Germline NLRP1 Mutations Cause Skin Inflammatory and Cancer Susceptibility Syndromes via Inflammasome Activation.

Inflammasome complexes function as key innate immune effectors that trigger inflammation in response to pathogen- and danger-associated signals. Here, we report that germline mutations in the inflammasome sensor NLRP1 cause two overlapping skin disorders: multiple self-healing palmoplantar carcinoma (MSPC) and familial keratosis lichenoides chronica (FKLC). We find that NLRP1 is the most prominent inflammasome sensor in human skin, and all pathogenic NLRP1 mutations are gain-of-function alleles that predispose to inflammasome activation. Mechanistically, NLRP1 mutations lead to increased self-oligomerization by disrupting the PYD and LRR domains, which are essential in maintaining NLRP1 as an inactive monomer. Primary keratinocytes from patients experience spontaneous inflammasome activation and paracrine IL-1 signaling, which is sufficient to cause skin inflammation and epidermal hyperplasia. Our findings establish a group of non-fever inflammasome disorders, uncover an unexpected auto-inhibitory function for the pyrin domain, and provide the first genetic evidence linking NLRP1 to skin inflammatory syndromes and skin cancer predisposition.

Sophisticated natural products as antibiotics.

In this Review, we explore natural product antibiotics that do more than simply inhibit an active site of an essential enzyme. We review these compounds to provide inspiration for the design of much-needed new antibacterial agents, and examine the complex mechanisms that have evolved to effectively target bacteria, including covalent binders, inhibitors of resistance, compounds that utilize self-promoted entry, those that evade resistance, prodrugs, target corrupters, inhibitors of 'undruggable' targets, compounds that form supramolecular complexes, and selective membrane-acting agents. These are exemplified by ß-lactams that bind covalently to inhibit transpeptidases and ß-lactamases, siderophore chimeras that hijack import mechanisms to smuggle antibiotics into the cell, compounds that are activated by bacterial enzymes to produce reactive molecules, and antibiotics such as aminoglycosides that corrupt, rather than merely inhibit, their targets. Some of these mechanisms are highly sophisticated, such as the preformed ß-strands of darobactins that target the undruggable ß-barrel chaperone BamA, or teixobactin, which binds to a precursor of peptidoglycan and then forms a supramolecular structure that damages the membrane, impeding the emergence of resistance. Many of the compounds exhibit more than one notable feature, such as resistance evasion and target corruption. Understanding the surprising complexity of the best antimicrobial compounds provides a roadmap for developing novel compounds to address the antimicrobial resistance crisis by mining for new natural products and inspiring us to design similarly sophisticated antibiotics.

Structural basis of NINJ1-mediated plasma membrane rupture in cell death.

Eukaryotic cells can undergo different forms of programmed cell death, many of which culminate in plasma membrane rupture as the defining terminal event1-7. 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-18 (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.

The dynamic mechanism of 4E-BP1 recognition and phosphorylation by mTORC1.

The activation of cap-dependent translation in eukaryotes requires multisite, hierarchical phosphorylation of 4E-BP by the 1 MDa kinase mammalian target of rapamycin complex 1 (mTORC1). To resolve the mechanism of this hierarchical phosphorylation at the atomic level, we monitored by NMR spectroscopy the interaction of intrinsically disordered 4E binding protein isoform 1 (4E-BP1) with the mTORC1 subunit regulatory-associated protein of mTOR (Raptor). The N-terminal RAIP motif and the C-terminal TOR signaling (TOS) motif of 4E-BP1 bind separate sites in Raptor, resulting in avidity-based tethering of 4E-BP1. This tethering orients the flexible central region of 4E-BP1 toward the mTORC1 kinase site for phosphorylation. The structural constraints imposed by the two tethering interactions, combined with phosphorylation-induced conformational switching of 4E-BP1, explain the hierarchy of 4E-BP1 phosphorylation by mTORC1. Furthermore, we demonstrate that mTORC1 recognizes both free and eIF4E-bound 4E-BP1, allowing rapid phosphorylation of the entire 4E-BP1 pool and efficient activation of translation. Finally, our findings provide a mechanistic explanation for the differential rapamycin sensitivity of the 4E-BP1 phosphorylation sites.

