Pore engineering as a general strategy to improve protein-based enzyme nanoreactor performance.

Enzyme nanoreactors are nanoscale compartments consisting of encapsulated enzymes and a selectively permeable barrier. Sequestration and co-localization of enzymes can increase catalytic activity, stability, and longevity, highly desirable features for many biotechnological and biomedical applications of enzyme catalysts. One promising strategy to construct enzyme nanoreactors is to repurpose protein nanocages found in nature. However, protein-based enzyme nanoreactors often exhibit decreased catalytic activity, partially caused by a mismatch of protein shell selectivity and the substrate requirements of encapsulated enzymes. No broadly applicable and modular protein-based nanoreactor platform is currently available. Here, we introduce a pore-engineered universal enzyme nanoreactor platform based on encapsulins - microbial self-assembling protein nanocompartments with programmable and selective enzyme packaging capabilities. We structurally characterize our protein shell designs via cryo-electron microscopy and highlight their polymorphic nature. Through fluorescence polarization assays, we show their improved molecular flux behavior and highlight their expanded substrate range via a number of proof-of-concept enzyme nanoreactor designs. This work lays the foundation for utilizing our encapsulin-based nanoreactor platform for future biotechnological and biomedical applications.

Point mutation in a virus-like capsid drives symmetry reduction to form tetrahedral cages.

Protein capsids are a widespread form of compartmentalization in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximizes the internal volume that can be enclosed within. Despite the strong preference for icosahedral symmetry, we show that simple point mutations in a virus-like capsid can drive the assembly of unique symmetry-reduced structures. Starting with the encapsulin from Myxococcus xanthus, a 180-mer bacterial capsid that adopts the well-studied viral HK97 fold, we use mass photometry and native charge detection mass spectrometry to identify a triple histidine point mutant that forms smaller dimorphic assemblies. Using cryoelectron microscopy, we determine the structures of a precedented 60-mer icosahedral assembly and an unexpected 36-mer tetrahedron that features significant geometric rearrangements around a new interaction surface between capsid protomers. We subsequently find that the tetrahedral assembly can be generated by triple-point mutation to various amino acids and that even a single histidine point mutation is sufficient to form tetrahedra. These findings represent a unique example of tetrahedral geometry when surveying all characterized encapsulins, HK97-like capsids, or indeed any virus-derived capsids reported in the Protein Data Bank, revealing the surprising plasticity of capsid self-assembly that can be accessed through minimal changes in the protein sequence.

The biosynthesis of the odorant 2-methylisoborneol is compartmentalized inside a protein shell.

Terpenoids are the largest class of natural products, found across all domains of life. One of the most abundant bacterial terpenoids is the volatile odorant 2-methylisoborneol (2-MIB), partially responsible for the earthy smell of soil and musty taste of contaminated water. Many bacterial 2-MIB biosynthetic gene clusters were thought to encode a conserved transcription factor, named EshA in the model soil bacterium Streptomyces griseus. Here, we revise the function of EshA, now referred to as Sg Enc, and show that it is a Family 2B encapsulin shell protein. Using cryo-electron microscopy, we find that Sg Enc forms an icosahedral protein shell and encapsulates 2-methylisoborneol synthase (2-MIBS) as a cargo protein. Sg Enc contains a cyclic adenosine monophosphate (cAMP) binding domain (CBD)-fold insertion and a unique metal-binding domain, both displayed on the shell exterior. We show that Sg Enc CBDs do not bind cAMP. We find that 2-MIBS cargo loading is mediated by an N-terminal disordered cargo-loading domain and that 2-MIBS activity and Sg Enc shell structure are not modulated by cAMP. Our work redefines the function of EshA and establishes Family 2B encapsulins as cargo-loaded protein nanocompartments involved in secondary metabolite biosynthesis.

A two-component quasi-icosahedral protein nanocompartment with variable shell composition and irregular tiling.

Protein shells or capsids are a widespread form of compartmentalization in nature. Viruses use protein capsids to protect and transport their genomes while many cellular organisms use protein shells for varied metabolic purposes. These protein-based compartments often exhibit icosahedral symmetry and consist of a small number of structural components with defined roles. Encapsulins are a prevalent protein-based compartmentalization strategy in prokaryotes. All encapsulins studied thus far consist of a single shell protein that adopts the viral HK97-fold. Here, we report the characterization of a Family 2B two-component encapsulin from Streptomyces lydicus. We show the differential assembly behavior of the two shell components and demonstrate their ability to co-assemble into mixed shells with variable shell composition. We determined the structures of both shell proteins using cryo-electron microscopy. Using 3D-classification and crosslinking studies, we highlight the irregular tiling of mixed shells. Our work expands the known assembly modes of HK97-fold proteins and lays the foundation for future functional and engineering studies on two-component encapsulins.

