Ralf Stehle

Structural basis for RNA recognition in roquin-mediated post-transcriptional gene regulation.

Roquin function in T cells is essential for the prevention of autoimmune disease. Roquin interacts with the 3′ untranslated regions (UTRs) of co-stimulatory receptors and controls T-cell activation and differentiation. Here we show that the N-terminal ROQ domain from mouse roquin adopts an extended winged-helix (WH) fold, which is sufficient for binding to the constitutive decay element (CDE) in the Tnf 3′ UTR. The crystal structure of the ROQ domain in complex with a prototypical CDE RNA stem-loop reveals tight recognition of the RNA stem and its triloop. Surprisingly, roquin uses mainly non-sequence-specific contacts to the RNA, thus suggesting a relaxed CDE consensus and implicating a broader spectrum of target mRNAs than previously anticipated. Consistently with this, NMR and binding experiments with CDE-like stem-loops together with cell-based assays confirm roquin-dependent regulation of relaxed CDE consensus motifs in natural 3′ UTRs.

Nonaqueous Microemulsions Based on N,N′-Alkylimidazolium Alkylsulfate Ionic Liquids

The ternary system composed of the ionic liquid surfactant (IL-S) 1-butyl-3-methylimidazolium dodecylsulfate ([Bmim][DodSO4]), the room temperature ionic liquid (RTIL) 1-ethyl-3-methylimidazolium ethylsulfate ([Emim][EtSO4]), and toluene has been investigated. Three major mechanisms guiding the structure of the isotropic phase were identified by means of conductometric experiments, which have been correlated to the presence of oil-in-IL, bicontinuous, and IL-in-oil microemulsions. IL-S forms micelles in toluene, which swell by adding RTIL as to be shown by dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) experiments. Therefore, it is possible to form water-free IL-in-oil reverse microemulsions ≤10 nm in size as a new type of nanoreactor.

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

Significance G proteins are molecular switches for cellular signaling induced by G-protein–coupled receptor activation. The Gα subunit is the central timer of signal transduction regulated by GTP hydrolysis, which returns the system to its inactive state. Although previous work has characterized the structural states of Gα during the GTPase cycle, we show here that Gα is highly dynamic in the apo and GDP-bound states but in complex with GTP is completely rigid and is locked in a defined domain orientation. These insights help demonstrate that the conformational plasticity of G proteins is a central feature of their switching functionality. Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein–coupled receptor (GPCR) activation. Agonist–receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDP-bound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape.

Structure of Rab11-FIP3-Rabin8 reveals simultaneous binding of FIP3 and Rabin8 effectors to Rab11

The small GTPase Rab11 and its effectors FIP3 and Rabin8 are essential to membrane-trafficking pathways required for cytokinesis and ciliogenesis. Although effector binding is generally assumed to be sequential and mutually exclusive, we show that Rab11 can simultaneously bind FIP3 and Rabin8. We determined crystal structures of human Rab11–GMPPNP–Rabin8 and Rab11–GMPPNP–FIP3–Rabin8. The structures reveal that the C-terminal domain of Rabin8 adopts a previously undescribed fold that interacts with Rab11 at an unusual effector-binding site neighboring the canonical FIP3-binding site. We show that Rab11–GMPPNP–FIP3–Rabin8 is more stable than Rab11–GMPPNP–Rabin8, owing to direct interaction between Rabin8 and FIP3 within the dual effector–bound complex. The data allow us to propose a model for how membrane-targeting complexes assemble at the trans-Golgi network and recycling endosomes, through multiple weak interactions that create high-avidity complexes.

Bilayer undulation dynamics in unilamellar phospholipid vesicles: Effect of temperature, cholesterol and trehalose

We report a combined dynamic light scattering (DLS) and neutron spin-echo (NSE) study on the local bilayer undulation dynamics of phospholipid vesicles composed of 1,2-dimyristoyl-glycero-3-phosphatidylcholine (DMPC) under the influence of temperature and the additives cholesterol and trehalose. The additives affect vesicle size and self-diffusion. Mechanical properties of the membrane and corresponding bilayer undulations are tuned by changing lipid headgroup or acyl chain properties through temperature or composition. On the local length scale, changes at the lipid headgroup influence the bilayer bending rigidity κ less than changes at the lipid acyl chain: We observe a bilayer softening around the main phase transition temperature Tm of the single lipid system, and stiffening when more cholesterol is added, in concordance with literature. Surprisingly, no effect on the mechanical properties of the vesicles is observed upon the addition of trehalose.

