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Im Zuge der steigenden Bedeutung der Proteomforschung und der »Molekularisierung« der Medizin werden neue, effizientere Plattformen zur Untersuchung von Proteinen und deren Wechselwirkungen notwendig. Hier bietet die Nanotechnologie, eine Wissenschaft mit Ursprüngen in der Physik und der Halbleiterindustrie, attraktive Lösungsperspektiven. Ein Bereich der Forschung am Institut für Biochemie der Universität Frankfurt um Prof. Dr. Robert Tampé widmet sich den Aspekten der Nanotechnologie zur Entwicklung von Protein-Chips für die Proteomforschung und Erzeugung von Mustern im Kleinstformat.
Major histocompatibility complex class I (MHC I) molecules present antigenic peptides to cytotoxic T cells to eliminate infected or cancerous cells. The transporter associated with antigen processing (TAP) shuttles proteasomally generated peptides into the ER for MHC I loading. As central part of the peptide-loading complex (PLC), TAP is targeted by viral factors, which inhibit peptide supply and thereby impact MHC I-mediated immune responses. However, it is still poorly understood how antigen presentation via different MHC I allotypes is affected by TAP inhibition. Here, we show that conditional expression of herpes simplex viral ICP47 suppresses surface presentation of HLA-A and HLA-C, but not of HLA-B, while the human cytomegaloviral US6 reduces surface levels of all MHC I allotypes. This marked difference in HLA-B antigen presentation is echoed by an enrichment of HLA-B allomorphs at US6-arrested PLC in comparison to ICP47-PLC. Although both viral factors prevent TAP-mediated peptide supply, our data imply that MHC I allomorphs favor different conformationally arrested states of the PLC, leading to differential downregulation of MHC I surface presentation. These findings will help understand MHC I biology in general and will even advance the targeted treatment of infections depending on patients’ allotypes.
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.
Polyacrylamide gel electrophoresis (PAGE) and immunoblotting (Western blotting) are the most common methods in life science. In conjunction with these methods, the polyhistidine-tag has proven to be a superb fusion tag for protein purification as well as specific protein detection by immunoblotting, which led to a vast amount of commercially available antibodies. Nevertheless, antibody batch-to-batch variations and nonspecific binding complicate the laborious procedure. The interaction principle applied for His-tagged protein purification by metal-affinity chromatography using N-nitrilotriacetic acid (NTA) was employed to develop small high-affinity lock-and-key molecules coupled to a fluorophore. These multivalent NTA probes allow specific detection of His-tagged proteins by fluorescence. Here, we report on HisQuick-PAGE as a fast and versatile immunoblot alternative, using such high-affinity fluorescent super-chelator probes. The procedure allows direct, fast, and ultra-sensitive in-gel detection and analysis of soluble proteins as well as intact membrane protein complexes and macromolecular ribonucleoprotein particles.
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 ABC transporter Mdl1p, a structural and functional homologue of the transporter associated with antigen processing (TAP) plays an important role in intracellular peptide transport from the mitochondrial matrix of Saccharomyces cerevisiae. To characterize the ATP hydrolysis cycle of Mdl1p, the nucleotide-binding domain (NBD) was overexpressed in Escherichia coli and purified to homogeneity. The isolated NBD was active in ATP binding and hydrolysis with a turnover of 25 ATP per minute and a Km of 0.6 mm and did not show cooperativity in ATPase activity. However, the ATPase activity was non-linearly dependent on protein concentration (Hill coefficient of 1.7), indicating that the functional state is a dimer. Dimeric catalytic transition states could be trapped either by incubation with orthovanadate or beryllium fluoride, or by mutagenesis of the NBD. The nucleotide composition of trapped intermediate states was determined using [alpha-32P]ATP and [gamma-32P]ATP. Three different dimeric intermediate states were isolated, containing either two ATPs, one ATP and one ADP, or two ADPs. Based on these experiments, it was shown that: (i) ATP binding to two NBDs induces dimerization, (ii) in all isolated dimeric states, two nucleotides are present, (iii) phosphate can dissociate from the dimer, (iv) both nucleotides are hydrolyzed, and (v) hydrolysis occurs in a sequential mode. Based on these data, we propose a processive-clamp model for the catalytic cycle in which association and dissociation of the NBDs depends on the status of bound nucleotides.
Nanopores are key in portable sequencing and research given their ability to transport elongated DNA or small bioactive molecules through narrow transmembrane channels. Transport of folded proteins could lead to similar scientific and technological benefits. Yet this has not been realised due to the shortage of wide and structurally defined natural pores. Here we report that a synthetic nanopore designed via DNA nanotechnology can accommodate folded proteins. Transport of fluorescent proteins through single pores is kinetically analysed using massively parallel optical readout with transparent silicon-on-insulator cavity chips vs. electrical recordings to reveal an at least 20-fold higher speed for the electrically driven movement. Pores nevertheless allow a high diffusive flux of more than 66 molecules per second that can also be directed beyond equillibria. The pores may be exploited to sense diagnostically relevant proteins with portable analysis technology, to create molecular gates for drug delivery, or to build synthetic cells.