SEB: a computational tool for symbolic derivation of the small-angle scattering from complex composite structures.

Analysis of small-angle scattering (SAS) data requires intensive modeling to infer and characterize the structures present in a sample. This iterative improvement of models is a time-consuming process. Presented here is Scattering Equation Builder (SEB), a C++ library that derives exact analytic expressions for the form factors of complex composite structures. The user writes a small program that specifies how the sub-units should be linked to form a composite structure and calls SEB to obtain an expression for the form factor. SEB supports e.g. Gaussian polymer chains and loops, thin rods and circles, solid spheres, spherical shells and cylinders, and many different options for how these can be linked together. The formalism behind SEB is presented and simple case studies are given, such as block copolymers with different types of linkage, as well as more complex examples, such as a random walk model of 100 linked sub-units, dendrimers, polymers and rods attached to the surfaces of geometric objects, and finally the scattering from a linear chain of five stars, where each star is built up of four diblock copolymers. These examples illustrate how SEB can be used to develop complex models and hence reduce the cost of analyzing SAS data.

Cyclodipeptide oxidase is an enzyme filament.

Modified cyclic dipeptides represent a widespread class of secondary metabolites with diverse pharmacological activities, including antibacterial, antifungal, and antitumor. Here, we report the structural characterization of the Streptomyces noursei enzyme AlbAB, a cyclodipeptide oxidase (CDO) carrying out α,ß-dehydrogenations during the biosynthesis of the antibiotic albonoursin. We show that AlbAB is a megadalton heterooligomeric enzyme filament containing covalently bound flavin mononucleotide cofactors. We highlight that AlbAB filaments consist of alternating dimers of AlbA and AlbB and that enzyme activity is crucially dependent on filament formation. We show that AlbA-AlbB interactions are highly conserved suggesting that other CDO-like enzymes are likely enzyme filaments. As CDOs have been employed in the structural diversification of cyclic dipeptides, our results will be useful for future applications of CDOs in biocatalysis and chemoenzymatic synthesis.

Structural basis for peroxidase encapsulation inside the encapsulin from the Gram-negative pathogen Klebsiella pneumoniae.

Encapsulins are self-assembling protein nanocompartments capable of selectively encapsulating dedicated cargo proteins, including enzymes involved in iron storage, sulfur metabolism, and stress resistance. They represent a unique compartmentalization strategy used by many pathogens to facilitate specialized metabolic capabilities. Encapsulation is mediated by specific cargo protein motifs known as targeting peptides (TPs), though the structural basis for encapsulation of the largest encapsulin cargo class, dye-decolorizing peroxidases (DyPs), is currently unknown. Here, we characterize a DyP-containing encapsulin from the enterobacterial pathogen Klebsiella pneumoniae. By combining cryo-electron microscopy with TP and TP-binding site mutagenesis, we elucidate the molecular basis for cargo encapsulation. TP binding is mediated by cooperative hydrophobic and ionic interactions as well as shape complementarity. Our results expand the molecular understanding of enzyme encapsulation inside protein nanocompartments and lay the foundation for rationally modulating encapsulin cargo loading for biomedical and biotechnological applications.

Structural and biochemical characterization of an encapsulin-associated rhodanese from Acinetobacter baumannii.

Rhodanese-like domains (RLDs) represent a widespread protein family canonically involved in sulfur transfer reactions between diverse donor and acceptor molecules. RLDs mediate these transsulfuration reactions via a transient persulfide intermediate, created by modifying a conserved cysteine residue in their active sites. RLDs are involved in various aspects of sulfur metabolism, including sulfide oxidation in mitochondria, iron-sulfur cluster biogenesis, and thio-cofactor biosynthesis. However, due to the inherent complexity of sulfur metabolism caused by the intrinsically high nucleophilicity and redox sensitivity of thiol-containing compounds, the physiological functions of many RLDs remain to be explored. Here, we focus on a single domain Acinetobacter baumannii RLD (Ab-RLD) associated with a desulfurase encapsulin which is able to store substantial amounts of sulfur inside its protein shell. We determine the 1.6 Å x-ray crystal structure of Ab-RLD, highlighting a homodimeric structure with a number of unusual features. We show through kinetic analysis that Ab-RLD exhibits thiosulfate sulfurtransferase activity with both cyanide and glutathione acceptors. Using native mass spectrometry and in vitro assays, we provide evidence that Ab-RLD can stably carry a persulfide and thiosulfate modification and may employ a ternary catalytic mechanism. Our results will inform future studies aimed at investigating the functional link between Ab-RLD and the desulfurase encapsulin.

