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Motivated by recent studies of two-phase lipid vesicles possessing 2D solid domains integrated within a fluid bilayer phase, we study the shape equilibria of closed vesicles possessing a single planar, circular inclusion.
Ammonia monooxygenase (AMO), a copper-dependent membrane enzyme, catalyzes the first and rate-limiting step of nitrification: the oxidation of ammonia to hydroxylamine. Despite its central role in the global nitrogen cycle and its biotechnological relevance, structural characterization of AMO has lagged behind that of its homolog, particulate methane monooxygenase (pMMO), due to the slow growth rates of ammonia-oxidizing bacteria and the instability of AMO upon purification. Recent cryoEM studies of Nitrosomonas europaea AMO and Methylococcus capsulatus (Bath) pMMO in native membranes revealed new structural features, including two adjacent copper-binding sites in the transmembrane region, Cu C and Cu D , believed to constitute the active site. Although multiple structures were determined under various conditions, simultaneous occupancy of Cu C and Cu D was never observed, leaving their potential functional interplay unresolved. Here we report the 2.6 Å resolution cryoEM structure of AMO from Nitrosospira briensis C-128 in isolated native membranes. This structure reveals the first instance of simultaneous copper occupancy of the Cu C and Cu D sites, along with occupancy of the periplasmic Cu B site. Electron paramagnetic resonance (EPR) spectroscopic data indicate that the Cu B site is primarily occupied by Cu(II), while Cu C and Cu D are primarily occupied by diamagnetic ions, presumably Cu(I). Notably, a lipid molecule is bound between the Cu C and Cu D sites, separating them by ∼8.0 Å. The results underscore the importance of studying these enzymes in their native environments across species to resolve conserved and divergent molecular features.
Indolicidin, a cationic antimicrobial peptide, interacts with lipid bilayers through electrostatic and hydrophobic interactions, disrupting microbial membranes. We investigated the depth-dependent localization of gold-nanoparticle-labeled indolicidin in a supported model membrane using X-ray fluorescence standing wave (SWXF) analysis. Liposomes composed of DMPC and DMPG were incubated with indolicidin labeled at its C-terminus with a 1.8 nm gold nanoparticle, then deposited onto a Si/Mo multilayer substrate via vesicle bursting. SWXF measurements revealed that at low peptide incubation concentrations (2–5 μM), gold-nanoparticle-labeled indolicidin remains primarily associated with the bilayer's outer leaflet. At higher concentrations (10 μM), the peptide penetrates deeper into the bilayer, with the labeled C-terminal region either localizing near the membrane's hydrophobic core or inducing membrane breakup. In conclusion, these findings suggest a concentration-dependent insertion mechanism.
Algae made our world possible, and it can help us make the future more sustainable; but we need to change the way we live and adopt new more efficient production systems, and we need to do that now. When the world was new, the atmosphere was mainly carbon dioxide, and no animal life was possible. Along came algae with the process of photosynthesis, and things began to change. Ancient cyanobacteria algae turned carbon dioxide into enormous sums of lipids, proteins and carbohydrates, while they secreted oxygen into the atmosphere. Over a billion years, as oxygen filled the air and algae filled the seas, animal life became possible. Eventually all that algae biomass became petroleum and natural gas, which for eons sat undisturbed in vast underground reservoirs, holding enormous sums of untapped energy. Less than 200 years ago humans learned to tap these energy reserves to create the world we know today, but in so doing, we have released millions of years of stored CO 2 back into the atmosphere. Algae can again help make the world a better place, but this will require new thinking and new ways of producing our food, feed and fuels. We need an algae revolution 2.0.
Carbon nanotube porins (CNTPs) are biomimetic membrane channels that demonstrate excellent biocompatibility and unique water and ion transport properties. Gating transport in CNTPs with external voltage could increase control over ion flow and selectivity. Herein, we used continuum modeling to probe the parameters that enable and further affect CNTP gating efficiency, including the size and composition of the supporting lipid membrane, slip flow in the carbon nanotube, and the intrinsic electronic properties of the nanotube. Our results show that the optimal gated CNTP device consists of a semiconducting CNTP inserted into a small membrane patch containing an internally conductive layer. Moreover, we demonstrate that the ionic transport modulated by gate voltages is controlled by the charge distribution along the CNTP under the external gate electric potential. The theoretical understanding developed in this study offers valuable guidance for the design of gated CNTP devices for nanofluidic studies, novel biomimetic membranes, and cellular interfaces in the future.
Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.
Programmed cell death (PCD) in filamentous fungi prevents cytoplasmic mixing following fusion between conspecific genetically distinct individuals (allorecognition) and serves as a defense mechanism against mycoparasitism, genome exploitation, and deleterious cytoplasmic elements (i.e., senescence plasmids). Recently, we identified regulator of cell death-1 ( rcd-1 ), a gene controlling PCD in germinated asexual spores in the filamentous fungus Neurospora crassa . rcd-1 alleles are highly polymorphic and fall into two haplogroups in N. crassa populations. Coexpression of alleles from the two haplogroups, rcd-1–1 and rcd-1–2 , is necessary and sufficient to trigger a cell death reaction. Here, we investigated the molecular bases of rcd-1 -dependent cell death. Based on in silico analyses, we found that RCD-1 is a remote homolog of the N-terminal pore-forming domain of gasdermin, the executioner protein of a highly inflammatory cell death reaction termed pyroptosis, which plays a key role in mammalian innate immunity. We show that RCD-1 localizes to the cell periphery and that cellular localization of RCD-1 was correlated with conserved positively charged residues on predicted amphipathic α-helices, as shown for murine gasdermin-D. Similar to gasdermin, RCD-1 binds acidic phospholipids in vitro, notably, cardiolipin and phosphatidylserine, and interacts with liposomes containing such lipids. The RCD-1 incompatibility system was reconstituted in human 293T cells, where coexpression of incompatible rcd-1–1/rcd-1–2 alleles triggered pyroptotic-like cell death. Oligomers of RCD-1 were associated with the cell death reaction, further supporting the evolutionary relationship between gasdermin and rcd-1 . This report documents an ancient transkingdom relationship of cell death execution modules involved in organismal defense.
Cis-prenyltransferase (cis-PTase) catalyzes the rate-limiting step in the synthesis of glycosyl carrier lipids required for protein glycosylation in the lumen of endoplasmic reticulum. Here, we report the crystal structure of the human NgBR/DHDDS complex, which represents an atomic resolution structure for any heterodimeric cis-PTase. The crystal structure sheds light on how NgBR stabilizes DHDDS through dimerization, participates in the enzyme’s active site through its C-terminal -RXG- motif, and how phospholipids markedly stimulate cis-PTase activity. Comparison of NgBR/DHDDS with homodimeric cis-PTase structures leads to a model where the elongating isoprene chain extends beyond the enzyme’s active site tunnel, and an insert within the α3 helix helps to stabilize this energetically unfavorable state to enable long-chain synthesis to occur. These data provide unique insights into how heterodimeric cis-PTases have evolved from their ancestral, homodimeric forms to fulfill their function in long-chain polyprenol synthesis.
S-acylation, also known as palmitoylation, is the most abundant form of protein lipidation in humans. This reversible posttranslational modification, which targets thousands of proteins, is catalyzed by 23 members of the DHHC family of integral membrane enzymes. DHHC enzymes use fatty acyl-CoA as the ubiquitous fatty acyl donor and become autoacylated at a catalytic cysteine; this intermediate subsequently transfers the fatty acyl group to a cysteine in the target protein. Protein S-acylation intersects with almost all areas of human physiology, and several DHHC enzymes are considered as possible therapeutic targets against diseases such as cancer. These efforts would greatly benefit from a detailed understanding of the molecular basis for this crucial enzymatic reaction. Here, in this study, we combine X-ray crystallography with all-atom molecular dynamics simulations to elucidate the structure of the precatalytic complex of human DHHC20 in complex with palmitoyl CoA. The resulting structure reveals that the fatty acyl chain inserts into a hydrophobic pocket within the transmembrane spanning region of the protein, whereas the CoA headgroup is recognized by the cytosolic domain through polar and ionic interactions. Biochemical experiments corroborate the predictions from our structural model. We show, using both computational and experimental analyses, that palmitoyl CoA acts as a bivalent ligand where the interaction of the DHHC enzyme with both the fatty acyl chain and the CoA headgroup is important for catalytic chemistry to proceed. This bivalency explains how, in the presence of high concentrations of free CoA under physiological conditions, DHHC enzymes can efficiently use palmitoyl CoA as a substrate for autoacylation.
