Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding.

Transmembrane ß barrel proteins are folded into the outer membrane (OM) of Gram-negative bacteria by the ß barrel assembly machinery (BAM) via a poorly understood process that occurs without known external energy sources. Here, we used single-particle cryo-EM to visualize the folding dynamics of a model ß barrel protein (EspP) by BAM. We found that BAM binds the highly conserved "ß signal" motif of EspP to correctly orient ß strands in the OM during folding. We also found that the folding of EspP proceeds via "hybrid-barrel" intermediates in which membrane integrated ß sheets are attached to the essential BAM subunit, BamA. The structures show an unprecedented deflection of the membrane surrounding the EspP intermediates and suggest that ß sheets progressively fold toward BamA to form a ß barrel. Along with in vivo experiments that tracked ß barrel folding while the OM tension was modified, our results support a model in which BAM harnesses OM elasticity to accelerate ß barrel folding.

Implications of conformational flexibility, lipid binding, and regulatory domains in cell-traversal protein CelTOS for apicomplexan migration.

Malaria and other apicomplexan-caused diseases affect millions of humans, agricultural animals, and pets. Cell traversal is a common feature used by multiple apicomplexan parasites to migrate through host cells and can be exploited to develop therapeutics against these deadly parasites. Here, we provide insights into the mechanism of the Cell-traversal protein for ookinetes and sporozoites (CelTOS), a conserved cell-traversal protein in apicomplexan parasites and malaria vaccine candidate. CelTOS has previously been shown to form pores in cell membranes to enable traversal of parasites through cells. We establish roles for the distinct protein regions of Plasmodium vivax CelTOS and examine the mechanism of pore formation. We further demonstrate that CelTOS dimer dissociation is required for pore formation, as disulfide bridging between monomers inhibits pore formation, and this inhibition is rescued by disulfide-bridge reduction. We also show that a helix-destabilizing amino acid, Pro127, allows CelTOS to undergo significant conformational changes to assemble into pores. The flexible C terminus of CelTOS is a negative regulator that limits pore formation. Finally, we highlight that lipid binding is a prerequisite for pore assembly as mutation of a phospholipids-binding site in CelTOS resulted in loss of lipid binding and abrogated pore formation. These findings identify critical regions in CelTOS and will aid in understanding the egress mechanism of malaria and other apicomplexan parasites as well as have implications for studying the function of other essential pore-forming proteins.

Transaldolase haploinsufficiency in subjects with acetaminophen-induced liver failure.

Transaldolase (TAL) is an enzyme in the pentose phosphate pathway (PPP) that generates NADPH for protection against oxidative stress. While deficiency of other PPP enzymes, such as transketolase (TKT), are incompatible with mammalian cell survival, mice lacking TAL are viable and develop progressive liver disease attributed to oxidative stress. Mice with homozygous or heterozygous TAL deficiency are predisposed to cirrhosis, hepatocellular carcinoma (HCC) and acetaminophen (APAP)-induced liver failure. Both mice and humans with complete TAL deficiency accumulate sedoheptulose 7-phosphate (S7P). Previous human studies relied on screening patients with S7P accumulation, thus excluding potentially pathogenic haploinsufficiency. Of note, mice with TAL haploinsufficiency are also predisposed to HCC and APAP-induced liver failure which are preventable with oral N-acetylcysteine (NAC) administration. Based on TALDO1 DNA sequencing, we detected functional TAL deficiency due to novel, heterozygous variations in two of 94 healthy adults and four of 27 subjects with APAP-induced liver failure (P = .022). The functional consequences of these variations were individually validated by site-directed mutagenesis of normal cDNA and loss of activity by recombinant enzyme. All four patients with TAL haplo-insufficiency with APAP-induced liver failure were successfully treated with NAC. We also document two novel variations in two of 15 children with previously unexplained liver cirrhosis. Examination of the National Center for Biotechnology Information databases revealed 274 coding region variations have been documented in 1125 TALDO1 sequences relative to 25 variations in 2870 TKT sequences (P < .0001). These findings suggest an unexpected prevalence and variety of genetic changes in human TALDO1 with relevance for liver injury that may be preventable by treatment with NAC.

