Remigiusz Worch

Cholesterol and Sphingomyelin Drive Ligand-independent T-cell Antigen Receptor Nanoclustering

Background: The TCR forms nanoclusters in the plasma membrane independent of ligand binding. Results: Membrane cholesterol and sphingomyelin facilitate TCR nanoclustering, thereby enhancing the avidity toward the ligand. Conclusion: The membrane lipid composition regulates the degree of TCR nanoclustering and thus T-cell sensitivity. Significance: This work contributes to the understanding of the consequences of specific lipid-membrane protein interactions. The T-cell antigen receptor (TCR) exists in monomeric and nanoclustered forms independently of antigen binding. Although the clustering is involved in the regulation of T-cell sensitivity, it is unknown how the TCR nanoclusters form. We show that cholesterol is required for TCR nanoclustering in T cells and that this clustering enhances the avidity but not the affinity of the TCR-antigen interaction. Investigating the mechanism of the nanoclustering, we found that radioactive photocholesterol specifically binds to the TCRβ chain in vivo. In order to reduce the complexity of cellular membranes, we used a synthetic biology approach and reconstituted the TCR in liposomes of defined lipid composition. Both cholesterol and sphingomyelin were required for the formation of TCR dimers in phosphatidylcholine-containing large unilamellar vesicles. Further, the TCR was localized in the liquid disordered phase in giant unilamellar vesicles. We propose a model in which cholesterol and sphingomyelin binding to the TCRβ chain causes TCR dimerization. The lipid-induced TCR nanoclustering enhances the avidity to antigen and thus might be involved in enhanced sensitivity of memory compared with naive T cells. Our work contributes to the understanding of the function of specific nonannular lipid-membrane protein interactions.

Focus on composition and interaction potential of single-pass transmembrane domains

Transmembrane domains (TMD) connect the inner with the outer world of a living cell. Single TMD containing (bitopic) receptors are of particular interest, because their oligomerization seems to be a common activation mechanism in cell signaling. We analyzed the composition of TMDs in bitopic proteins within the proteomes of 12 model organisms. The average number of strongly polar and charged residues decreases during evolution, while the occurrence of a dimerization motif, GxxxG, remains unchanged. This may reflect the avoidance of unspecific binding within a growing receptor interaction network. In addition, we propose a new experimental approach for studying helix–helix interactions in giant plasma membrane vesicles using scanning fluorescence cross‐correlation spectroscopy. Measuring eGFP/mRFP tagged versions of cytokine receptors confirms the homotypic interactions of the erythropoietin receptor in contrast to the Interleukin‐4 receptor chains. As a proof of principle, by swapping the TMDs, the interaction potential of erythropoietin receptor was partially transferred to Interleukin‐4 receptor α and vice versa. Non‐interacting receptors can therefore serve as host molecules for TMDs whose oligomerization capability must be assessed. Computational analysis of the free energy gain resulting from TMD dimer formation strongly corroborates the experimental findings, potentially allowing in silico pre‐screening of interacting pairs.

Single cell analysis of ligand binding and complex formation of interleukin-4 receptor subunits.

Interleukin-4 (IL-4) is an important class I cytokine involved in adaptive immunity. IL-4 binds with high affinity to the single-pass transmembrane receptor IL-4Rα. Subsequently, IL-4Rα/IL-4 is believed to engage a second receptor chain, either IL-2Rγ or IL-13Rα1, to form type I or II receptor complexes, respectively. This ternary complex formation then triggers downstream signaling via intracellular Janus kinases bound to the cytoplasmic receptor tails. Here, we study the successive steps of complex formation at the single cell level with confocal fluorescence imaging and correlation spectroscopy. We characterize binding and signaling of fluorescently labeled IL-4 by flow cytometry of IL-4-dependent BaF3 cells. The affinity to ectopically expressed IL-4Rα was then measured by single-color fluorescence correlation spectroscopy in adherent HEK293T cells that express the components of the type II IL-4R but not type I. Finally, IL-4-induced complex formation was tested by dual-color fluorescence cross-correlation spectroscopy. The data provide evidence for codiffusion of IL-4-A647 bound IL-4Rα and the type II subunit IL-13Rα1 fused to enhanced green fluorescent protein, whereas type I complexes containing IL-2Rγ and JAK3 were not detected at the cell surface. This behavior may reflect hitherto undefined differences in the mode of receptor activation between type I (lymphoid) and type II (epithelial) receptor expressing cells.