Magnetosomes are lipid-bound organelles that direct the biomineralization of magnetic nanoparticles in magnetotactic bacteria. Magnetosome membranes are not uniform in size and can grow in a biomineralization-dependent manner. However, the underlying mechanisms of magnetosome membrane growth regulation remain unclear. Using cryoelectron tomography, we systematically examined mutants with defects at various stages of magnetosome formation to identify factors involved in controlling membrane growth. We found that a conserved serine protease, MamE, plays a key role in magnetosome membrane growth regulation. When the protease activity of MamE is disrupted, magnetosome membrane growth is restricted, which, in turn, limits the size of the magnetite particles. Consistent with this finding, the upstream regulators of MamE protease activity, MamO and MamM, are also required for magnetosome membrane growth. We then used a combination of candidate and comparative proteomics approaches to identify Mms6 and MamD as two MamE substrates. Mms6 does not appear to participate in magnetosome membrane growth. However, in the absence of MamD, magnetosome membranes grow to a larger size than the wild type. Furthermore, when the cleavage of MamD by MamE protease is blocked, magnetosome membrane growth and biomineralization are severely inhibited, phenocopying the MamE protease-inactive mutant. We therefore propose that the growth of magnetosome membranes is controlled by a protease-mediated switch through processing of MamD. Overall, our work shows that, like many eukaryotic systems, bacteria control the growth and size of biominerals by manipulating the physical properties of intracellular organelles.
G-protein metallochaperone MeaB in bacteria [methylmalonic aciduria type A (MMAA) in humans] is responsible for facilitating the delivery of adenosylcobalamin (AdoCbl) to methylmalonyl-CoA mutase (MCM), the only AdoCbl-dependent enzyme in humans. Genetic defects in the switch III region of MMAA lead to the genetic disorder methylmalonic aciduria in which the body is unable to process certain lipids. Here, we present a crystal structure of Methylobacterium extorquens MeaB bound to a nonhydrolyzable guanosine triphosphate (GTP) analog guanosine-5'-[(β,γ)-methyleno]triphosphate (GMPPCP) with the Cbl-binding domain of its target mutase enzyme (MeMCM cbl ). This structure provides an explanation for the stimulation of the GTP hydrolyase activity of MeaB afforded by target protein binding. We find that upon MCM cbl association, one protomer of the MeaB dimer rotates ~180°, such that the inactive state of MeaB is converted to an active state in which the nucleotide substrate is now surrounded by catalytic residues. Importantly, it is the switch III region that undergoes the largest change, rearranging to make direct contacts with the terminal phosphate of GMPPCP. These structural data additionally provide insights into the molecular basis by which this metallochaperone contributes to AdoCbl delivery without directly binding the cofactor. Our data suggest a model in which GTP-bound MeaB stabilizes a conformation of MCM that is open for AdoCbl insertion, and GTP hydrolysis, as signaled by switch III residues, allows MCM to close and trap its cofactor. Substitutions of switch III residues destabilize the active state of MeaB through loss of protein:nucleotide and protein:protein interactions at the dimer interface, thus uncoupling GTP hydrolysis from AdoCbl delivery.
Eukaryotes originated prior to the establishment of modern marine oxygen (O 2 ) levels. According to the body fossil and lipid biomarker records, modern (crown) microbial eukaryote lineages began diversifying in the ocean no later than ~800 Ma. While it has long been predicted that increasing atmospheric O 2 levels facilitated the early diversification of microbial eukaryotes, the O 2 levels needed to permit this diversification remain unconstrained. Using time-resolved geochemical parameter and gene sequence information from a model marine oxygen minimum zone spanning a range of dissolved O 2 levels and redox states, we show that microbial eukaryote taxonomic richness and phylogenetic diversity remain the same until O 2 declines to around 2 to 3% of present atmospheric levels, below which these diversity metrics become significantly reduced. Our observations suggest that increasing O 2 would have only directly promoted early crown-eukaryote diversity if atmospheric O 2 was below 2 to 3% of modern levels when crown-eukaryotes originated and then later met or surpassed this range as crown-eukaryotes diversified. If atmospheric O 2 was already consistently at or above 2 to 3% of modern levels by the time that crown-eukaryotes originated, then the subsequent diversification of modern microbial eukaryotes was not directly driven by atmospheric oxygenation.
