Biology subjects

Giessen, T. W.

Publications and source records attributed to Giessen, T. W..

At least 19 recordsLinked to original sources

bioRxiv2026

Bacterial Geosmin Biosynthesis is Compartmentalized Inside a Two-Component Protein Shell

Terpenoids are ubiquitous, structurally diverse natural products found across the three domains of life. One of the most commonly observed and well-studied terpenoids is the volatile, petrichoric compound geosmin, responsible for the earthy smell of soil to which humans are receptive down to a few parts per trillion. Bioinformatic analysis of bacterial geosmin synthase (GeoA)-encoding gene clusters reveals that many are colocalized with genes encoding Family 2B encapsulin shell proteins. Here, we focus on the encapsulin-encoding geosmin gene cluster of the model myxobacterium Myxococcus xanthus and show that the two shell proteins form a two-component Family 2B encapsulin with GeoA as the internalized cargo. Structural analysis highlights that the shell contains characteristic, external cyclic adenosine monophosphate (cAMP) binding-fold domains (CBDs), and that mixed shells can adopt a previously unobserved closed two-fold pore conformation, potentially important in cargo function modulation and regulation. We show that GeoA cargo loading is mediated by repeating, short cargo loading peptides (CLPs), and that GeoA encapsulation provides benefits for enzyme activity and stability. This work expands the short list of Family 2B encapsulins known to be involved in specialized metabolite biosynthesis, and provides insights into a putative mode of cargo protein regulation via pore state modulation.

biochemistry↗

bioRxiv2026

Evidence that the Kuenenia stuttgartiensis encapsulin does not protect against NO damage

A paradigm shift is underway in microbiology: many prokaryotes, long considered to lack the compartmentalization present in all eukaryotic life, have been found to possess a great diversity of protein based intracellular compartments. Notably, the genomes of many marine and freshwater anaerobic ammonium oxidizing (anammox) bacteria encode one of these compartmentalization strategies; encapsulin nanocompartments. These systems structure suggests a role for anammox encapsulins in the anammox metabolism, a process of global biogeochemical significance, which results in the loss of biologically available nitrogen from aquatic environments. Here we test if the most common anammox encapsulin architecture could provide a mechanism to detoxify NO, one of the reactive intermediates produced in the core anammox metabolism. Through experiments in which the Kuenenia stuttgartiensis encapsulin was heterologously expressed by an inducible plasmid in E. coli, we show evidence that suggests the K. stuttgartiensis encapsulin provides no protection from NO.

microbiology↗

bioRxiv2026

A permeable protein nanocage enables facile cargo loading and cytosolic protein delivery

The cytosolic delivery of therapeutic proteins remains one of the most persistent challenges in modern drug delivery. Here, we report the discovery and characterization of an encapsulin-based protein nanocage, QtEnc, with unexpected permeability properties and the ability to internalize cargo proteins in vitro, fundamentally departing from existing protein nanocage cargo loading paradigms. This permeability enables simple, rapid, and single-step post-assembly cargo loading, accommodating cargos as large as 482 kDa, and allowing multiplexed cargo co-encapsulation with tunable ratios. Leveraging this property, we develop a modular QtEnc-based NanoCarrier (QtEncNC) with a pH-responsive cargo detachment module and an endosomal escape module, enabling low pH-triggered cargo release from assembled shells and subsequent endosomal escape for cytosolic delivery. Using a cytotoxic protein, BLF1, as a proof-of-concept QtEncNC payload, we demonstrate efficient cytosolic protein delivery in HeLa cells. These findings establish QtEncNC as a versatile and modular platform for cytosolic protein delivery with broad biomedical potential.

bioengineering↗

bioRxiv2026

Directed evolution of multimeric proteins is enabled by dual-compensatory gene duplication

Gene duplication has played a critical role in the evolutionary history of proteins, enabling complex multimers to emerge from simpler precursors. Yet in protein engineering, current methods for directed evolution do not exploit gene duplication, hampering access to the vast array of diverse variants that are only enriched in the presence of a wild-type copy. We establish a directed evolution strategy for multimeric proteins that harnesses gene duplication to compensate for metabolic burden and self-assembly fitness, allowing previously inaccessible variants to be enriched. Starting from a homomeric 240-mer capsid, gene duplication enables selection of both extreme homomeric variants and obligate heteromers. This strategy significantly expands engineering access to diverse high-performing variants, while also supporting a plausible model for evolutionary diversification of higher-order multimers in nature.

evolutionary biology↗

bioRxiv2025

Engineering Spatial Control of Bacterial Organelles

Bacteria were once thought to lack organelles, but it is now clear they confine cellular reactions using an array of membrane- and protein-based compartments. A central question, however, is how bacterial organelles are organized in the cell, and whether their spatial control can be engineered. Here, we show that a two-protein system (McdAB) that positions carboxysomes - CO2-fixing organelles found in autotrophic bacteria - can be repurposed to provide programmable spatial control to diverse organelles in Escherichia coli. McdAB not only restores proper assembly and positioning of heterologously produced carboxysomes in E. coli, but can also be reprogrammed to spatially organize all other known types of bacterial organelles, including encapsulins, biomolecular condensates, and even membrane-bound organelles. Programmable spatial organization of bacterial organelles establishes a new design principle for synthetic biology, where the location of reactions is as tunable as their content. Our work paves the way for more efficient biocatalysis in engineered microbes.

microbiology↗

bioRxiv2024

Structural and biochemical characterization of a widespread enterobacterial peroxidase encapsulin

