Refine
Document Type
- Article (7)
Language
- English (7) (remove)
Has Fulltext
- yes (7)
Is part of the Bibliography
- no (7) (remove)
Keywords
- structural biology (7) (remove)
Institute
- Biochemie und Chemie (7) (remove)
Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot, J. L., and Engelman, D. M. (1990) Biochemistry 29, 4031–4037) is postulated to proceed in 2 steps: partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two, and the first three transmembrane (TM) segments, as well as a construct comprising TM1–TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin, A., Cohen, L. S., Arshava, B., Tantry, S., Becker, J. M., Zerbe, O., and Naider, F. (2009) Biophys. J. 96, 3187–3196), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123, and TM127 adopt predominantly native-like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior.
Determining the structure and mechanisms of all individual functional modules of cells at high molecular detail has often been seen as equal to understanding how cells work. Recent technical advances have led to a flush of high-resolution structures of various macromolecular machines, but despite this wealth of detailed information, our understanding of cellular function remains incomplete. Here, we discuss present-day limitations of structural biology and highlight novel technologies that may enable us to analyze molecular functions directly inside cells. We predict that the progression toward structural cell biology will involve a shift toward conceptualizing a 4D virtual reality of cells using digital twins. These will capture cellular segments in a highly enriched molecular detail, include dynamic changes, and facilitate simulations of molecular processes, leading to novel and experimentally testable predictions. Transferring biological questions into algorithms that learn from the existing wealth of data and explore novel solutions may ultimately unveil how cells work.
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:
The synergetic effects of combining structural biology and epr spectroscopy on membrane proteins
(2017)
Protein structures as provided by structural biology such as X-ray crystallography, cryo-electron microscopy and NMR spectroscopy are key elements to understand the function of a protein on the molecular level. Nonetheless, they might be error-prone due to crystallization artifacts or, in particular in case of membrane-imbedded proteins, a mostly artificial environment. In this review, we will introduce different EPR spectroscopy methods as powerful tools to complement and validate structural data gaining insights in the dynamics of proteins and protein complexes such that functional cycles can be derived. We will highlight the use of EPR spectroscopy on membrane-embedded proteins and protein complexes ranging from receptors to secondary active transporters as structural information is still limited in this field and the lipid environment is a particular challenge.
A(syn)-U/T and G(syn)-C+ Hoogsteen (HG) base pairs (bps) are energetically more disfavored relative to Watson–Crick (WC) bps in A-RNA as compared to B-DNA by >1 kcal/mol for reasons that are not fully understood. Here, we used NMR spectroscopy, optical melting experiments, molecular dynamics simulations and modified nucleotides to identify factors that contribute to this destabilization of HG bps in A-RNA. Removing the 2′-hydroxyl at single purine nucleotides in A-RNA duplexes did not stabilize HG bps relative to WC. In contrast, loosening the A-form geometry using a bulge in A-RNA reduced the energy cost of forming HG bps at the flanking sites to B-DNA levels. A structural and thermodynamic analysis of purine-purine HG mismatches reveals that compared to B-DNA, the A-form geometry disfavors syn purines by 1.5–4 kcal/mol due to sugar-backbone rearrangements needed to sterically accommodate the syn base. Based on MD simulations, an additional penalty of 3–4 kcal/mol applies for purine-pyrimidine HG bps due to the higher energetic cost associated with moving the bases to form hydrogen bonds in A-RNA versus B-DNA. These results provide insights into a fundamental difference between A-RNA and B-DNA duplexes with important implications for how they respond to damage and post-transcriptional modifications.
In bacteria, the regulation of gene expression by cis-acting transcriptional riboswitches located in the 5'-untranslated regions of messenger RNA requires the temporal synchronization of RNA synthesis and ligand binding-dependent conformational refolding. Ligand binding to the aptamer domain of the riboswitch induces premature termination of the mRNA synthesis of ligand-associated genes due to the coupled formation of 3'-structural elements acting as terminators. To date, there has been no high resolution structural description of the concerted process of synthesis and ligand-induced restructuring of the regulatory RNA element. Here, we show that for the guanine-sensing xpt-pbuX riboswitch from Bacillus subtilis, the conformation of the full-length transcripts is static: it exclusively populates the functional off-state but cannot switch to the on-state, regardless of the presence or absence of ligand. We show that only the combined matching of transcription rates and ligand binding enables transcription intermediates to undergo ligand-dependent conformational refolding.
Mechanism of Na+-dependent citrate transport from the structure of an asymmetrical CitS dimer
(2015)
The common human pathogen Salmonella enterica takes up citrate as a nutrient via the sodium symporter SeCitS. Uniquely, our 2.5 Å x-ray structure of the SeCitS dimer shows three different conformations of the active protomer. One protomer is in the outside-facing state. Two are in different inside-facing states. All three states resolve the substrates in their respective binding environments. Together with comprehensive functional studies on reconstituted proteoliposomes, the structures explain the transport mechanism in detail. Our results indicate a six-step process, with a rigid-body 31° rotation of a helix bundle that translocates the bound substrates by 16 Å across the membrane. Similar transport mechanisms may apply to a wide variety of related and unrelated secondary transporters, including important drug targets.