Constructing efficient cell factories for product synthesis is frequently hampered by competing pathways and/or insufficient precursor supply. This is particularly evident in the case of triterpenoid biosynthesis in Yarrowia lipolytica, where squalene biosynthesis is tightly coupled to cytosolic biosynthesis of sterols essential for cell viability. Here, we addressed this problem by reconstructing the complete squalene biosynthetic pathway, starting from acetyl-CoA, in the peroxisome, thus harnessing peroxisomal acetyl-CoA pool and sequestering squalene synthesis in this organelle from competing cytosolic reactions. This strategy led to increasing the squalene levels by 1,300-fold relatively to native cytosolic synthesis. Subsequent enhancement of the peroxisomal acetyl-CoA supply by two independent approaches, 1) converting cellular lipid pool to peroxisomal acetyl-CoA and 2) establishing an orthogonal acetyl-CoA shortcut from CO 2 -derived acetate in the peroxisome, further significantly improved local squalene accumulation. Using these approaches, we constructed squalene-producing strains capable of yielding 32.8 g/L from glucose, and 31.6 g/L from acetate by employing a cofeeding strategy, in bioreactor fermentations. Our findings provide a feasible strategy for protecting intermediate metabolites that can be claimed by multiple reactions by engineering peroxisomes in Y. lipolytica as microfactories for the production of such intermediates and in particular acetyl-CoA-derived metabolites.
Antibody-derived T-cell receptor (TCR) agonists are commonly used to activate T cells. While antibodies can trigger TCRs regardless of clonotype, they bypass native T cell signal integration mechanisms that rely on monovalent, membrane-associated, and relatively weakly binding ligand in the context of cellular adhesion. Commonly used antibodies and their derivatives bind much more strongly than native peptide major histocompatibility complex (pMHC) ligands bind their cognate TCRs. Because ligand dwell time is a critical parameter that tightly correlates with physiological function of the TCR signaling system, there is a general need, both in research and therapeutics, for universal TCR ligands with controlled kinetic binding parameters. To this end, we have introduced point mutations into recombinantly expressed α-TCRβ H57 Fab to modulate the dwell time of monovalent Fab binding to TCR. When tethered to a supported lipid bilayer via DNA complementation, these monovalent Fab’-DNA ligands activate T cells with potencies well-correlated with their TCR binding dwell time. Single-molecule tracking studies in live T cells reveal that individual binding events between Fab'-DNA ligands and TCRs elicit local signaling responses closely resembling native pMHC. The unique combination of high on- and off-rates of the H57 R97L mutant enables direct observations of cooperative interplay between ligand binding and TCR-proximal condensation of the linker for activation of T cells, which is not readily visualized with pMHC. This work provides insights into how T cells integrate kinetic information from TCR ligands and introduces a method to develop affinity panels for polyclonal T cells, such as cells from a human patient.
RAS is the most frequently mutated oncogene in cancer. RAS proteins show high sequence similarities in their G-domains but are significantly different in their C-terminal hypervariable regions (HVR). These regions interact with the cell membrane via lipid anchors that result from posttranslational modifications (PTM) of cysteine residues. KRAS4b is unique as it has only one cysteine that undergoes PTM, C185. Small molecule covalent modification of C185 would block any form of prenylation and subsequently inhibit attachment of KRAS4b to the cell membrane, blocking its biological activity. We translated this concept to the discovery and development of disulfide tethering screen hits into irreversible covalent modifiers of C185. These compounds inhibited proliferation of KRAS4b-driven mouse embryonic fibroblasts, but not cells driven by N-myristoylated KRAS4b that harbor a C185S mutation and are not dependent on C185 prenylation. Top–down proteomics was used to confirm target engagement in cells. These compounds bind in a pocket formed when the HVR folds back between helix 3 and 4 in the G-domain (HVR-α3-α4). This interaction can happen in the absence of small molecules as predicted by molecular dynamics simulations and is stabilized in the presence of C185 binders as confirmed by small-angle X-ray scattering and solution NMR. NOESY-HSQC, an NMR approach that measures internuclear distances of 6 Å or less, and structure analysis identified the critical residues and interactions that define the HVR-α3-α4 pocket. Further development of compounds that bind to this pocket could be the basis of a new approach to targeting KRAS cancers.
