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.

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.

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.

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.

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.

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.

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.

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.

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.

Exploring the Extreme Acid Tolerance of a Dynamic Protein Nanocage.

Encapsulins are microbial protein nanocages capable of efficient self-assembly and cargo enzyme encapsulation. Due to their favorable properties, including high thermostability, protease resistance, and robust heterologous expression, encapsulins have become popular bioengineering tools for applications in medicine, catalysis, and nanotechnology. Resistance against physicochemical extremes like high temperature and low pH is a highly desirable feature for many biotechnological applications. However, no systematic search for acid-stable encapsulins has been carried out, while the influence of pH on encapsulin shells has so far not been thoroughly explored. Here, we report on a newly identified encapsulin nanocage from the acid-tolerant bacterium Acidipropionibacterium acidipropionici. Using transmission electron microscopy, dynamic light scattering, and proteolytic assays, we demonstrate its extreme acid tolerance and resilience against proteases. We structurally characterize the novel nanocage using cryo-electron microscopy, revealing a dynamic five-fold pore that displays distinct "closed" and "open" states at neutral pH but only a singular "closed" state under strongly acidic conditions. Further, the "open" state exhibits the largest pore in an encapsulin shell reported to date. Non-native protein encapsulation capabilities are demonstrated, and the influence of external pH on internalized cargo is explored. Our results expand the biotechnological application range of encapsulin nanocages toward potential uses under strongly acidic conditions and highlight pH-responsive encapsulin pore dynamics.

Heterologous expression and purification of encapsulins in Streptomyces coelicolor.

In recent years a large number of encapsulin nanocompartment-encoding operons have been identified in bacterial and archaeal genomes. Encapsulin-encoding genes and operons from GC-rich Gram-positive bacteria, particularly of the phylum Actinobacteria, are often difficult to overexpress and purify in a soluble form using standard Escherichia coli expression systems. Here, we present a protocol to heterologously overexpress encapsulin nanocompartments and encapsulin-containing operons in Streptomyces coelicolor. Successful encapsulin production begins with the transfer of a Streptomyces expression plasmid, encoding the gene(s) of interest, via conjugation to the model actinobacterium S. coelicolor. After growing the conjugated S. coelicolor culture to the optimal optical density, protein production is induced by the addition of the inducer thiostrepton, followed by expression in liquid culture for 1-3 days. Cells are lysed and encapsulin proteins purified using ammonium sulfate precipitation and size exclusion chromatography. The method outlined in this protocol can be utilized to improve cargo loading and overall soluble expression of encapsulin systems when compared to expression in E. coli.•Clone an encapsulin-encoding gene or operon into a Streptomyces expression vector.•Transfer the Streptomyces expression vector to S. coelicolor via conjugation.•Heterologously express and purify empty or cargo-loaded encapsulins from S. coelicolor.

Pore structure controls stability and molecular flux in engineered protein cages.

Protein cages are a common architectural motif used by living organisms to compartmentalize and control biochemical reactions. While engineered protein cages have featured in the construction of nanoreactors and synthetic organelles, relatively little is known about the underlying molecular parameters that govern stability and flux through their pores. In this work, we systematically designed 24 variants of the Thermotoga maritima encapsulin cage, featuring pores of different sizes and charges. Twelve pore variants were successfully assembled and purified, including eight designs with exceptional thermal stability. While negatively charged mutations were better tolerated, we were able to form stable assemblies covering a full range of pore sizes and charges, as observed in seven new cryo-EM structures at 2.5- to 3.6-Å resolution. Molecular dynamics simulations and stopped-flow experiments revealed the importance of considering both pore size and charge, together with flexibility and rate-determining steps, when designing protein cages for controlling molecular flux.

Triggered Reversible Disassembly of an Engineered Protein Nanocage*.