Encapsulins are self-assembling protein compartments found in prokaryotes and specifically encapsulate dedicated cargo enzymes. The most abundant encapsulin cargo class are Dye-decolorizing Peroxidases (DyPs). It has been previously suggested that DyP encapsulins are involved in oxidative stress resistance and bacterial pathogenicity due to DyPs inherent ability to reduce and detoxify hydrogen peroxide while oxidizing a broad range of organic co-substrates. Here, we report the structural and biochemical analysis of a DyP encapsulin widely found across enterobacteria. Using bioinformatic approaches, we show that this DyP encapsulin is encoded by a conserved transposon-associated operon, enriched in enterobacterial pathogens. Through low pH and peroxide exposure experiments, we highlight the stability of this DyP encapsulin under harsh conditions and show that DyP catalytic activity is highest at low pH. We determine the structure of the DyP-loaded shell and free DyP via cryo-electron microscopy, revealing the structural basis for DyP cargo loading and peroxide preference. Our work lays the foundation to further explore the substrate range and physiological functions of enterobacterial DyP encapsulins.

biochemistry↗

bioRxiv2024

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.

synthetic biology↗

bioRxiv2024

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.

biochemistry↗

bioRxiv2024

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.

biochemistry↗

bioRxiv2024

Structural and biochemical characterization of an encapsulin-associated rhodanesefrom 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 [A] 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.

biochemistry↗

bioRxiv2024

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. Significance statementViral capsids are cage-like protein assemblies that preferentially adopt icosahedral symmetry to maximise their internal volume for housing genetic material. This icosahedral preference extends to encapsulins, a widespread family of bacterial protein cages which evolved from viral capsids. Counter to this fundamental geometric preference, the formation of well-defined tetrahedral cages from a single amino acid substitution in an encapsulin reveals the surprising geometric flexibility of a common viral protein fold. These findings suggest that protein oligomerisation is far more permissive than intuitively expected, where serendipitous interactions between proteins arising from minimal mutations can cascade to form vast architectural changes. The ability to redesign protein architectures through simple mutations should enable biotechnological advances in vaccine development, drug delivery, and enzymatic biomanufacturing.

biochemistry↗

bioRxiv2023

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 ,{beta}-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.

biochemistry↗

bioRxiv2023

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.

biochemistry↗

bioRxiv2023

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 dyneins 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.

microbiology↗

bioRxiv2023

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 [A] 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.

biochemistry↗

bioRxiv2023

A widespread proteinaceous sulfur storage compartment in bacteria

Intracellular compartmentalization is essential for all cells and enables the regulation and optimization of metabolism1. One of the main functions of subcellular compartments is the storage of nutrients2-4. As bacteria do generally not possess membrane-bound organelles, they often have to rely on functionally analogous protein-based compartments2,5-7. Encapsulin nanocompartments are one of the most prevalent protein-based compartmentalization strategies found in prokaryotes5,8. Here we show that desulfurase encapsulins represent a novel sulfur storage compartment in bacteria able to sequester large amounts of crystalline elemental sulfur. We determined the 1.78 [A] cryo-EM structure of a 24 nm desulfurase-loaded encapsulin highlighting the molecular details of the protein shell and desulfurase encapsulation. We found that elemental sulfur crystals can be formed inside encapsulin shells in a desulfurase-dependent manner with L-cysteine acting as the sulfur donor. Intracellular sulfur accumulation can be influenced by the concentration and type of sulfur source in growth media. The selectively permeable protein shell allows the long-term intracellular storage of redox-labile elemental sulfur by excluding cellular reducing agents from its interior. We found that encapsulation substantially improves desulfurase activity and stability while also preventing substrate inhibition. These findings represent the first example of a dedicated and widespread storage system for the essential element sulfur in bacteria and provide the basis for understanding how this novel protein-based storage compartment is integrated within bacterial metabolism.

biochemistry↗

bioRxiv2022

Exploring the Extreme Acid Tolerance of a Dynamic Protein Nanocage

Encapsulins are protein nanocages capable of efficient self-assembly and cargo enzyme encapsulation. They are found in a wide variety of bacteria and archaea, including many extremophiles, and are involved in iron and sulfur homeostasis, oxidative stress resistance, and secondary metabolite production. Resistance against physicochemical extremes like high temperature and low pH is a key adaptation of many extremophiles and also represents a highly desirable feature for many biotechnological applications. However, no systematic characterization of 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 (AaEnc) 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. AaEnc is the first characterized highly acid stable encapsulin with a unique pH-dependent dynamic pore and its molecular characterization provides novel mechanistic details underlying the pH stability of large dynamic protein complexes.

biochemistry↗

bioRxiv2022

Engineered protein nanocages for concurrent RNA and protein packaging in vivo

Protein nanocages have emerged as an important engineering platform for biotechnological and biomedical applications. Among naturally occurring protein cages, encapsulin nanocompartments have recently gained prominence due to their favorable physico-chemical properties, ease of shell modification, and highly efficient and selective intrinsic protein packaging capabilities. Here, we expand encapsulin function by designing and characterizing encapsulins for concurrent RNA and protein encapsulation in vivo. Our strategy is based on modifying encapsulin shells with nucleic acid binding peptides without disrupting the native protein packaging mechanism. We show that our engineered encapsulins reliably self-assemble in vivo, are capable of efficient size-selective in vivo RNA packaging, can simultaneously load multiple functional RNAs, and can be used for concurrent in vivo packaging of RNA and protein. Our engineered encapsulation platform has potential for co-delivery of therapeutic RNAs and proteins to elicit synergistic effects, and as a modular tool for other biotechnological applications.

synthetic biology↗