Rhoptry Proteins ROP5 and ROP18 Are Major Murine Virulence Factors in Genetically Divergent South American Strains of Toxoplasma gondii.

Toxoplasma gondii has evolved a number of strategies to evade immune responses in its many hosts. Previous genetic mapping of crosses between clonal type 1, 2, and 3 strains of T. gondii, which are prevalent in Europe and North America, identified two rhoptry proteins, ROP5 and ROP18, that function together to block innate immune mechanisms activated by interferon gamma (IFNg) in murine hosts. However, the contribution of these and other virulence factors in more genetically divergent South American strains is unknown. Here we utilized a cross between the intermediately virulent North American type 2 ME49 strain and the highly virulent South American type 10 VAND strain to map the genetic basis for differences in virulence in the mouse. Quantitative trait locus (QTL) analysis of this new cross identified one peak that spanned the ROP5 locus on chromosome XII. CRISPR-Cas9 mediated deletion of all copies of ROP5 in the VAND strain rendered it avirulent and complementation confirmed that ROP5 is the major virulence factor accounting for differences between type 2 and type 10 strains. To extend these observations to other virulent South American strains representing distinct genetic populations, we knocked out ROP5 in type 8 TgCtBr5 and type 4 TgCtBr18 strains, resulting in complete loss of virulence in both backgrounds. Consistent with this, polymorphisms that show strong signatures of positive selection in ROP5 were shown to correspond to regions known to interface with host immunity factors. Because ROP5 and ROP18 function together to resist innate immune mechanisms, and a significant interaction between them was identified in a two-locus scan, we also assessed the role of ROP18 in the virulence of South American strains. Deletion of ROP18 in South American type 4, 8, and 10 strains resulted in complete attenuation in contrast to a partial loss of virulence seen for ROP18 knockouts in previously described type 1 parasites. These data show that ROP5 and ROP18 are conserved virulence factors in genetically diverse strains from North and South America, suggesting they evolved to resist innate immune defenses in ancestral T. gondii strains, and they have subsequently diversified under positive selection.

Isoprenoid biosynthesis inhibition disrupts Rab5 localization and food vacuolar integrity in Plasmodium falciparum.

The antimalarial agent fosmidomycin is a validated inhibitor of the nonmevalonate isoprenoid biosynthesis (methylerythritol 4-phosphate [MEP]) pathway in the malaria parasite, Plasmodium falciparum. Since multiple classes of prenyltransferase inhibitors kill P. falciparum, we hypothesized that protein prenylation was one of the essential functions of this pathway. We found that MEP pathway inhibition with fosmidomycin reduces protein prenylation, confirming that de novo isoprenoid biosynthesis produces the isoprenyl substrates for protein prenylation. One important group of prenylated proteins is small GTPases, such as Rab family members, which mediate cellular vesicular trafficking. We have found that Rab5 proteins dramatically mislocalize upon fosmidomycin treatment, consistent with a loss of protein prenylation. Fosmidomycin treatment caused marked defects in food vacuolar morphology and integrity, consistent with a defect in Rab-mediated vesicular trafficking. These results provide insights to the biological functions of isoprenoids in malaria parasites and may assist the rational selection of secondary agents that will be useful in combination therapy with new isoprenoid biosynthesis inhibitors.

Cryo-EM structures of membrane-bound dynamin in a post-hydrolysis state primed for membrane fission.

Dynamin assembles as a helical polymer at the neck of budding endocytic vesicles, constricting the underlying membrane as it progresses through the GTPase cycle to sever vesicles from the plasma membrane. Although atomic models of the dynamin helical polymer bound to guanosine triphosphate (GTP) analogs define earlier stages of membrane constriction, there are no atomic models of the assembled state post-GTP hydrolysis. Here, we used cryo-EM methods to determine atomic structures of the dynamin helical polymer assembled on lipid tubules, akin to necks of budding endocytic vesicles, in a guanosine diphosphate (GDP)-bound, super-constricted state. In this state, dynamin is assembled as a 2-start helix with an inner lumen of 3.4 nm, primed for spontaneous fission. Additionally, by cryo-electron tomography, we trapped dynamin helical assemblies within HeLa cells using the GTPase-defective dynamin K44A mutant and observed diverse dynamin helices, demonstrating that dynamin can accommodate a range of assembled complexes in cells that likely precede membrane fission.