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.
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.
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.
The heterotetrameric human transfer RNA (tRNA) splicing endonuclease (TSEN) catalyzes the excision of intronic sequences from precursor tRNAs (pre-tRNAs)1. Mutations in TSEN and its associated RNA kinase CLP1 are linked to the neurodegenerative disease pontocerebellar hypoplasia (PCH)2–8. The three-dimensional (3D) assembly of TSEN/CLP1, the mechanism of substrate recognition, and the molecular details of PCH-associated mutations are not fully understood. Here, we present cryo-electron microscopy structures of human TSEN with intron-containing pre-tRNATyrgta and pre-tRNAArgtct. TSEN exhibits broad structural homology to archaeal endonucleases9 but has evolved additional regulatory elements that are involved in handling and positioning substrate RNA. Essential catalytic residues of subunit TSEN34 are organized for the 3’ splice site which emerges from a bulge-helix configuration. The triple-nucleotide bulge at the intron/3’-exon boundary is stabilized by an arginine tweezer motif of TSEN2 and an interaction with the proximal minor groove of the helix. TSEN34 and TSEN54 define the 3’ splice site by holding the tRNA body in place. TSEN54 adapts a bipartite fold with a flexible central region required for CLP1 binding. PCH-associated mutations are located far from pre-tRNA binding interfaces explaining their negative impact on structural integrity of TSEN without abrogating its catalytic activity in vitro10. Our work defines the molecular framework of pre-tRNA recognition and cleavage by TSEN and provides a structural basis to better understand PCH in the future.
The ATP-binding cassette transporter TAPL translocates polypeptides from the cytosol into the lysosomal lumen. TAPL can be divided into two functional units: coreTAPL, active in ATP-dependent peptide translocation, and the N-terminal membrane spanning domain, TMD0, responsible for cellular localization and interaction with the lysosomal associated membrane proteins LAMP-1 and LAMP-2. Although the structure and function of ABC transporters were intensively analyzed in the past, the knowledge about accessory membrane embedded domains is limited. Therefore, we expressed the TMD0 of TAPL via a cell-free expression system and confirmed its correct folding by NMR and interaction studies. In cell as well as cell-free expressed TMD0 forms oligomers, which were assigned as dimers by PELDOR spectroscopy and static light scattering. By NMR spectroscopy of uniformly and selectively isotope labeled TMD0 we performed a complete backbone and partial side chain assignment. Accordingly, TMD0 has a four transmembrane helix topology with a short helical segment in a lysosomal loop. The topology of TMD0 was confirmed by paramagnetic relaxation enhancement with paramagnetic stearic acid as well as by nuclear Overhauser effects with c6-DHPC and cross-peaks with water.
The ATP-binding cassette half-transporter Mdl1 from Saccharomyces cerevisiae has been proposed to be involved in the quality control of misassembled respiratory chain complexes by exporting degradation products generated by the m-AAA proteases from the matrix. Direct functional or structural data of the transport complex are, however, not known so far. After screening expression in various hosts, Mdl1 was overexpressed 100-fold to 1% of total mitochondrial membrane protein in S. cerevisiae. Based on detergent screens, Mdl1 was solubilized and purified to homogeneity. Mdl1 showed a high binding affinity for MgATP (Kd = 0.26 μm) and an ATPase activity with a Km of 0.86 mm (Hill coefficient of 0.98) and a turnover rate of 2.6 ATP/s. Mutagenesis of the conserved glutamate downstream of the Walker B motif (E599Q) or the conserved histidine of the H-loop (H631A) abolished ATP hydrolysis, whereas ATP binding was not affected. Mdl1 reconstituted into liposomes showed an ATPase activity similar to the solubilized complex. By single particle electron microscopy, a first three-dimensional structure of the mitochondrial ATP-binding cassette transporter was derived at 2.3-nm resolution, revealing a homodimeric complex in an open conformation.
Members of the ATP‐binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP‐binding cassette in the nucleotide‐binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs:
Jeder Mensch kämpft täglich erfolgreich mit Krankheitserregern, ohne dass er sich der komplexen molekularen Vorgänge dabei bewusst wäre. Wie in einem Hollywood-Streifen geht es rasant zur Sache. Ist das Immunsystem angeschlagen oder trifft es auf starke Gegner, kann eine Infektion binnen weniger Tage außer Kontrolle geraten und lebensbedrohliche Reaktionen hervorrufen. Der menschliche Organismus benötigt eine effiziente Verteidigungsstrategie gegen die Eindringlinge und muss, ebenso wie der britische Geheimdienst im Bond-Film, in die Ausbildung geübter Agenten investieren, Agenten mit Doppel-Null-Status. Agenten wie James Bond.