Dynamics and Interaction of Interleukin-4 Receptor Subunits in Living Cells

It has long been established that dimerization of Interleukin-4 receptor (IL-4R) subunits is a pivotal step for JAK/STAT signal transduction. However, ligand-induced complex formation at the surface of living cells has been challenging to observe. Here we report an experimental assay employing trisNTA dyes for orthogonal, external labeling of eGFP-tagged receptor constructs that allows the quantification of receptor heterodimerization by dual-color fluorescence cross-correlation spectroscopy. Fluorescence cross-correlation spectroscopy analysis at the plasma membrane shows that IL-4R subunit dimerization is indeed a strictly ligand-induced process. Under conditions of saturating cytokine occupancy, we determined intramembrane dissociation constants (K(d,2D)) of 180 and 480 receptors per μm(2) for the type-2 complexes IL-4:IL-4Rα/IL-13Rα1 and IL-13:IL-13Rα1/IL-4Rα, respectively. For the lower affinity type-1 complex IL-4:IL-4Rα/IL-2Rγ, we estimated a K(d,2D) of ∼1000 receptors per μm(2). The receptor densities required for effective dimerization thus exceed the typical, average expression levels by several orders of magnitude. In addition, we find that all three receptor subunits accumulate rapidly within a subpopulation of early sorting and recycling endosomes stably anchored just beneath the plasma membrane (cortical endosomes, CEs). The receptors, as well as labeled IL-4 and trisNTA ligands are specifically trafficked into CEs by a constitutive internalization mechanism. This may compensate for the inherent weak affinities that govern ligand-induced receptor dimerization at the plasma membrane. Consistently, activated receptors are also concentrated at the CEs. Our observations thus suggest that receptor trafficking may play an important role for the regulation of IL-4R-mediated JAK/STAT signaling.

Effect of temperature on the formation of liquid phase-separating giant unilamellar vesicles (GUV)

Giant unilamellar vesicles (GUVs) are widely used as model systems to study both, lipid and membrane protein behavior. During their preparation by the commonly applied electroformation method, a number of issues must be considered to avoid the production of artifacts due to a poor lipid hydration and protein degradation. Here we focus on the effect of preparation temperature on GUVs composed of the most commonly used domain-forming mixture dioleoylelphospatidylcholine/shingomyelin/cholesterol (DOPC/SM/chol) (2/2/1). Lower production temperatures are generally preferable when aiming at a functional reconstitution of proteins into the membrane. On the other hand, lower growth temperature is suspected to alter the lipid composition and the yield of phase-separating vesicles. By confocal imaging, we find that vesicles prepared significantly above and below the melting temperature T(m) have the same overall morphology, similar size distributions of vesicles and a similar area coverage by liquid-ordered (L(o)) domains. However, a large population analysis indeed reveals a different overall yield of phase-separating vesicles. Two-focus scanning fluorescence correlation spectroscopy measurements did not show any divergence of lipid analog mobility in (L(o)) and (L(d)) phases in vesicles prepared at different temperatures, indicating that the lowered growth temperature did not influence the lipid organization within the two phases. Moreover, the expected advantages of lower preparation temperature for proteo-GUVs could be exemplified by the reconstitution of voltage dependent anion channel (VDAC) into DOPC/SM/chol GUVs, which aggregates at high, but not at low preparation temperatures.

Stacking efficiency and flexibility analysis of aromatic amino acids in cap‐binding proteins

Recognition of the ribonucleic acid 5′ termini (RNA 5′ cap) by a wide class of cap‐binding proteins is largely accomplished by cation‐π stacking that involves the positively charged 7‐methylguanine ring and aromatic amino acid side chains. Quantum calculations of the stacking energy were performed by means of MP2 perturbation method for binary and ternary associates composed of the 7‐methylguanine moiety and tryptophan, tyrosine, or phenylalanine, in their spatial orientations known from the crystalline cap‐protein complexes. The results clearly pointed to an enhancement of the stacking energy due to a net positive charge in the cap guanine moiety and allowed analysis of a role of various amino acids in stabilization of the complexes. Conformational flexibility of the aromatic amino acids taking part in binding ligands to a wide class of RNA‐recognizing proteins, including the cap‐binding proteins, was determined by regional order neural network (RONN) algorithm that provides results close to those of the crystallographic B‐factors analysis. Interestingly, some of the tyrosines that are classified in general as “rigid” showed high flexibility when engaged in binding the cap to nuclear cap‐binding protein complex CBC and to viral methyltransferase VP39. Parallel analyses of the binding energy and flexibility of the protein fragments engaged in the binding leads to understanding differences in molecular mechanisms of the cap recognition by various proteins, CBC compared with the eukaryotic initiation factor eIF4E, and enzymes vs. other protein factors. Proteins 2008.