An enzyme containing microemulsion based on skin friendly oil and surfactant as decontamination medium for organo phosphates: Phase behavior, structure, and enzyme activity

The present contribution presents a microemulsion system containing cosmetic oil and sugar surfactant and the enzyme diisopropyl fluorophosphatase (DFPase) as active agent for the decontamination of human skin. The bicontinuous structure and the physical properties of the microemulsion are characterized by dynamic light scattering and small angle neutron scattering. The DFPase from the squid Loligo vulgaris is catalyzing the hydrolysis of highly toxic organophosphates. The effect of the enzyme on the structure of the microemulsion is investigated. Moreover, the enzyme/microemulsion system is also studied with respect to its activity using nuclear magnetic resonance spectroscopy leading to promising results. A fast decomposition of the nerve agent sarin is achieved.

The DFPase from Loligo vulgaris in sugar surfactant-based bicontinuous microemulsions: structure, dynamics, and enzyme activity

The enzyme diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris is of great interest because of its ability to catalyze the hydrolysis of highly toxic organophosphates. In this work, the enzyme structure in solution (native state) was studied by use of different scattering methods. The results are compared with those from hydrodynamic model calculations based on the DFPase crystal structure. Bicontinuous microemulsions made of sugar surfactants are discussed as host systems for the DFPase. The microemulsion remains stable in the presence of the enzyme, which is shown by means of scattering experiments. Moreover, activity assays reveal that the DFPase still has high activity in this complex reaction medium. To complement the scattering experiments cryo-SEM was also employed to study the microemulsion structure.

Influence of charge density on bilayer bending rigidity in lipid vesicles: A combined dynamic light scattering and neutron spin-echo study

We report a combined dynamic light scattering and neutron spin-echo study on vesicles composed of the uncharged stabilizing lipid 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). Mechanical properties of a model membrane and thus the corresponding bilayer undulation dynamics can be specifically tuned by changing its composition through lipid headgroup or acyl chain properties. We compare the undulation dynamics in lipid vesicles composed of DMPC/DOTAP to vesicles composed of a mixture of the uncharged helper lipid DMPC with the also uncharged reference lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). We have performed dynamic light scattering on the lipid mixtures to investigate changes in lipid vesicle size and the corresponding center-of-mass diffusion. We study lipid translational diffusion in the membrane plane and local bilayer undulations using neutron spin-echo spectroscopy, on two distinct time scales, namely around 25 ns and around 150 ns. Finally, we calculate the respective bilayer bending rigidities κ for both types of lipid vesicles. We find that on the local length scale inserting lipid headgroup charge into the membrane influences the bilayer undulation dynamics and bilayer bending rigidity κ less than inserting lipid acyl chain unsaturation: We observe a bilayer softening with increasing inhomogenity of the lipid mixture, which could be caused by a hydrophobic mismatch between the acyl chains of the respective lipid components, causing a lateral phase segregation (domain formation) in the membrane plane.Graphical abstract

Molecular architecture and dynamics of ASH1 mRNA recognition by its mRNA-transport complex

mRNA localization is an essential mechanism of gene regulation and is required for processes such as stem-cell division, embryogenesis and neuronal plasticity. It is not known which features in the cis-acting mRNA localization elements (LEs) are specifically recognized by motor-containing transport complexes. To the best of our knowledge, no high-resolution structure is available for any LE in complex with its cognate protein complex. Using X-ray crystallography and complementary techniques, we carried out a detailed assessment of an LE of the ASH1 mRNA from yeast, its complex with its shuttling RNA-binding protein She2p, and its highly specific, cytoplasmic complex with She3p. Although the RNA alone formed a flexible stem loop, She2p binding induced marked conformational changes. However, only joining by the unstructured She3p resulted in specific RNA recognition. The notable RNA rearrangements and joint action of a globular and an unfolded RNA-binding protein offer unprecedented insights into the step-wise maturation of an mRNA-transport complex.

Inhibition of Canonical NF-κB Signaling by a Small Molecule Targeting NEMO-Ubiquitin Interaction.

The IκB kinase (IKK) complex acts as the gatekeeper of canonical NF-κB signaling, thereby regulating immunity, inflammation and cancer. It consists of the catalytic subunits IKKα and IKKβ and the regulatory subunit NEMO/IKKγ. Here, we show that the ubiquitin binding domain (UBAN) in NEMO is essential for IKK/NF-κB activation in response to TNFα, but not IL-1β stimulation. By screening a natural compound library we identified an anthraquinone derivative that acts as an inhibitor of NEMO-ubiquitin binding (iNUB). Using biochemical and NMR experiments we demonstrate that iNUB binds to NEMOUBAN and competes for interaction with methionine-1-linked linear ubiquitin chains. iNUB inhibited NF-κB activation upon UBAN-dependent TNFα and TCR/CD28, but not UBAN-independent IL-1β stimulation. Moreover, iNUB was selectively killing lymphoma cells that are addicted to chronic B-cell receptor triggered IKK/NF-κB activation. Thus, iNUB disrupts the NEMO-ubiquitin protein-protein interaction interface and thereby inhibits physiological and pathological NF-κB signaling.