Encapsulins.

Subcellular compartmentalization is a defining feature of all cells. In prokaryotes, compartmentalization is generally achieved via protein-based strategies. The two main classes of microbial protein compartments are bacterial microcompartments and encapsulin nanocompartments. Encapsulins self-assemble into proteinaceous shells with diameters between 24 and 42 nm and are defined by the viral HK97-fold of their shell protein. Encapsulins have the ability to encapsulate dedicated cargo proteins, including ferritin-like proteins, peroxidases, and desulfurases. Encapsulation is mediated by targeting sequences present in all cargo proteins. Encapsulins are found in many bacterial and archaeal phyla and have been suggested to play roles in iron storage, stress resistance, sulfur metabolism, and natural product biosynthesis. Phylogenetic analyses indicate that they share a common ancestor with viral capsid proteins. Many pathogens encode encapsulins, and recent evidence suggests that they may contribute toward pathogenicity. The existing information on encapsulin structure, biochemistry, biological function, and biomedical relevance is reviewed here.

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.

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.

In Vitro Synthesis and Reconstitution Using Mammalian Cell-Free Lysates Enables the Systematic Study of the Regulation of LINC Complex Assembly.

Understanding the structure and structure-function relationships of membrane proteins is a fundamental problem in biomedical research. Given the difficulties inherent to performing mechanistic biochemical and biophysical studies of membrane proteins in vitro, we previously developed a facile HeLa cell-based cell-free expression (CFE) system that enables the efficient reconstitution of full-length (FL) functional inner nuclear membrane Sad1/UNC-84 (SUN) proteins (i.e., SUN1 and SUN2) in supported lipid bilayers. Here, we provide evidence that suggests that the reconstitution of CFE-synthesized FL membrane proteins in supported lipid bilayers occurs primarily through the fusion of endoplasmic reticulum-derived microsomes present within our CFE reactions with our supported lipid bilayers. In addition, we demonstrate the ease with which our synthetic biology platform can be used to investigate the impact of the chemical environment on the ability of CFE-synthesized FL SUN proteins reconstituted in supported lipid bilayers to interact with the luminal domain of the KASH protein nesprin-2. Moreover, we use our platform to study the molecular requirements for the homo- and heterotypic interactions between SUN1 and SUN2. Finally, we show that our platform can be used to simultaneously reconstitute three different CFE-synthesized FL membrane proteins in a single supported lipid bilayer. Overall, these results establish our HeLa cell-based CFE and supported lipid bilayer reconstitution platform as a powerful tool for performing mechanistic dissections of the oligomerization and function of FL membrane proteins in vitro. While our platform is not a substitute for cell-based studies, it does provide important mechanistic insights into the biology of difficult-to-study membrane proteins.

Advances in encapsulin nanocompartment biology and engineering.

Compartmentalization is an essential feature of all cells. It allows cells to segregate and coordinate physiological functions in a controlled and ordered manner. Different mechanisms of compartmentalization exist, with the most relevant to prokaryotes being encapsulation via self-assembling protein-based compartments. One widespread example of such is that of encapsulins-cage-like protein nanocompartments able to compartmentalize specific reactions, pathways, and processes in bacteria and archaea. While still relatively nascent bioengineering tools, encapsulins exhibit many promising characteristics, including a number of defined compartment sizes ranging from 24 to 42 nm, straightforward expression, the ability to self-assemble via the Hong Kong 97-like fold, marked physical robustness, and internal and external handles primed for rational genetic and molecular manipulation. Moreover, encapsulins allow for facile and specific encapsulation of native or heterologous cargo proteins via naturally or rationally fused targeting peptide sequences. Taken together, the attributes of encapsulins promise substantial customizability and broad usability. This review discusses recent advances in employing engineered encapsulins across various fields, from their use as bionanoreactors to targeted delivery systems and beyond. A special focus will be provided on the rational engineering of encapsulin systems and their potential promise as biomolecular research tools.

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.

A Catalytic Nanoreactor Based on in Vivo Encapsulation of Multiple Enzymes in an Engineered Protein Nanocompartment.

Bacterial protein compartments concentrate and sequester enzymes, thereby regulating biochemical reactions. Here, we generated a new functional nanocompartment in Escherichia coli by engineering the MS2 phage capsid protein to encapsulate multiple cargo proteins. Sequestration of multiple proteins in MS2-based capsids was achieved by SpyTag/SpyCatcher protein fusions that covalently crosslinked with the interior surface of the capsid. Further, the functional two-enzyme indigo biosynthetic pathway could be targeted to the engineered capsids, leading to a 60 % increase in indigo production in vivo. The enzyme-loaded particles could be purified in their active form and showed enhanced long-term stability in vitro (about 95 % activity after seven days) compared with free enzymes (about 5 % activity after seven days). In summary, this engineered in vivo encapsulation system provides a simple and versatile way for generating highly stable multi-enzyme nanoreactors for in vivo and in vitro applications.