The antibiotic darobactin mimics a ß-strand to inhibit outer membrane insertase.

Antibiotics that target Gram-negative bacteria in new ways are needed to resolve the antimicrobial resistance crisis1-3. Gram-negative bacteria are protected by an additional outer membrane, rendering proteins on the cell surface attractive drug targets4,5. The natural compound darobactin targets the bacterial insertase BamA6-the central unit of the essential BAM complex, which facilitates the folding and insertion of outer membrane proteins7-13. BamA lacks a typical catalytic centre, and it is not obvious how a small molecule such as darobactin might inhibit its function. Here we resolve the mode of action of darobactin at the atomic level using a combination of cryo-electron microscopy, X-ray crystallography, native mass spectrometry, in vivo experiments and molecular dynamics simulations. Two cyclizations pre-organize the darobactin peptide in a rigid ß-strand conformation. This creates a mimic of the recognition signal of native substrates with a superior ability to bind to the lateral gate of BamA. Upon binding, darobactin replaces a lipid molecule from the lateral gate to use the membrane environment as an extended binding pocket. Because the interaction between darobactin and BamA is largely mediated by backbone contacts, it is particularly robust against potential resistance mutations. Our results identify the lateral gate as a functional hotspot in BamA and will allow the rational design of antibiotics that target this bacterial Achilles heel.

Regulation of α-synuclein by chaperones in mammalian cells.

Neurodegeneration in patients with Parkinson's disease is correlated with the occurrence of Lewy bodies-intracellular inclusions that contain aggregates of the intrinsically disordered protein α-synuclein1. The aggregation propensity of α-synuclein in cells is modulated by specific factors that include post-translational modifications2,3, Abelson-kinase-mediated phosphorylation4,5 and interactions with intracellular machineries such as molecular chaperones, although the underlying mechanisms are unclear6-8. Here we systematically characterize the interaction of molecular chaperones with α-synuclein in vitro as well as in cells at the atomic level. We find that six highly divergent molecular chaperones commonly recognize a canonical motif in α-synuclein, consisting of the N terminus and a segment around Tyr39, and hinder the aggregation of α-synuclein. NMR experiments9 in cells show that the same transient interaction pattern is preserved inside living mammalian cells. Specific inhibition of the interactions between α-synuclein and the chaperone HSC70 and members of the HSP90 family, including HSP90ß, results in transient membrane binding and triggers a remarkable re-localization of α-synuclein to the mitochondria and concomitant formation of aggregates. Phosphorylation of α-synuclein at Tyr39 directly impairs the interaction of α-synuclein with chaperones, thus providing a functional explanation for the role of Abelson kinase in Parkinson's disease. Our results establish a master regulatory mechanism of α-synuclein function and aggregation in mammalian cells, extending the functional repertoire of molecular chaperones and highlighting new perspectives for therapeutic interventions for Parkinson's disease.

Author Correction: A new antibiotic selectively kills Gram-negative pathogens.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A new antibiotic selectively kills Gram-negative pathogens.

The current need for novel antibiotics is especially acute for drug-resistant Gram-negative pathogens1,2. These microorganisms have a highly restrictive permeability barrier, which limits the penetration of most compounds3,4. As a result, the last class of antibiotics that acted against Gram-negative bacteria was developed in the 1960s2. 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.

Author Correction: Chimeric peptidomimetic antibiotics against Gram-negative bacteria.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Chimeric peptidomimetic antibiotics against Gram-negative bacteria.