The potential of a protein-engineered His tag to immobilize macromolecules in a predictable orientation at metal-chelating lipid interfaces was investigated using recombinant 20 S proteasomes His-tagged in various positions. Electron micrographs demonstrated that the orientation of proteasomes bound to chelating lipid films could be controlled via the location of their His tags: proteasomes His-tagged at their sides displayed exclusively side-on views, while proteasomes His-tagged at their ends displayed exclusively end-on views. The activity of proteasomes immobilized at chelating lipid interfaces was well preserved. In solution, His-tagged proteasomes hydrolyzed casein at rates comparable with wild-type proteasomes, unless the His tags were located in the vicinity of the N termini of α-subunits. The N termini of α-subunits might partly occlude the entrance channel in α-rings through which substrates enter the proteasome for subsequent degradation. A combination of electron micrographs and atomic force microscope topographs revealed a propensity of vertically oriented proteasomes to crystallize in two dimensions on fluid lipid films. The oriented immobilization of His-tagged proteins at biocompatible lipid interfaces will assist structural studies as well as the investigation of biomolecular interaction via a wide variety of surface-sensitive techniques including single-molecule analysis.
The transporter associated with antigen processing (TAP) plays a key role in adaptive immunity by translocating proteasomal degradation products from the cytosol into the endoplasmic reticulum lumen for subsequent loading onto major histocompatibility (MHC) class I molecules. For functional and structural analysis of this ATP-binding cassette complex, we established the overexpression of TAP in the methylotrophic yeast Pichia pastoris. Screening of optimal solubilization and purification conditions allowed the isolation of the heterodimeric transport complex, yielding 30 mg of TAP/liter of culture. Detailed analysis of TAP function in the membrane, solubilized, purified, and reconstituted states revealed a direct influence of the native lipid environment on activity. TAP-associated phospholipids, essential for function, were profiled by liquid chromatography Fourier transform mass spectrometry. The antigen translocation activity is stimulated by phosphatidylinositol and -ethanolamine, whereas cholesterol has a negative effect on TAP activity.
Sandra Posch, Camilo Aponte-Santamaría, Richard Schwarzl, Andreas Karner, Matthias Radtke, Frauke Gräter, Tobias Obser, Gesa König, Maria A. Brehm, Hermann J. Gruber, Roland R. Netz, Carsten Baldauf, Reinhard Schneppenheim, Robert Tampé, Peter Hinterdorfer
Mutual A domain interactions in the force sensing protein von Willebrand factor
Journal of Structural Biology, Volume 197, Issue 1, January 2017, Pages 57-64. https://doi.org/10.1016/j.jsb.2016.04.012
We here give information for a deeper understanding of single molecule force spectroscopy (SMFS) data through the example of the blood protein von Willebrand factor (VWF). It is also shown, how fitting of rupture forces versus loading rate profiles in the molecular dynamics (MD) loading-rate range can be used to demonstrate the qualitative agreement between SMFS and MD simulations. The recently developed model by Bullerjahn, Sturm, and Kroy (BSK) was used for this demonstration. Further, Brownian dynamics (BD) simulations, which can be utilized to estimate the lifetimes of intramolecular VWF interactions under physiological shear, are described. For interpretation and discussion of the methods and data presented here, we would like to directly point the reader to the related research paper, “Mutual A domain interactions in the force sensing protein von Willebrand Factor” (Posch et al., 2016).
Cytotoxic T lymphocytes eliminate infected cells upon surface display of antigenic peptides on major histocompatibility complex I molecules. To promote immune evasion, UL49.5 of several varicelloviruses interferes with the pathway of major histocompatibility complex I antigen processing. However, the inhibition mechanism has not been elucidated yet. Within the macromolecular peptide-loading complex we identified the transporter associated with antigen processing (TAP1 and TAP2) as the prime target of UL49.5. Moreover, we determined the active oligomeric state and crucial elements of the viral factor. Remarkably, the last two residues of the cytosolic tail of UL49.5 are essential for endoplasmic reticulum (ER)-associated proteasomal degradation of TAP. However, this process strictly requires additional signaling of an upstream regulatory element in the ER lumenal domain of UL49.5. Within this new immune evasion mechanism, we show for the first time that additive elements of a small viral factor and their signaling across the ER membrane are essential for targeted degradation of a multi-subunit membrane complex.
Photoresponsive hydrogels can be employed to coordinate the organization of proteins in three dimensions (3D) and thus to spatiotemporally control their physiochemical properties by light. However, reversible and user-defined tethering of proteins and protein complexes to biomaterials pose a considerable challenge as this is a cumbersome process, which, in many cases, does not support the precise localization of biomolecules in the z direction. Here, we report on the 3D patterning of proteins with polyhistidine tags based on in-situ two-photon lithography. By exploiting a two-photon activatable multivalent chelator head, we established the protein mounting of hydrogels with micrometer precision. In the presence of photosensitizers, a substantially enhanced two-photon activation of the developed tool inside hydrogels was detected, enabling the user-defined 3D protein immobilization in hydrogels with high specificity, micrometer-scale precision, and under mild light doses. Our protein-binding strategy allows the patterning of a wide variety of proteins and offers the possibility to dynamically modify the biofunctional properties of materials at defined subvolumes in 3D.