Cryo-EM structure of the extracellular domain of murine Thrombopoietin Receptor in complex with Thrombopoietin.

Thrombopoietin (Tpo) is the primary regulator of megakaryocyte and platelet numbers and is required for haematopoetic stem cell maintenance. Tpo functions by binding its receptor (TpoR, a homodimeric Class I cytokine receptor) and initiating cell proliferation or differentiation. Here we characterise the murine Tpo:TpoR signalling complex biochemically and structurally, using cryo-electron microscopy. Tpo uses opposing surfaces to recruit two copies of receptor, forming a 1:2 complex. Although it binds to the same, membrane-distal site on both receptor chains, it does so with significantly different affinities and its highly glycosylated C-terminal domain is not required. In one receptor chain, a large insertion, unique to TpoR, forms a partially structured loop that contacts cytokine. Tpo binding induces the juxtaposition of the two receptor chains adjacent to the cell membrane. The therapeutic agent romiplostim also targets the cytokine-binding site and the characterisation presented here supports the future development of improved TpoR agonists.

Structures of the interleukin 11 signalling complex reveal gp130 dynamics and the inhibitory mechanism of a cytokine variant.

Interleukin (IL-)11, an IL-6 family cytokine, has pivotal roles in autoimmune diseases, fibrotic complications, and solid cancers. Despite intense therapeutic targeting efforts, structural understanding of IL-11 signalling and mechanistic insights into current inhibitors are lacking. Here we present cryo-EM and crystal structures of the human IL-11 signalling complex, including the complex containing the complete extracellular domains of the shared IL-6 family ß-receptor, gp130. We show that complex formation requires conformational reorganisation of IL-11 and that the membrane-proximal domains of gp130 are dynamic. We demonstrate that the cytokine mutant, IL-11 Mutein, competitively inhibits signalling in human cell lines. Structural shifts in IL-11 Mutein underlie inhibition by altering cytokine binding interactions at all three receptor-engaging sites and abrogating the final gp130 binding step. Our results reveal the structural basis of IL-11 signalling, define the molecular mechanisms of an inhibitor, and advance understanding of gp130-containing receptor complexes, with potential applications in therapeutic development.

Phosphorylation-dependent pseudokinase domain dimerization drives full-length MLKL oligomerization.

The necroptosis pathway is a lytic, pro-inflammatory mode of cell death that is widely implicated in human disease, including renal, pulmonary, gut and skin inflammatory pathologies. The precise mechanism of the terminal steps in the pathway, where the RIPK3 kinase phosphorylates and triggers a conformation change and oligomerization of the terminal pathway effector, MLKL, are only emerging. Here, we structurally identify RIPK3-mediated phosphorylation of the human MLKL activation loop as a cue for MLKL pseudokinase domain dimerization. MLKL pseudokinase domain dimerization subsequently drives formation of elongated homotetramers. Negative stain electron microscopy and modelling support nucleation of the MLKL tetramer assembly by a central coiled coil formed by the extended, ~80 Å brace helix that connects the pseudokinase and executioner four-helix bundle domains. Mutational data assert MLKL tetramerization as an essential prerequisite step to enable the release and reorganization of four-helix bundle domains for membrane permeabilization and cell death.

The structure of the PA28-20S proteasome complex from Plasmodium falciparum and implications for proteostasis.

The activity of the proteasome 20S catalytic core is regulated by protein complexes that bind to one or both ends. The PA28 regulator stimulates 20S proteasome peptidase activity in vitro, but its role in vivo remains unclear. Here, we show that genetic deletion of the PA28 regulator from Plasmodium falciparum (Pf) renders malaria parasites more sensitive to the antimalarial drug dihydroartemisinin, indicating that PA28 may play a role in protection against proteotoxic stress. The crystal structure of PfPA28 reveals a bell-shaped molecule with an inner pore that has a strong segregation of charges. Small-angle X-ray scattering shows that disordered loops, which are not resolved in the crystal structure, extend from the PfPA28 heptamer and surround the pore. Using single particle cryo-electron microscopy, we solved the structure of Pf20S in complex with one and two regulatory PfPA28 caps at resolutions of 3.9 and 3.8 Å, respectively. PfPA28 binds Pf20S asymmetrically, strongly engaging subunits on only one side of the core. PfPA28 undergoes rigid body motions relative to Pf20S. Molecular dynamics simulations support conformational flexibility and a leaky interface. We propose lateral transfer of short peptides through the dynamic interface as a mechanism facilitating the release of proteasome degradation products.

A visual approach to proteomics.

Cryo-electron tomography is an emerging imaging technique that has unique potential for molecular cell biology. At the present resolution of 4-5 nm, large supramolecular structures can be studied in unperturbed cellular environments and, in the future, it will become possible to map molecular landscapes inside cells in a more comprehensive manner. 'Visual proteomics' aims to complement and extend mass-spectrometry-based inventories, and to provide a quantitative description of the macromolecular interactions that underlie cellular functions.

Optically transparent porous medium for nondestructive studies of microbial biofilm architecture and transport dynamics.

We describe a novel and noninvasive, microscopy-based method for visualizing the structure and dynamics of microbial biofilms, individual fluorescent microbial cells, and inorganic colloids within a model porous medium. Biofilms growing in flow cells packed with granules of an amorphous fluoropolymer could be visualized as a consequence of refractive index matching between the solid fluoropolymer grains and the aqueous immersion medium. In conjunction with the capabilities of confocal microscopy for nondestructive optical sectioning, the use of amorphous fluoropolymers as a solid matrix permits observation of organisms and dynamic processes to a depth of 2 to 3 mm, whereas sediment biofilms growing in sand-filled flow cells can only be visualized in the region adjacent to the flow cell wall. This method differs fundamentally from other refractive index-matching applications in that optical transparency was achieved by matching a solid phase to water (and not vice versa), thereby permitting real-time microscopic studies of particulate-containing, low-refractive-index media such as biological and chromatographic systems.

Interactions between laponite and microbial biofilms in porous media: implications for colloid transport and biofilm stability.

Quartz sand columns and sand-filled microscope flow cells were used to investigate the transport characteristics of the clay colloid laponite, and a biofilm-forming bacterium, Pseudomonas aeruginosa SG81. Separate experiments were performed with each particle to determine their individual transport characteristics in clean sand columns. In a second set of experiments, bacterial biofilms were formed prior to introduction of the clay colloids. In the independent transport experiments, bacteria and laponite each conformed to known physicochemical principles. A sodium chloride concentration of 7 x 10(-2) M caused complete retention of the laponite within the sand columns. P. aeruginosa SG81 was generally less influenced by ionic strength effects; it showed relatively low mobility at all ionic strengths tested and some (albeit reduced) mobility when introduced to the columns in 1M NaCl, the highest concentration tested, but nevertheless showed reproducible trends. Under conditions favourable to laponite retention and biofilm stability (7 x 10(-2) MNaCl), laponite suspensions were able to remobilise a portion of the attached bacterial biomass. At low ionic strength, the profile of laponite elution was also altered in the presence of a P. aeruginosa biofilm. These observations suggest that while a reduction in ionic strength has a dominant influence on the mobilisation of biological and inorganic colloids, the presence of laponite and biomass can have a distinct influence on the mobility of both types of colloids. Since these events are likely to occur in subsurface environments, our results suggest that colloid-biofilm interactions will have implications for colloid-bound contaminant transport and the remobilisation of pathogens.