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A hallmark of inflammasome activation is the ASC speck, a micrometre-sized structure formed by the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), which consists of a pyrin domain (PYD) and a caspase recruitment domain (CARD). Here we show that assembly of the ASC speck involves oligomerization of ASC PYD into filaments and cross-linking of these filaments by ASC CARD . ASC mutants with a non-functional CARD only assemble filaments but not specks, and moreover disrupt endogenous specks in primary macrophages. Systematic site-directed mutagenesis of ASC PYD is used to identify oligomerization-deficient ASC mutants and demonstrate that ASC speck formation is required for efficient processing of IL-1β, but dispensable for gasdermin-D cleavage and pyroptosis induction. Our results suggest that the oligomerization of ASC creates a multitude of potential caspase-1 activation sites, thus serving as a signal amplification mechanism for inflammasome-mediated cytokine production. Inflammasomes regulate IL-1β family maturation and pyroptosis. Here the authors show that ASC oligomerization and the formation of ASC specks are needed for IL-1β processing, but are not required for pyroptosis, indicating distinct inflammasome regulatory pathways.

The 70 kDa heat shock protein (Hsp70) family of molecular chaperones ensures protein biogenesis and homeostasis, driven by ATP hydrolysis. Here, we introduce in-cyclo NMR , an experimental setup that combines high-resolution NMR spectroscopy with an ATP recovery and a phosphate removal system. In-cyclo NMR simultaneously resolves kinetic rates and structural information along functional cycles of ATP-driven molecular machines. We benchmark the method on the nucleotide binding domain (NBD) of the human Hsp70 chaperone BiP. The protein cycles through ATP binding, hydrolysis, and two parallel pathways of product release. We determine the kinetic rates of all eleven underlying elementary reactions and show these to match independent measurements. The two product release pathways regulate the cycle duration dependent on the products concentration. Under physiological conditions, they are both used. The in-cyclo NMR method will serve as a platform for studies of ATP-driven functional cycles at a remarkable level of detail. Mas et al introduce in-cyclo NMR to quantify all states and kinetic steps of Hsp70 chaperones ATPase cycle. In-cyclo NMR will enable studies of other molecular machines at an unprecedented level of detail.

Eukaryotic cells can undergo different forms of programmed cell death, many of which culminate in plasma membrane rupture as the defining terminal event 1 – 7 . Plasma membrane rupture was long thought to be driven by osmotic pressure, but it has recently been shown to be in many cases an active process, mediated by the protein ninjurin-1 8 (NINJ1). Here we resolve the structure of NINJ1 and the mechanism by which it ruptures membranes. Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane α-helices. Filament directionality and stability is defined by two amphipathic α-helices that interlink adjacent filament subunits. The NINJ1 filament features a hydrophilic side and a hydrophobic side, and molecular dynamics simulations show that it can stably cap membrane edges. The function of the resulting supramolecular arrangement was validated by site-directed mutagenesis. Our data thus suggest that, during lytic cell death, the extracellular α-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. The membrane protein NINJ1 is therefore an interactive component of the eukaryotic cell membrane that functions as an in-built breaking point in response to activation of cell death. Structural, biochemical and mutagenesis studies indicate that, in dying cells, the membrane protein NINJ1 assembles into filaments, disrupting the cell membrane.

The current need for novel antibiotics is especially acute for drug-resistant Gram-negative pathogens 1 , 2 . These microorganisms have a highly restrictive permeability barrier, which limits the penetration of most compounds 3 , 4 . As a result, the last class of antibiotics that acted against Gram-negative bacteria was developed in the 1960s 2 . We reason that useful compounds can be found in bacteria that share similar requirements for antibiotics with humans, and focus on Photorhabdus symbionts of entomopathogenic nematode microbiomes. Here we report a new antibiotic that we name darobactin, which was obtained using a screen of Photorhabdus isolates. Darobactin is coded by a silent operon with little production under laboratory conditions, and is ribosomally synthesized. Darobactin has an unusual structure with two fused rings that form post-translationally. The compound is active against important Gram-negative pathogens both in vitro and in animal models of infection. Mutants that are resistant to darobactin map to BamA, an essential chaperone and translocator that folds outer membrane proteins. Our study suggests that bacterial symbionts of animals contain antibiotics that are particularly suitable for development into therapeutics. Bacterial symbionts of animals may contain antibiotics that are particularly suitable for development into therapeutics; one such compound, darobactin, is active against important Gram-negative pathogens both in vitro and in animal models of infection.

Polymyxins are last-resort antibiotics with potent activity against multi-drug resistant pathogens. They interact with lipopolysaccharide (LPS) in bacterial membranes, but mechanistic details at the molecular level remain unclear. Here, we characterize the interaction of polymyxins with native, LPS-containing outer membrane patches of Escherichia coli by high-resolution atomic force microscopy imaging, along with structural and biochemical assays. We find that polymyxins arrange LPS into hexagonal assemblies to form crystalline structures. Formation of the crystalline structures is correlated with the antibiotic activity, and absent in polymyxin-resistant strains. Crystal lattice parameters alter with variations of the LPS and polymyxin molecules. Quantitative measurements show that the crystalline structures decrease membrane thickness and increase membrane area as well as stiffness. Together, these findings suggest the formation of rigid LPS–polymyxin crystals and subsequent membrane disruption as the mechanism of polymyxin action and provide a benchmark for optimization and de novo design of LPS-targeting antimicrobials. Manioglu et al use high-resolution atomic force microscopy to resolve how polymyxins interact with the bacterial membrane. Polymyxins arrange the bacterial lipids into regular hexagonal structures that stiffen the membrane and lead to rupture.