Acetyl-TAG (3-acetyl-1,2-diacylglycerol), unique triacylglycerols (TAG) possessing an acetate group at the sn -3 position, exhibit valuable properties, such as reduced viscosity and freezing points. Previous attempts to engineer acetyl-TAG production in oilseed crops did not achieve the high levels found in naturally producing Euonymus seeds. Here, we demonstrate the successful generation of camelina and pennycress transgenic lines accumulating nearly pure acetyl-TAG at 93 mol% and 98 mol%, respectively. These ultrahigh acetyl-TAG synthesizing lines were created using gene-edited FATTY ACID ELONGASE1 ( FAE1 ) mutant lines as an improved genetic background to increase levels of acetyl-CoA available for acetyl-TAG synthesis mediated by the expression of EfDAcT, a high-activity diacylglycerol acetyltransferase isolated from Euonymus fortunei . Combining EfDAcT expression with suppression of the competing TAG-synthesizing enzyme DGAT1 further enhanced acetyl-TAG accumulation. These ultrahigh levels of acetyl-TAG exceed those in earlier engineered oilseeds and are equivalent or greater than those in Euonymus seeds. Imaging of lipid localization in transgenic seeds revealed that the low amounts of residual TAG were mostly confined to the embryonic axis. Similar spatial distributions of specific TAG and acetyl-TAG molecular species, as well as their probable diacylglycerol (DAG) precursors, provide additional evidence that acetyl-TAG and TAG are both synthesized from the same tissue-specific DAG pools. Remarkably, this ultrahigh production of acetyl-TAG in transgenic seeds exhibited minimal negative effects on seed properties, highlighting the potential for production of designer oils required for economical biofuel industries.
Removing glutamate from the synaptic cleft is vital for proper function of the brain. Excitatory amino acid transporters mediate this process by uptaking the neurotransmitter from the synaptic cleft back to the cell after its release. The archaeal homolog, Glt Ph , an aspartate transporter fromPyrococcus horikoshii, presents the best structurally characterized model for this family of transporters. In order to transport, Glt Ph undergoes elevator-like conformational changes between inward-facing (IF) and outward-facing (OF) states. Here, we characterize, at an atomic level, the OF⇌IF transition of Glt Ph in differentapo/bound states using a combination of ensemble-based enhanced sampling techniques, employing more than two thousand of coupled simulation replicas of membrane-embedded Glt Ph . The resulting free-energy profiles portray the transition ofapo/bound states as a complex four-stage process, while sodium binding alone locks the structure in one of its states. Along the transition, the transport domain (TD) disengages from the scaffold domain (SD), allowing it to move as a piston sliding vertically with respect to the membrane during the elevator-like motion of TD. Lipid interactions with residues comprising the SD–TD interface directly influence the large-scale conformational changes and, consequently, the energetics of transport. Structural intermediates formed during the transition leak water molecules and may correlate to the uncoupled Cl − ion conductance observed experimentally in both prokaryotic and mammalian glutamate transporters. Mechanistic insights obtained from our study provide a structural framework for better development of therapeutic for neurological disorders.
Methane- and ammonia-oxidizing bacteria play key roles in the global carbon and nitrogen cycles, respectively. These bacteria use homologous copper membrane monooxygenases to accomplish the defining chemical transformations of their metabolisms: the oxidations of methane to methanol by particulate methane monooxygenase (pMMO) and ammonia to hydroxylamine by ammonia monooxygenase (AMO), enzymes of prime interest for applications in mitigating climate change. However, investigations of these enzymes have been hindered by the need for disruptive detergent solubilization prior to structure determination, confounding studies of pMMO and precluding studies of AMO. Here, we overcome these challenges by using cryoEM to visualize pMMO and AMO directly in their native membrane arrays at 2.4 to 2.8 Å resolution. These structures reveal details of the copper centers, numerous bound lipids, and previously unobserved components, including identifiable and distinct supernumerary helices interacting with pMMO and AMO, suggesting a widespread role for these helices in copper membrane monooxygenases. Comparisons between these structures, their metallocofactors, and their unexpected protein–protein interactions highlight features that may govern activity or the formation of higher-order arrays in native membranes. The ability to obtain molecular insights within the native membrane will enable further understanding of these environmentally important enzymes.