A synthetic adenylation-domain-based tRNA-aminoacylation catalyst.

The incorporation of non-proteinogenic amino acids represents a major challenge for the creation of functionalized proteins. The ribosomal pathway is limited to the 20-22 proteinogenic amino acids while nonribosomal peptide synthetases (NRPSs) are able to select from hundreds of different monomers. Introduced herein is a fusion-protein-based design for synthetic tRNA-aminoacylation catalysts based on combining NRPS adenylation domains and a small eukaryotic tRNA-binding domain (Arc1p-C). Using rational design, guided by structural insights and molecular modeling, the adenylation domain PheA was fused with Arc1p-C using flexible linkers and achieved tRNA-aminoacylation with both proteinogenic and non-proteinogenic amino acids. The resulting aminoacyl-tRNAs were functionally validated and the catalysts showed broad substrate specificity towards the acceptor tRNA. Our strategy shows how functional tRNA-aminoacylation catalysts can be created for bridging the ribosomal and nonribosomal worlds. This opens up new avenues for the aminoacylation of tRNAs with functional non-proteinogenic amino acids.

The tRNA-dependent biosynthesis of modified cyclic dipeptides.

In recent years it has become apparent that aminoacyl-tRNAs are not only crucial components involved in protein biosynthesis, but are also used as substrates and amino acid donors in a variety of other important cellular processes, ranging from bacterial cell wall biosynthesis and lipid modification to protein turnover and secondary metabolite assembly. In this review, we focus on tRNA-dependent biosynthetic pathways that generate modified cyclic dipeptides (CDPs). The essential peptide bond-forming catalysts responsible for the initial generation of a CDP-scaffold are referred to as cyclodipeptide synthases (CDPSs) and use loaded tRNAs as their substrates. After initially discussing the phylogenetic distribution and organization of CDPS gene clusters, we will focus on structural and catalytic properties of CDPSs before turning to two recently characterized CDPS-dependent pathways that assemble modified CDPs. Finally, possible applications of CDPSs in the rational design of structural diversity using combinatorial biosynthesis will be discussed before concluding with a short outlook.

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.

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 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.

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.

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.

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.

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.

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 tRNA-dependent two-enzyme pathway for the generation of singly and doubly methylated ditryptophan 2,5-diketopiperazines.

A large number of bioactive natural products containing a 2,5-diketopiperazine (DKP) moiety have been isolated from various microbial sources. Especially tryptophan-containing cyclic dipeptides (CDPs) show great structural and functional diversity, while little is known about their biosynthetic pathways. Here, we describe the bioinformatic analysis of a cyclodipeptide synthase (CDPS)-containing gene cluster from Actinosynnema mirum spanning 2.9 kb that contains two putative DKP-modifying enzymes. We establish the biosynthetic pathway leading to two methylated ditryptophan CDPs through in vivo and in vitro analyses. Our studies identify the first CDPS (Amir_4627) that shows high substrate specificity synthesizing only one main product, cyclo(Trp-Trp) (cWW). It is the first member of the CDPS family that can form ditryptophan DKPs and the first prokaryotic CDPS whose main product constituents differ from the four amino acids (Phe, Leu, Tyr, and Met) usually found in CDPS-dependent CDPs. We show that after cWW formation a S-adenosyl-l-methionine-dependent N-methyltransferase (Amir_4628) conducts two successive methylations at the DKP-ring nitrogens and additionally show that it is able to methylate four other phenylalanine-containing CDPs. This makes Amir_4628 the first identified DKP-ring-modifying methyltransferase. The large number of known modifying enzymes of bacterial and fungal origin known to act upon Trp-containing DKPs makes the identification of a potent catalyst for cWW formation, encoded by a small gene, valuable for combinatorial in vivo as well as chemoenzymatic approaches, with the aim of generating derivatives of known CDP natural products or entirely new chemical entities with potentially improved or new biological activities.

Two [4Fe-4S] clusters containing radical SAM enzyme SkfB catalyze thioether bond formation during the maturation of the sporulation killing factor.

The sporulation killing factor (SKF) is a 26-residue ribosomally assembled and posttranslationally modified sactipeptide. It is produced by Bacillus subtilis 168 and plays a key role in its sporulation. Like all sactipeptides, SKF contains a thioether bond, which links the cysteine residue Cys4 with the α-carbon of the methionine residue Met12. In this study we demonstrate that this bond is generated by the two [4Fe-4S] clusters containing radical SAM enzyme SkfB, which is encoded in the skf operon. By mutational analysis of both cluster-binding sites, we were able to postulate a mechanism for thioether generation which is in agreement with that of AlbA. Furthermore, we were able to show that thioether bond formation is specific toward hydrophobic amino acids at the acceptor site. Additionally we demonstrate that generation of the thioether linkage is leader-peptide-dependent, suggesting that this reaction is the first step in SKF maturation.