The helical hairpin structure of the influenza fusion peptide can be seen on a hydrophobic moment map.

An assignment of the helical hairpin of the influenza fusion peptide has been made based on the hydrophobic moments, represented in a form of two‐dimensional map. Such assignment holds for all serotypes, even for the cases of mutations altering the amino acid character. Similar results are obtained for the experimentally developed hydrophobicity scales, whose values reflect the transfer energies between aqueous and membrane environments. A distinct, however still structure‐related hydrophobic map corresponds to a helical and contiguous HIV gp41 fp. The method may be used as a simple tool for sequence‐based prediction of structures adopted by viral fusion peptides.

Three conserved C-terminal residues of influenza fusion peptide alter its behavior at the membrane interface

The N-terminal fragment of the viral hemagglutinin HA2 subunit is termed a fusion peptide (HAfp). The 23-amino acid peptide (HAfp1-23) contains three C-terminal W21-Y22-G23 residues which are highly conserved among serotypes of influenza A and has been shown to form a tight helical hairpin very distinct from the boomerang structure of HAfp1-20. We studied the effect of peptide length on fusion properties, structural dynamics, and binding to the membrane interface. We developed a novel fusion visualization assay based on FLIM microscopy on giant unilamellar vesicles (GUV). By means of molecular dynamics simulations and spectroscopic measurements, we show that the presence of the three C-terminal W21-Y22-G23 residues promotes the hairpin formation, which orients perpendicularly to the membrane plane and induces more disorder in the surrounding lipids than the less structured HAfp1-20. Moreover, we report cholesterol-enriched domain formation induced exclusively by the longer fusion peptide.

New Insight into Metal Ion-Driven Catalysis of Nucleic Acids by Influenza PA-Nter

PA subunit of influenza RNA-dependent RNA polymerase deserves constantly increasing attention due to its essential role in influenza life cycle. N-terminal domain of PA (PA-Nter) harbors endonuclease activity, which is indispensable in viral transcription and replication. Interestingly, existing literature reports on in vitro ion preferences of the enzyme are contradictory. Some show PA-Nter activity exclusively with Mn2+, whereas others report Mg2+ as a natural cofactor. To clarify it, we performed a series of experiments with varied ion concentrations and substrate type. We observed cleavage in the presence of both ions, with a slight preference for manganese, however PA-Nter activity highly depended on the amount of residual, co-purified ions. Furthermore, to quantify cleavage reaction rate, we applied fluorescence cross-correlation spectroscopy (FCCS), providing highly sensitive and real-time monitoring of single molecules. Using nanomolar ssDNA in the regime of enzyme excess, we estimated the maximum reaction rate at 0.81± 0.38 and 1.38± 0.34 nM/min for Mg2+ and Mn2+, respectively. However, our calculations of PA-Nter ion occupancy, based on thermodynamic data, suggest Mg2+ to be a canonical metal in PA-Nter processing of RNA in vivo. Presented studies constitute a step toward better understanding of PA-Nter ion-dependent activity, which will possibly contribute to new successful inhibitor design in the future.

NOVEL WAY OF CAPPING mRNA TRIMER AND STUDIES OF ITS INTERACTION WITH HUMAN NUCLEAR CAP-BINDING COMPLEX

Binding of mRNA 5′ cap by the nuclear cap-binding complex (CBC) is crucial for a wide variety of mRNA metabolic events. The interaction involving the CBP20 subunit of CBC is mediated by numerous hydrogen bonds and by stacking of the tyrosine sidechains with two first bases of the capped mRNA. To examine a possible role of a longer mRNA chain in the CBC-cap recognition, we have synthesized an mRNA tetramer using a novel way of capping an RNA trimer and determined its affinity for CBC by fluorescence titration.