Gasdermin‐D (GSDMD), a member of the gasdermin protein family, mediates pyroptosis in human and murine cells. Cleaved by inflammatory caspases, GSDMD inserts its N‐terminal domain (GSDMDNterm) into ...cellular membranes and assembles large oligomeric complexes permeabilizing the membrane. So far, the mechanisms of GSDMDNterm insertion, oligomerization, and pore formation are poorly understood. Here, we apply high‐resolution (≤ 2 nm) atomic force microscopy (AFM) to describe how GSDMDNterm inserts and assembles in membranes. We observe GSDMDNterm inserting into a variety of lipid compositions, among which phosphatidylinositide (PI(4,5)P2) increases and cholesterol reduces insertion. Once inserted, GSDMDNterm assembles arc‐, slit‐, and ring‐shaped oligomers, each of which being able to form transmembrane pores. This assembly and pore formation process is independent on whether GSDMD has been cleaved by caspase‐1, caspase‐4, or caspase‐5. Using time‐lapse AFM, we monitor how GSDMDNterm assembles into arc‐shaped oligomers that can transform into larger slit‐shaped and finally into stable ring‐shaped oligomers. Our observations translate into a mechanistic model of GSDMDNterm transmembrane pore assembly, which is likely shared within the gasdermin protein family.
Synopsis
Gasdermin‐D, which mediates pyroptosis in human and murine cells, is directly observed inserting into lipid membranes and assembling arc‐, slit‐ and ring‐shaped oligomers. The observations translate into a mechanistic model of gasdermin‐D assembling transmembrane lytic pores.
High‐resolution time‐lapse imaging of gasdermin‐D pore formation.
Gasdermin‐D assembles arc‐, slit‐ and ring‐shaped oligomers.
Arc‐ and slit‐shaped pores transform into stable ring‐shaped pores.
Phosphatidylinositide (PI(4,5)P2) increases gasdermin‐D pore formation.
Cholesterol reduces gasdermin‐D pore formation.
High‐resolution atomic force microscopy shows how a pyroptosis‐mediating gasdermin protein inserts into lipid membranes and assembles arc‐, slit‐ and ring‐shaped oligomers.
The current need for novel antibiotics is especially acute for drug-resistant Gram-negative pathogens
. These microorganisms have a highly restrictive permeability barrier, which limits the ...penetration of most compounds
. As a result, the last class of antibiotics that acted against Gram-negative bacteria was developed in the 1960s
. 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.
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
-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
regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors.
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.
The voltage-dependent anion channel (VDAC) is an essential protein in the eukaryotic outer mitochondrial membrane, providing the pore for substrate diffusion. Three high-resolution structures of the ...isoform 1 of VDAC in detergent micelles and bicelles have recently been published, using solution NMR and X-ray crystallography. They resolve longstanding discussions about the membrane topology of VDAC and provide the first eukaryotic β-barrel membrane protein structure. The structure contains a surprising feature that had not been observed in an integral membrane protein before: A parallel β-strand pairing and thus an odd number of strands. The studies also give a structural and functional basis for the voltage gating mechanism of VDAC and its modulation by NADH; however, they do not fully explain these functions yet. With the
de novo structure of VDAC-1, as well as those of half a dozen other proteins, the number of integral membrane protein structures solved by solution NMR has doubled in the past two years. Numerous further structural and functional studies on many different membrane proteins show that solution NMR has become an important tool for membrane protein molecular biology.
Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous ...triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD), which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms.
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.
In two-component systems, the information gathered by histidine kinases (HKs) are relayed to cognate response regulators (RRs). Thereby, the phosphoryl group of the auto-phosphorylated HK is ...transferred to the receiver (Rec) domain of the RR to allosterically activate its effector domain. In contrast, multi-step phosphorelays comprise at least one additional Rec (Rec
) domain that is typically part of the HK and acts as an intermediary for phosphoryl-shuttling. While RR Rec domains have been studied extensively, little is known about discriminating features of Rec
domains. Here we study the Rec
domain of the hybrid HK CckA by X-ray crystallography and NMR spectroscopy. Strikingly, all active site residues of the canonical Rec-fold are pre-arranged for phosphoryl-binding and BeF
binding does not alter secondary or quaternary structure, indicating the absence of allosteric changes, the hallmark of RRs. Based on sequence-covariation and modeling, we analyze the intra-molecular DHp/Rec association in hybrid HKs.
The most abundant protein of the mitochondrial outer membrane is the voltage-dependent anion channel (VDAC), which facilitates the exchange of ions and molecules between mitochondria and cytosol and ...is regulated by interactions with other proteins and small molecules. VDAC has been studied extensively for more than three decades, and last year three independent investigations revealed a structure of VDAC-1 exhibiting 19 transmembrane β-strands, constituting a unique structural class of β-barrel membrane proteins. Here, we provide a historical perspective on VDAC research and give an overview of the experimental design used to obtain these structures. Furthermore, we validate the protein refolding approach and summarize the biochemical and biophysical evidence that links the 19-stranded structure to the native form of VDAC.
Several recent atomic-resolution studies have resolved how chaperones interact with their client proteins. In some cases, molecular chaperones recognize and bind their clients in conformational ...ensembles that are locally highly dynamic and interconvert, while in other cases clients bind in unique conformations. The presence of a locally dynamic client ensemble state has important consequences, both for the interpretation of experimental data and for the functionality of chaperones, as local dynamics facilitate rapid client release, folding on and from the chaperone surface, and client recognition without shape complementarity. Facilitated by the local dynamics, at least some chaperones appear to specifically recognize energetically frustrated sites of partially folded client proteins, such that the release of frustration contributes to the interaction affinity.
Chaperone-bound clients can populate multiconformational states.
The client conformers can interconvert dynamically.
Describing the multiconformational states requires appropriate biophysical approaches.
The dynamics allows folding on and from the chaperone surface.
For native clients, the dynamics facilitate recognition of frustration without shape complementarity.
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