Point mutation in a virus-like capsid drives symmetry reduction to form tetrahedral cages.

Protein capsids are a widespread form of compartmentalisation in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximises the internal volume that can be enclosed within. Despite the strong preference for icosahedral symmetry, we show that simple point mutations in a virus-like capsid can drive the assembly of novel symmetry-reduced structures. Starting with the encapsulin from Myxococcus xanthus, a 180-mer bacterial capsid that adopts the well-studied viral HK97 fold, we use mass photometry and native charge detection mass spectrometry to identify a triple histidine point mutant that forms smaller dimorphic assemblies. Using cryo-EM, we determine the structures of a precedented 60-mer icosahedral assembly and an unprecedented 36-mer tetrahedron that features significant geometric rearrangements around a novel interaction surface between capsid protomers. We subsequently find that the tetrahedral assembly can be generated by triple point mutation to various amino acids, and that even a single histidine point mutation is sufficient to form tetrahedra. These findings represent the first example of tetrahedral geometry across all characterised encapsulins, HK97-like capsids, or indeed any virus-derived capsids reported in the Protein Data Bank, revealing the surprising plasticity of capsid self-assembly that can be accessed through minimal changes in protein sequence.

A widespread bacterial protein compartment sequesters and stores elemental sulfur.

Subcellular compartments often serve to store nutrients or sequester labile or toxic compounds. As bacteria mostly do not possess membrane-bound organelles, they often have to rely on protein-based compartments. Encapsulins are one of the most prevalent protein-based compartmentalization strategies found in prokaryotes. Here, we show that desulfurase encapsulins can sequester and store large amounts of crystalline elemental sulfur. We determine the 1.78-angstrom cryo-EM structure of a 24-nanometer desulfurase-loaded encapsulin. Elemental sulfur crystals can be formed inside the encapsulin shell in a desulfurase-dependent manner with l-cysteine as the sulfur donor. Sulfur accumulation can be influenced by the concentration and type of sulfur source in growth medium. The selectively permeable protein shell allows the storage of redox-labile elemental sulfur by excluding cellular reducing agents, while encapsulation substantially improves desulfurase activity and stability. These findings represent an example of a protein compartment able to accumulate and store elemental sulfur.

Striatal dopamine signals reflect perceived cue-action-outcome associations in mice.

Striatal dopamine drives associative learning by acting as a teaching signal. Much work has focused on simple learning paradigms, including Pavlovian and instrumental learning. However, higher cognition requires that animals generate internal concepts of their environment, where sensory stimuli, actions and outcomes become flexibly associated. Here, we performed fiber photometry dopamine measurements across the striatum of male mice as they learned cue-action-outcome associations based on implicit and changing task rules. Reinforcement learning models of the behavioral and dopamine data showed that rule changes lead to adjustments of learned cue-action-outcome associations. After rule changes, mice discarded learned associations and reset outcome expectations. Cue- and outcome-triggered dopamine signals became uncoupled and dependent on the adopted behavioral strategy. As mice learned the new association, coupling between cue- and outcome-triggered dopamine signals and task performance re-emerged. Our results suggest that dopaminergic reward prediction errors reflect an agent's perceived locus of control.

Structural basis for peroxidase encapsulation in a protein nanocompartment.

Encapsulins are self-assembling protein nanocompartments capable of selectively encapsulating dedicated cargo proteins, including enzymes involved in iron storage, sulfur metabolism, and stress resistance. They represent a unique compartmentalization strategy used by many pathogens to facilitate specialized metabolic capabilities. Encapsulation is mediated by specific cargo protein motifs known as targeting peptides (TPs), though the structural basis for encapsulation of the largest encapsulin cargo class, dye-decolorizing peroxidases (DyPs), is currently unknown. Here, we characterize a DyP-containing encapsulin from the enterobacterial pathogen Klebsiella pneumoniae. By combining cryo-electron microscopy with TP mutagenesis, we elucidate the molecular basis for cargo encapsulation. TP binding is mediated by cooperative hydrophobic and ionic interactions as well as shape complementarity. Our results expand the molecular understanding of enzyme encapsulation inside protein nanocompartments and lay the foundation for rationally modulating encapsulin cargo loading for biomedical and biotechnological applications.

