Encapsulin cargo loading: progress and potential

Jones, Jesse A.; Benisch, Robert; Giessen, