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[Nachruf] Hugo Fasold
(2018)
Living matter is defined by metastability, implying a tightly balanced synthesis and turnover of cellular components. The first step of eukaryotic protein degradation via the ubiquitin-proteasome system (UPS) leads to peptides, which are subsequently degraded to single amino acids by an armada of proteases. A small fraction of peptides, however, escapes further cytosolic destruction and is transported by ATP-binding cassette (ABC) transporters into the endoplasmic reticulum (ER) and lysosomes. The ER-resident heterodimeric transporter associated with antigen processing (TAP) is a crucial component in adaptive immunity for the transport and loading of peptides onto major histocompatibility complex class I (MHC I) molecules. Although the function of the lysosomal resident homodimeric TAPL-like (TAPL) remains, until today, only loosely defined, an involvement in immune defense is anticipated since it is highly expressed in dendritic cells and macrophages. Here, we compare the gene organization and the function of single domains of both peptide transporters. We highlight the structural organization, the modes of substrate binding and translocation as well as physiological functions of both organellar transporters.
Molecular analysis of the ribosome recycling factor ABCE1 bound to the 30S post-splitting complex
(2020)
Ribosome recycling by the twin-ATPase ABCE1 is a key regulatory process in mRNA translation and surveillance and in ribosome-associated protein quality control in Eukarya and Archaea. Here, we captured the archaeal 30S ribosome post-splitting complex at 2.8 Å resolution by cryo-electron microscopy. The structure reveals the dynamic behavior of structural motifs unique to ABCE1, which ultimately leads to ribosome splitting. More specifically, we provide molecular details on how conformational rearrangements of the iron–sulfur cluster domain and hinge regions of ABCE1 are linked to closure of its nucleotide-binding sites. The combination of mutational and functional analyses uncovers an intricate allosteric network between the ribosome, regulatory domains of ABCE1, and its two structurally and functionally asymmetric ATP-binding sites. Based on these data, we propose a refined model of how signals from the ribosome are integrated into the ATPase cycle of ABCE1 to orchestrate ribosome recycling.
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.
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 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 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 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.
The transporter associated with antigen processing (TAP) translocates antigenic peptides from the cytosol into the endoplasmic reticular lumen for subsequent loading onto major histocompatibility complex (MHC) class I molecules. These peptide-MHC complexes are inspected at the cell surface by cytotoxic T-lymphocytes. Assembly of the functional peptide transport and loading complex depends on intra- and intermolecular packing of transmembrane helices (TMs). Here, we have examined the membrane topology of human TAP1 within an assembled and functional transport complex by cysteine-scanning mutagenesis. The accessibility of single cysteine residues facing the cytosol or endoplasmic reticular lumen was probed by a minimally invasive approach using membrane-impermeable, thiol-specific fluorophores in semipermeabilized “living” cells. TAP1 contains ten transmembrane segments, which place the N and C termini in the cytosol. The transmembrane domain consists of a translocation core of six TMs, a building block conserved among most ATP-binding cassette transporters, and a unique additional N-terminal domain of four TMs, essential for tapasin binding and assembly of the peptide-loading complex. This study provides a first map of the structural organization of the TAP machinery within the macromolecular MHCI peptide-loading 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.