Phosphorus is an essential nutrient taken up by organisms in the form of inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi sensing and signaling cascades, enabling them to stably maintain cellular Pi concentrations. Pi homeostasis is regulated by inositol pyrophosphate signaling molecules (PP-InsPs), which are sensed by SPX domain-containing proteins. In plants, PP-InsP-bound SPX receptors inactivate Myb coiled-coil (MYB-CC) Pi starvation response transcription factors (PHRs) by an unknown mechanism. Here we report that a InsP 8 –SPX complex targets the plant-unique CC domain of PHRs. Crystal structures of the CC domain reveal an unusual four-stranded anti-parallel arrangement. Interface mutations in the CC domain yield monomeric PHR1, which is no longer able to bind DNA with high affinity. Mutation of conserved basic residues located at the surface of the CC domain disrupt interaction with the SPX receptor in vitro and in planta, resulting in constitutive Pi starvation responses. Together, our findings suggest that InsP 8 regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors. Plants regulate phosphate homeostasis via the interaction of PHR transcription factors with SPX receptors bound to inositol pyrophosphate signaling molecules. Here the authors show that inositol pyrophosphate-bound SPX interacts with the coiled-coil domain of PHR, which regulates the oligomerization and activity of the transcription factor.

The voltage-dependent anion channel (VDAC) mediates trafficking of small molecules and ions across the eukaryotic outer mitochondrial membrane. VDAC also interacts with antiapoptotic proteins from the Bcl-2 family, and this interaction inhibits release of apoptogenic proteins from the mitochondrion. We present the nuclear magnetic resonance (NMR) solution structure of recombinant human VDAC-1 reconstituted in detergent micelles. It forms a 19-stranded β barrel with the first and last strand parallel. The hydrophobic outside perimeter of the barrel is covered by detergent molecules in a beltlike fashion. In the presence of cholesterol, recombinant VDAC-1 can form voltage-gated channels in phospholipid bilayers similar to those of the native protein. NMR measurements revealed the binding sites of VDAC-1 for the Bcl-2 protein Bcl-xL, for reduced β-nicotinamide adenine dinucleotide, and for cholesterol. Bcl-xL interacts with the VDAC barrel laterally at strands 17 and 18.

Skp is a bacterial chaperone that prevents aggregation of outer membrane proteins as they traverse the periplasmic space. The conformation and dynamics of Skp in complex with two OMPs are now examined by NMR spectroscopy. The analyses reveal that Skp provides a scaffold for its substrates, which in turn populate a dynamic conformational ensemble. The biogenesis of integral outer-membrane proteins (OMPs) in Gram-negative bacteria requires molecular chaperones that prevent the aggregation of OMP polypeptides in the aqueous periplasmic space. How these energy-independent chaperones interact with their substrates is not well understood. We have used high-resolution NMR spectroscopy to examine the conformation and dynamics of the Escherichia coli periplasmic chaperone Skp and two of its complexes with OMPs. The Skp trimer constitutes a flexible architectural scaffold that becomes more rigid upon substrate binding. The OMP substrates populate a dynamic conformational ensemble with structural interconversion rates on the submillisecond timescale. The global lifetime of the chaperone–substrate complex is seven orders of magnitude longer, emerging from the short local lifetimes by avidity. The dynamic state allows for energy-independent substrate release and provides a general paradigm for the conformation of OMP polypeptides bound to energy-independent chaperones.

Gasdermins are main effectors of pyroptosis, an inflammatory form of cell death. Released by proteolysis, the N-terminal gasdermin domain assembles large oligomers to punch lytic pores into the cell membrane. While the endpoint of this reaction, the fully formed pore, has been well characterized, the assembly and pore-forming mechanisms remain largely unknown. To resolve these mechanisms, we characterize mouse gasdermin-A3 by high-resolution time-lapse atomic force microscopy. We find that gasdermin-A3 oligomers assemble on the membrane surface where they remain attached and mobile. Once inserted into the membrane gasdermin-A3 grows variable oligomeric stoichiometries and shapes, each able to open transmembrane pores. Molecular dynamics simulations resolve how the membrane-inserted amphiphilic β-hairpins and the structurally adapting hydrophilic head domains stabilize variable oligomeric conformations and open the pore. The results show that without a vertical collapse gasdermin pore formation propagates along a set of multiple parallel but connected reaction pathways to ensure a robust cellular response. Gasdermin-A3 pore formation propagates along diverse pathways. It begins with membrane attachment and oligomeric pre-assembly. Once inserted in the membrane, the oligomers re-assemble into various shapes and sizes, which open their lytic pores.

Many proteins involved in eukaryotic phosphate homeostasis are regulated by SPX domains. In yeast, the vacuolar transporter chaperone (VTC) complex contains two such domains, but mechanistic details of its regulation are not well understood. Here, we show at the atomic level how inositol pyrophosphates interact with SPX domains of subunits Vtc2 and Vtc3 to control the activity of the VTC complex. Vtc2 inhibits the catalytically active VTC subunit Vtc4 by homotypic SPX–SPX interactions via the conserved helix α1 and the previously undescribed helix α7. Binding of inositol pyrophosphates to Vtc2 abrogates this interaction, thus activating the VTC complex. Accordingly, VTC activation is also achieved by site-specific point mutations that disrupt the SPX–SPX interface. Structural data suggest that ligand binding induces reorientation of helix α1 and exposes the modifiable helix α7, which might facilitate its post-translational modification in vivo. The variable composition of these regions within the SPX domain family might contribute to the diversified SPX functions in eukaryotic phosphate homeostasis. Pipercevic et al resolve how inositol molecules activate the VTC protein complex. The VTC complex stores phosphate in yeast and is controlled by SPX domains. The inositol molecules break an interaction between SPX domains to activate the complex.