There is an urgent need for new antibiotics against Gram-negative pathogens that are resistant to carbapenem and third-generation cephalosporins, against which antibiotics of last resort have lost most of their efficacy. Here we describe a class of synthetic antibiotics inspired by scaffolds derived from natural products. These chimeric antibiotics contain a ß-hairpin peptide macrocycle linked to the macrocycle found in the polymyxin and colistin family of natural products. They are bactericidal and have a mechanism of action that involves binding to both lipopolysaccharide and the main component (BamA) of the ß-barrel folding complex (BAM) that is required for the folding and insertion of ß-barrel proteins into the outer membrane of Gram-negative bacteria. Extensively optimized derivatives show potent activity against multidrug-resistant pathogens, including all of the Gram-negative members of the ESKAPE pathogens1. These derivatives also show favourable drug properties and overcome colistin resistance, both in vitro and in vivo. The lead candidate is currently in preclinical toxicology studies that-if successful-will allow progress into clinical studies that have the potential to address life-threatening infections by the Gram-negative pathogens, and thus to resolve a considerable unmet medical need.

Pseudocontact Shifts in Biomolecular NMR Spectroscopy.

Paramagnetic centers in biomolecules, such as specific metal ions that are bound to a protein, affect the nuclei in their surrounding in various ways. One of these effects is the pseudocontact shift (PCS), which leads to strong chemical shift perturbations of nuclear spins, with a remarkably long range of 50 Å and beyond. The PCS in solution NMR is an effect originating from the anisotropic part of the dipole-dipole interaction between the magnetic momentum of unpaired electrons and nuclear spins. The PCS contains spatial information that can be exploited in multiple ways to characterize structure, function, and dynamics of biomacromolecules. It can be used to refine structures, magnify effects of dynamics, help resonance assignments, allows for an intermolecular positioning system, and gives structural information in sensitivity-limited situations where all other methods fail. Here, we review applications of the PCS in biomolecular solution NMR spectroscopy, starting from early works on natural metalloproteins, following the development of non-natural tags to chelate and attach lanthanoid ions to any biomolecular target to advanced applications on large biomolecular complexes and inside living cells. We thus hope to not only highlight past applications but also shed light on the tremendous potential the PCS has in structural biology.

Outer membrane permeability: Antimicrobials and diverse nutrients bypass porins in Pseudomonas aeruginosa.

Gram-negative bacterial pathogens have an outer membrane that restricts entry of molecules into the cell. Water-filled protein channels in the outer membrane, so-called porins, facilitate nutrient uptake and are thought to enable antibiotic entry. Here, we determined the role of porins in a major pathogen, Pseudomonas aeruginosa, by constructing a strain lacking all 40 identifiable porins and 15 strains carrying only a single unique type of porin and characterizing these strains with NMR metabolomics and antimicrobial susceptibility assays. In contrast to common assumptions, all porins were dispensable for Pseudomonas growth in rich medium and consumption of diverse hydrophilic nutrients. However, preferred nutrients with two or more carboxylate groups such as succinate and citrate permeated poorly in the absence of porins. Porins provided efficient translocation pathways for these nutrients with broad and overlapping substrate selectivity while efficiently excluding all tested antibiotics except carbapenems, which partially entered through OprD. Porin-independent permeation of antibiotics through the outer-membrane lipid bilayer was hampered by carboxylate groups, consistent with our nutrient data. Together, these results challenge common assumptions about the role of porins by demonstrating porin-independent permeation of the outer-membrane lipid bilayer as a major pathway for nutrient and drug entry into the bacterial cell.

RNA-bound PGC-1α controls gene expression in liquid-like nuclear condensates.

Plasticity of cells, tissues, and organs is controlled by the coordinated transcription of biological programs. However, the mechanisms orchestrating such context-specific transcriptional networks mediated by the dynamic interplay of transcription factors and coregulators are poorly understood. The peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a prototypical master regulator of adaptive transcription in various cell types. We now uncovered a central function of the C-terminal domain of PGC-1α to bind RNAs and assemble multiprotein complexes including proteins that control gene transcription and RNA processing. These interactions are important for PGC-1α recruitment to chromatin in transcriptionally active liquid-like nuclear condensates. Notably, such a compartmentalization of active transcription mediated by liquid-liquid phase separation was observed in mouse and human skeletal muscle, revealing a mechanism by which PGC-1α regulates complex transcriptional networks. These findings provide a broad conceptual framework for context-dependent transcriptional control of phenotypic adaptations in metabolically active tissues.