N-acetylcysteine reduces disease activity by blocking mammalian target of rapamycin in T cells from systemic lupus erythematosus patients: a randomized, double-blind, placebo-controlled trial.

OBJECTIVE: Systemic lupus erythematosus (SLE) patients exhibit T cell dysfunction, which can be regulated through mitochondrial transmembrane potential (Δψm) and mammalian target of rapamycin (mTOR) by glutathione (GSH). This randomized, double-blind, placebo-controlled study was undertaken to examine the safety, tolerance, and efficacy of the GSH precursor N-acetylcysteine (NAC). METHODS: A total of 36 SLE patients received either daily placebo or 1.2 gm, 2.4 gm, or 4.8 gm of NAC. Disease activity was evaluated monthly by the British Isles Lupus Assessment Group (BILAG) index, the SLE Disease Activity Index (SLEDAI), and the Fatigue Assessment Scale (FAS) before, during, and after a 3-month treatment period. Mitochondrial transmembrane potential and mTOR were assessed by flow cytometry. Forty-two healthy subjects matched to patients for age, sex, and ethnicity were studied as controls. RESULTS: NAC up to 2.4 gm/day was tolerated by all patients, while 33% of those receiving 4.8 gm/day had reversible nausea. Placebo or NAC 1.2 gm/day did not influence disease activity. Considered together, 2.4 gm and 4.8 gm NAC reduced the SLEDAI score after 1 month (P = 0.0007), 2 months (P = 0.0009), 3 months (P = 0.0030), and 4 months (P = 0.0046); the BILAG score after 1 month (P = 0.029) and 3 months (P = 0.009); and the FAS score after 2 months (P = 0.0006) and 3 months (P = 0.005). NAC increased Δψm (P = 0.0001) in all T cells, profoundly reduced mTOR activity (P = 0.0009), enhanced apoptosis (P = 0.0004), reversed expansion of CD4-CD8- T cells (mean ± SEM 1.35 ± 0.12-fold change; P = 0.008), stimulated FoxP3 expression in CD4+CD25+ T cells (P = 0.045), and reduced anti-DNA production (P = 0.049). CONCLUSION: This pilot study suggests that NAC safely improves lupus disease activity by blocking mTOR in T lymphocytes.

Synthesis and Effect of Conformationally Locked Carbocyclic Guanine Nucleotides on Dynamin.

Guanine nucleotides can flip between a North and South conformation in the ribose moiety. To test the enzymatic activity of GTPases bound to nucleotides in the two conformations, we generated methanocarba guanine nucleotides in the North or South envelope conformations, i.e., (N)-GTP and (S)-GTP, respectively. With dynamin as a model system, we examined the effects of (N)-GTP and (S)-GTP on dynamin-mediated membrane constriction, an activity essential for endocytosis. Dynamin membrane constriction and fission activity are dependent on GTP binding and hydrolysis, but the effect of the conformational state of the GTP nucleotide on dynamin activity is not known. After reconstituting dynamin-mediated lipid tubulation and membrane constriction in vitro, we observed via cryo-electron microscopy (cryo-EM) that (N)-GTP, but not (S)-GTP, enables the constriction of dynamin-decorated lipid tubules. These findings suggest that the activity of dynamin is dependent on the conformational state of the GTP nucleotide. However, a survey of nucleotide ribose conformations associated with dynamin structures in nature shows almost exclusively the (S)-conformation. The explanation for this mismatch of (N) vs. (S) required for GTP analogues in a dynamin-mediated process will be addressed in future studies.

The structure and spontaneous curvature of clathrin lattices at the plasma membrane.