Protein nanocages play crucial roles in sub-cellular compartmentalization and spatial control in all domains of life and have been used as biomolecular tools for applications in biocatalysis, drug delivery, and bionanotechnology. The ability to control their assembly state under physiological conditions would further expand their practical utility. To gain such control, we introduced a peptide capable of triggering conformational change at a key structural position in the largest known encapsulin nanocompartment. We report the structure of the resulting engineered nanocage and demonstrate its ability to disassemble and reassemble on demand under physiological conditions. We demonstrate its capacity for in vivo encapsulation of proteins of choice while also demonstrating in vitro cargo loading capabilities. Our results represent a functionally robust addition to the nanocage toolbox and a novel approach for controlling protein nanocage disassembly and reassembly under mild conditions.

Large-scale computational discovery and analysis of virus-derived microbial nanocompartments.

Encapsulins are a class of microbial protein compartments defined by the viral HK97-fold of their capsid protein, self-assembly into icosahedral shells, and dedicated cargo loading mechanism for sequestering specific enzymes. Encapsulins are often misannotated and traditional sequence-based searches yield many false positive hits in the form of phage capsids. Here, we develop an integrated search strategy to carry out a large-scale computational analysis of prokaryotic genomes with the goal of discovering an exhaustive and curated set of all HK97-fold encapsulin-like systems. We find over 6,000 encapsulin-like systems in 31 bacterial and four archaeal phyla, including two novel encapsulin families. We formulate hypotheses about their potential biological functions and biomedical relevance, which range from natural product biosynthesis and stress resistance to carbon metabolism and anaerobic hydrogen production. An evolutionary analysis of encapsulins and related HK97-type virus families shows that they share a common ancestor, and we conclude that encapsulins likely evolved from HK97-type bacteriophages.

Structure and function of Spc42 coiled-coils in yeast centrosome assembly and duplication.

Centrosomes and spindle pole bodies (SPBs) are membraneless organelles whose duplication and assembly is necessary for bipolar mitotic spindle formation. The structural organization and functional roles of major proteins in these organelles can provide critical insights into cell division control. Spc42, a phosphoregulated protein with an N-terminal dimeric coiled-coil (DCC), assembles into a hexameric array at the budding yeast SPB core, where it functions as a scaffold for SPB assembly. Here, we present in vitro and in vivo data to elucidate the structural arrangement and biological roles of Spc42 elements. Crystal structures reveal details of two additional coiled-coils in Spc42: a central trimeric coiled-coil and a C-terminal antiparallel DCC. Contributions of the three Spc42 coiled-coils and adjacent undetermined regions to the formation of an ∼145 Šhexameric lattice in an in vitro lipid monolayer assay and to SPB duplication and assembly in vivo reveal structural and functional redundancy in Spc42 assembly. We propose an updated model that incorporates the inherent symmetry of these Spc42 elements into a lattice, and thereby establishes the observed sixfold symmetry. The implications of this model for the organization of the central SPB core layer are discussed.

Design considerations in coiled-coil fusion constructs for the structural determination of a problematic region of the human cardiac myosin rod.

X-ray structural determination of segments of the myosin rod has proved difficult because of the strong salt-dependent aggregation properties and repeating pattern of charges on the surface of the coiled-coil that lead to the formation of paracrystals. This problem has been resolved in part through the use of globular assembly domains that improve protein folding and prevent aggregation. The primary consideration now in designing coiled-coil fusion constructs for myosin is deciding where to truncate the coiled-coil and which amino acid residues to include from the folding domain. This is especially important for myosin that contains numerous regions of low predicted coiled-coil propensity. Here we describe the strategy adopted to determine the structure of the region that extends from Arg1677 - Leu1797 that included two areas that do not show a strong sequence signature of a conventional left-handed coiled coil or canonical heptad repeat. This demonstrates again that, with careful choice of fusion constructs, overlapping structures exhibit very similar conformations for the myosin rod fragments in the canonical regions. However, conformational variability is seen around Leu1706 which is a hot spot for cardiomyopathy mutations suggesting that this might be important for function.