Chaperone-Bound Clients: The Importance of Being Dynamic.

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.

Hybrid histidine kinase activation by cyclic di-GMP-mediated domain liberation.

Cytosolic hybrid histidine kinases (HHKs) constitute major signaling nodes that control various biological processes, but their input signals and how these are processed are largely unknown. In Caulobacter crescentus, the HHK ShkA is essential for accurate timing of the G1-S cell cycle transition and is regulated by the corresponding increase in the level of the second messenger c-di-GMP. Here, we use a combination of X-ray crystallography, NMR spectroscopy, functional analyses, and kinetic modeling to reveal the regulatory mechanism of ShkA. In the absence of c-di-GMP, ShkA predominantly adopts a compact domain arrangement that is catalytically inactive. C-di-GMP binds to the dedicated pseudoreceiver domain Rec1, thereby liberating the canonical Rec2 domain from its central position where it obstructs the large-scale motions required for catalysis. Thus, c-di-GMP cannot only stabilize domain interactions, but also engage in domain dissociation to allosterically invoke a downstream effect. Enzyme kinetics data are consistent with conformational selection of the ensemble of active domain constellations by the ligand and show that autophosphorylation is a reversible process.

Mechanism of membrane pore formation by human gasdermin-D.

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.

Acquisition of enzymatic progress curves in real time by quenching-free ion exchange chromatography.

We describe a quenching-free, 'online' ion exchange chromatography (oIEC) method for the quantitative analysis of enzymatic reactions in real-time. We show that separate quenching of the ongoing reaction performed conventionally is not required, since enzymatic reactions are interrupted upon immobilization of the reaction compounds by binding to the stationary phase of the ion exchange column. The reaction mix samples are directly injected into the column, thereby improving data consistency and allowing automation of the process. The method allows reliable and efficient acquisition of enzymatic progress curves by automatic loading of aliquots of an ongoing reaction at predefined timepoints. We demonstrate the applicability of this method for a variety of enzymatic reactions. SUBJECT: Enzymatic assays and analysis.

Molecular chaperones and their denaturing effect on client proteins.

Advanced NMR methods combined with biophysical techniques have recently provided unprecedented insight into structure and dynamics of molecular chaperones and their interaction with client proteins. These studies showed that several molecular chaperones are able to dissolve aggregation-prone polypeptides in aqueous solution. Furthermore, chaperone-bound clients often feature fluid-like backbone dynamics and chaperones have a denaturing effect on clients. Interestingly, these effects that chaperones have on client proteins resemble the effects of known chaotropic substances. Following this analogy, chaotropicity could be a fruitful concept to describe, quantify and rationalize molecular chaperone function. In addition, the observations raise the possibility that at least some molecular chaperones might share functional similarities with chaotropes. We discuss these concepts and outline future research in this direction.

Protocol for High-Yield Production of Photo-Leucine-Labeled Proteins in Escherichia coli.

UV-cross-linking mass spectrometry is an emerging technique to obtain structural information of biomacromolecules and their complexes in vivo and in vitro. In particular, certain photo-reactive amino acids (pA) such as photo-leucine (pLeu) and photo-methionine can provide unique short-distance information on the structural core regions of proteins. Here, we present a protocol for high-yield incorporation of pLeu in proteins recombinantly expressed in Escherichia coli. The protein of interest is expressed at high cell densities, which reduces the required amount of the pA by a factor of 10, as compared to the standard protocols, while maintaining high incorporation rates. For the two chaperones, trigger factor and SecB, up to 3 mg of pLeu-labeled protein were thus obtained from 100 mL of cell culture, with label incorporation rates of up to 34%. For trigger factor, UV-induced cross-linking leads to the identification of 12 cross-links that are in agreement with the published three-dimensional structures. The accessibility of milligram amounts of pLeu-labeled proteins at low costs will be highly useful to address structural biology questions.