Clathrin-mediated endocytosis is the primary pathway for receptor and cargo internalization in eukaryotic cells. It is characterized by a polyhedral clathrin lattice that coats budding membranes. The mechanism and control of lattice assembly, curvature, and vesicle formation at the plasma membrane has been a matter of long-standing debate. Here, we use platinum replica and cryoelectron microscopy and tomography to present a structural framework of the pathway. We determine the shape and size parameters common to clathrin-mediated endocytosis. We show that clathrin sites maintain a constant surface area during curvature across multiple cell lines. Flat clathrin is present in all cells and spontaneously curves into coated pits without additional energy sources or recruited factors. Finally, we attribute curvature generation to loosely connected and pentagon-containing flat lattices that can rapidly curve when a flattening force is released. Together, these data present a universal mechanistic model of clathrin-mediated endocytosis.

Lupus-associated endogenous retroviral LTR polymorphism and epigenetic imprinting promote HRES-1/RAB4 expression and mTOR activation.

Overexpression and long terminal repeat (LTR) polymorphism of the HRES­1/Rab4 human endogenous retrovirus locus have been associated with T cell activation and disease manifestations in systemic lupus erythematosus (SLE). Although genomic DNA methylation is diminished overall in SLE, its role in HRES-1/Rab4 expression is unknown. Therefore, we determined how lupus-associated polymorphic rs451401 alleles of the LTR regulate transcription from the HRES-1/Rab4 promoter and thus affect T cell activation. The results showed that cytosine-119 is hypermethylated while cytosine-51 of the promoter and the LTR enhancer are hypomethylated in SLE. Pharmacologic or genetic inactivation of DNA methyltransferase 1 augmented the expression of HRES-1/Rab4. The minimal promoter was selectively recognized by metabolic stress sensor NRF1 when cytosine-119 but not cytosine-51 was methylated, and NRF1 stimulated HRES-1/Rab4 expression in human T cells. In turn, IRF2 and PSIP1 bound to the LTR enhancer and exerted control over HRES-1/Rab4 expression in rs451401 genotype- and methylation-dependent manners. The LTR enhancer conferred markedly greater expression of HRES-1/Rab4 in subjects with rs451401CC over rs451401GG alleles that in turn promoted mechanistic target of rapamycin (mTOR) activation upon T cell receptor stimulation. HRES-1/Rab4 alone robustly activated mTOR in human T cells. These findings identify HRES-1/Rab4 as a methylation- and rs451401 allele-dependent transducer of environmental stress and controller of T cell activation.

Dynamin regulates the dynamics and mechanical strength of the actin cytoskeleton as a multifilament actin-bundling protein.

The dynamin GTPase is known to bundle actin filaments, but the underlying molecular mechanism and physiological relevance remain unclear. Our genetic analyses revealed a function of dynamin in propelling invasive membrane protrusions during myoblast fusion in vivo. Using biochemistry, total internal reflection fluorescence microscopy, electron microscopy and cryo-electron tomography, we show that dynamin bundles actin while forming a helical structure. At its full capacity, each dynamin helix captures 12-16 actin filaments on the outer rim of the helix. GTP hydrolysis by dynamin triggers disassembly of fully assembled dynamin helices, releasing free dynamin dimers/tetramers and facilitating Arp2/3-mediated branched actin polymerization. The assembly/disassembly cycles of dynamin promote continuous actin bundling to generate mechanically stiff actin super-bundles. Super-resolution and immunogold platinum replica electron microscopy revealed dynamin along actin bundles at the fusogenic synapse. These findings implicate dynamin as a unique multifilament actin-bundling protein that regulates the dynamics and mechanical strength of the actin cytoskeletal network.

Structural Insights into the Mechanism of Dynamin Superfamily Proteins.

Dynamin superfamily proteins (DSPs) mediate membrane fission and fusion necessary for endocytosis, organelle biogenesis and maintenance, as well as for bacterial cytokinesis. They also function in the innate immune response to pathogens and in organizing the cytoskeleton. In this review, we summarize the current understanding of the molecular mechanism of DSPs, with emphasis on the structural basis of function. Studies from the past decade on the structure and mechanism of DSPs enable comparative analysis of shared mechanisms and unique features of this protein family.