Cyclodipeptide oxidase is an enzyme filament.

Modified cyclic dipeptides represent a widespread class of secondary metabolites with diverse pharmacological activities, including antibacterial, antifungal, and antitumor. Here, we report the structural characterization of the Streptomyces noursei enzyme AlbAB, a cyclodipeptide oxidase (CDO) carrying out α,ß-dehydrogenations during the biosynthesis of the antibiotic albonoursin. We show that AlbAB is a megadalton heterooligomeric enzyme filament containing covalently bound flavin mononucleotide cofactors. We highlight that AlbAB filaments consist of alternating dimers of AlbA and AlbB and that enzyme activity is crucially dependent on filament formation. We show that AlbA-AlbB interactions are highly conserved suggesting that all CDO-like enzymes are likely enzyme filaments. Our work represents the first structural characterization of a CDO. As CDOs have been employed in the structural diversification of cyclic dipeptides, our results will be useful for future applications of CDOs in biocatalysis and chemoenzymatic synthesis.

Structure and heterogeneity of a highly cargo-loaded encapsulin shell.

Encapsulins are self-assembling protein nanocompartments able to selectively encapsulate dedicated cargo enzymes. Encapsulins are widespread across bacterial and archaeal phyla and are involved in oxidative stress resistance, iron storage, and sulfur metabolism. Encapsulin shells exhibit icosahedral geometry and consist of 60, 180, or 240 identical protein subunits. Cargo encapsulation is mediated by the specific interaction of targeting peptides or domains, found in all cargo proteins, with the interior surface of the encapsulin shell during shell self-assembly. Here, we report the 2.53 Å cryo-EM structure of a heterologously produced and highly cargo-loaded T3 encapsulin shell from Myxococcus xanthus and explore the systems' structural heterogeneity. We find that exceedingly high cargo loading results in the formation of substantial amounts of distorted and aberrant shells, likely caused by a combination of unfavorable steric clashes of cargo proteins and shell conformational changes. Based on our cryo-EM structure, we determine and analyze the targeting peptide-shell binding mode. We find that both ionic and hydrophobic interactions mediate targeting peptide binding. Our results will guide future attempts at rationally engineering encapsulins for biomedical and biotechnological applications.

Dose-dependent in vivo effects of formulated moxidectin on seedling emergence of temperate grassland species.

Sheep function as effective endozoochorous seed vectors in grasslands. Recent laboratory-based studies showed that this important function can be impaired by macrocyclic lactone anthelmintics, which are used to control parasites and enter into the environment mainly via faeces; however, there is a lack of in vivo studies. We conducted a seed-feeding experiment with sheep that included four temperate grassland species from four different families (Achillea ptarmica, Asteraceae; Agrostis capillaris, Poaceae; Dianthus deltoides, Caryophyllaceae; Plantago lanceolata, Plantaginaceae). A series of three feeding trials was carried out after one of two groups of sheep received a single administration of a common oral formulation of the macrocyclic lactone moxidectin. Faeces were collected to determine seedling emergence rate and emergence timing as well as moxidectin concentration via HPLC. Seedling emergence differed significantly between the anthelmintic-treated sheep and the control group. This impact depended on time of seed uptake after anthelmintic administration. Number of emerging seedlings was significantly reduced (27.1 %) when faeces moxidectin concentrations were high (on average 3153 ng g-1; 1 d post treatment) and significantly increased (up to 68.8 %) when moxidectin concentrations were low (≤86 ng g-1; 7, 14 d pt). Mean emergence time was significantly lowered at low moxidectin concentrations. These results demonstrate dose-related effects of deworming on seedling emergence which might affect endozoochory and eventually plant population dynamics in grasslands.

HIV-1 binds dynein directly to hijack microtubule transport machinery.

Viruses exploit host cytoskeletal elements and motor proteins for trafficking through the dense cytoplasm. Yet the molecular mechanism that describes how viruses connect to the motor machinery is unknown. Here, we demonstrate the first example of viral microtubule trafficking from purified components: HIV-1 hijacking microtubule transport machinery. We discover that HIV-1 directly binds to the retrograde microtubule-associated motor, dynein, and not via a cargo adaptor, as previously suggested. Moreover, we show that HIV-1 motility is supported by multiple, diverse dynein cargo adaptors as HIV-1 binds to dynein light and intermediate chains on dynein's tail. Further, we demonstrate that multiple dynein motors tethered to rigid cargoes, like HIV-1 capsids, display reduced motility, distinct from the behavior of multiple motors on membranous cargoes. Our results introduce a new model of viral trafficking wherein a pathogen opportunistically 'hijacks' the microtubule transport machinery for motility, enabling multiple transport pathways through the host cytoplasm.

