex, implicating a built-in asymmetry of the complex. However, slow ATP hydrolysis in the newly generated canonic site (D500E) strictly depends on the formation of a posthydrolysis state in the consensus site, indicating an allosteric coupling of both active sites.
ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on 31P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain. Based on these results, we propose a model for the coupled ATPase-adenylate kinase mechanism, involving the canonical and an additional nucleotide-binding site. We extend these findings to other prokaryotic ABC exporters, namely LmrA and TmrAB, suggesting that the coupled activities are a general feature of ABC exporters.
Ribosome recycling orchestrated by the ATP binding cassette (ABC) protein ABCE1 can be considered as the final—or the first—step within the cyclic process of protein synthesis, connecting translation termination and mRNA surveillance with re-initiation. An ATP-dependent tweezer-like motion of the nucleotide-binding domains in ABCE1 transfers mechanical energy to the ribosome and tears the ribosome subunits apart. The post-recycling complex (PRC) then re-initiates mRNA translation. Here, we probed the so far unknown architecture of the 1-MDa PRC (40S/30S·ABCE1) by chemical cross-linking and mass spectrometry (XL-MS). Our study reveals ABCE1 bound to the translational factor-binding (GTPase) site with multiple cross-link contacts of the helix–loop–helix motif to the S24e ribosomal protein. Cross-linking of the FeS cluster domain to the ribosomal protein S12 substantiates an extreme lever-arm movement of the FeS cluster domain during ribosome recycling. We were thus able to reconstitute and structurally analyse a key complex in the translational cycle, resembling the link between translation initiation and ribosome recycling.
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.
Antigen presentation via major histocompatibility complex class I (MHC I) molecules is essential to mount an adaptive immune response against pathogens and cancerous cells. To this end, the transporter associated with antigen processing (TAP) delivers snippets of the cellular proteome, resulting from proteasomal degradation, into the ER lumen. After peptide loading and editing by the peptide-loading complex (PLC), stable peptide-MHC I complexes are released for cell surface presentation. Since the process of MHC I trafficking is poorly defined, we established an approach to control antigen presentation by introduction of a photo-caged amino acid in the catalytic ATP-binding site of TAP. By optical control, we initiate TAP-dependent antigen translocation, thus providing new insights into TAP function within the PLC and MHC I trafficking in living cells. Moreover, this versatile approach has the potential to be applied in the study of other cellular pathways controlled by P-loop ATP/GTPases.
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.
Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I–restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL49.5 proteins block TAP as well, these data indicate that UL49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL49.5. Taken together, these results classify the UL49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms.
Coevolution of viruses and their hosts represents a dynamic molecular battle between the immune system and viral factors that mediate immune evasion. After the abandonment of smallpox vaccination, cowpox virus infections are an emerging zoonotic health threat, especially for immunocompromised patients. Here we delineate the mechanistic basis of how cowpox viral CPXV012 interferes with MHC class I antigen processing. This type II membrane protein inhibits the coreTAP complex at the step after peptide binding and peptide-induced conformational change, in blocking ATP binding and hydrolysis. Distinct from other immune evasion mechanisms, TAP inhibition is mediated by a short ER-lumenal fragment of CPXV012, which results from a frameshift in the cowpox virus genome. Tethered to the ER membrane, this fragment mimics a high ER-lumenal peptide concentration, thus provoking a trans-inhibition of antigen translocation as supply for MHC I loading. These findings illuminate the evolution of viral immune modulators and the basis of a fine-balanced regulation of antigen processing.