Membrane Lipid Screen to Identify Molecular Targets of Biomolecules.

Proteins that bind to and disrupt cell membranes may target specific phospholipids. Here we describe a protocol to identify the lipid targets of proteins and biomolecules. First, we describe a screen to identify lipids in membranes that are specifically bound by the biomolecule of interest. Second, we describe a method for determining if the presence of these lipids within membranes is necessary for membrane disruption. The methods described here were used to determine that the malaria vaccine candidate CelTOS disrupts cell membranes by specifically targeting phosphatidic acid (Jimah et al., 2016). This protocol has a companion protocol: 'Liposome disruption assay to examine lytic properties of biomolecules' which can be applied to examine the ability of the biomolecule to disrupt membranes composed of the lipid target identified by following this protocol (Jimah et al., 2017).

Liposome Disruption Assay to Examine Lytic Properties of Biomolecules.

Proteins may have three dimensional structural or amino acid features that suggest a role in targeting and disrupting lipids within cell membranes. It is often necessary to experimentally investigate if these proteins and biomolecules are able to disrupt membranes in order to conclusively characterize the function of these biomolecules. Here, we describe an in vitro assay to evaluate the membrane lytic properties of proteins and biomolecules. Large unilamellar vesicles (liposomes) containing carboxyfluorescein at fluorescence-quenching concentrations are treated with the biomolecule of interest. A resulting increase in fluorescence due to leakage of the dye from liposomes and subsequent dilution in the buffer demonstrates that the biomolecule is sufficient for disrupting liposomes and membranes. Additionally, since liposome disruption may occur via pore-formation or via general solubilization of lipids similar to detergents, we provide a method to distinguish between these two mechanisms. Pore-formation can be identified and evaluated by examining the blockade of carboxyfluorescein release with dextran molecules that fit the pore. The methods described here were used to determine that the malaria vaccine candidate CelTOS and proapoptotic Bax disrupt liposomes by pore formation (Saito et al., 2000; Jimah et al., 2016). Since membrane lipid binding by a biomolecule precedes membrane disruption, we recommend the companion protocol: Jimah et al., 2017.

Prediction of New Stabilizing Mutations Based on Mechanistic Insights from Markov State Models.

Protein stabilization is fundamental to enzyme function and evolution, yet understanding the determinants of a protein's stability remains a challenge. This is largely due to a shortage of atomically detailed models for the ensemble of relevant protein conformations and their relative populations. For example, the M182T substitution in TEM ß-lactamase, an enzyme that confers antibiotic resistance to bacteria, is stabilizing but the precise mechanism remains unclear. Here, we employ Markov state models (MSMs) to uncover how M182T shifts the distribution of different structures that TEM adopts. We find that M182T stabilizes a helix that is a key component of a domain interface. We then predict the effects of other mutations, including a novel stabilizing mutation, and experimentally test our predictions using a combination of stability measurements, crystallography, NMR, and in vivo measurements of bacterial fitness. We expect our insights and methodology to provide a valuable foundation for protein design.

Malaria parasite CelTOS targets the inner leaflet of cell membranes for pore-dependent disruption.

Apicomplexan parasites contain a conserved protein CelTOS that, in malaria parasites, is essential for traversal of cells within the mammalian host and arthropod vector. However, the molecular role of CelTOS is unknown because it lacks sequence similarity to proteins of known function. Here, we determined the crystal structure of CelTOS and discovered CelTOS resembles proteins that bind to and disrupt membranes. In contrast to known membrane disruptors, CelTOS has a distinct architecture, specifically binds phosphatidic acid commonly present within the inner leaflet of plasma membranes, and potently disrupts liposomes composed of phosphatidic acid by forming pores. Microinjection of CelTOS into cells resulted in observable membrane damage. Therefore, CelTOS is unique as it achieves nearly universal inner leaflet cellular activity to enable the exit of parasites from cells during traversal. By providing novel molecular insight into cell traversal by apicomplexan parasites, our work facilitates the design of therapeutics against global pathogens.