Diagnostic performance of a 3D double-echo steady-state sequence at 3 T using radial reformats for detecting and grading rotator cuff tears: a pilot diagnostic accuracy study with magnetic resonance imaging and arthroscopic correlation.

BACKGROUND: In diagnosing rotator cuff tears (RCTs), magnetic resonance imaging (MRI) is the imaging modality of choice, and its accuracy is improving constantly. PURPOSE: To evaluate the diagnostic performance of a high-resolution 3D double-echo steady-state (DESS) sequence with radial and paracoronal 3-T MRI regarding the grading of RCTs in correlation with conventional 2D MRI and arthroscopic findings. MATERIAL AND METHODS: We retrospectively compared arthroscopic findings of RCTs with preoperative MRI, including conventional 2D sequences and radial and paracoronal DESS images in 20 patients. Two observers evaluated supraspinatus (SSP), infraspinatus (ISP), and subscapularis (SSC) tendon tears using a grading system. For statistical analysis, arthroscopy was used as the reference standard. RESULTS: Inter-observer agreement for detecting and grading SSP, ISP, and SSC tendon tears on radial and paracoronal sliced 3D DESS MRI was excellent (intraclass-correlation [ICC] = 0.92-0.98; all P < 0.001). Regarding the detection of SSP lesions, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 93.8%, 50%, 88.2%, and 66.7% for both radial and paracoronal DESS imaging. A sensitivity of 100%, specificity of 61.1%, PPV of 22.2%, and NPV of 100% were noted for detecting ISP tendon tears using radially reformatted DESS images. Regarding detecting SSC tendon tears using radially reformatted DESS images, sensitivity, specificity, PPV, and NPV were 100%, 81.3%, 50%, and 100%, respectively. The results with standard 2D MRI were similar. CONCLUSION: The DESS technique with radially reformatted images provided excellent sensitivity and high inter-observer agreement in detecting RCTs. It showed a moderate to high correlation between MRI and arthroscopy for diagnosing SSP and SSC tendon tears.

Electrically Tunable Reflective Metasurfaces with Continuous and Full-Phase Modulation for High-Efficiency Wavefront Control at Visible Frequencies.

All-dielectric optical metasurfaces can locally control the amplitude and phase of light at the nanoscale, enabling arbitrary wavefront shaping. However, lack of postfabrication tunability has limited the true potential of metasurfaces for many applications. Here, we utilize a thin liquid crystal (LC) layer as a tunable medium surrounding the metasurface to achieve a phase-only spatial light modulator (SLM) with high reflection in the visible frequency, exhibiting active and continuous resonance tuning with associated 2π phase control and uncoupled amplitude. Dynamic wavefront shaping is demonstrated by programming 96 individually addressable electrodes with a small pixel pitch of ∼1 µm. The small pixel size is facilitated by the reduced LC thickness, strongly suppressing cross-talk among pixels. This device is used to demonstrate dynamic beam steering with a wide field-of-view and high absolute diffraction efficiencies. We believe that our demonstration may help realize next-generation, high-resolution SLMs, with wide applications in dynamic holography, tunable optics, and light detection and ranging (LiDAR), to mention a few.

Structure and heterogeneity of a highly cargo-loaded encapsulin shell.

Encapsulins are self-assembling protein nanocompartments able to selectively encapsulate dedicated cargo enzymes. Encapsulins are widespread across bacterial and archaeal phyla and are involved in oxidative stress resistance, iron storage, and sulfur metabolism. Encapsulin shells exhibit icosahedral geometry and consist of 60, 180, or 240 identical protein subunits. Cargo encapsulation is mediated by the specific interaction of targeting peptides or domains, found in all cargo proteins, with the interior surface of the encapsulin shell during shell self-assembly. Here, we report the 2.53 Å cryo-EM structure of a heterologously produced and highly cargo-loaded T3 encapsulin shell from Myxococcus xanthus and explore the systems' structural heterogeneity. We find that exceedingly high cargo loading results in the formation of substantial amounts of distorted and aberrant shells, likely caused by a combination of unfavorable steric clashes of cargo proteins and shell conformational changes. Based on our cryo-EM structure, we determine and analyze the targeting peptide-shell binding mode. We find that both ionic and hydrophobic interactions mediate targeting peptide binding. Our results will guide future attempts at rationally engineering encapsulins for biomedical and biotechnological applications.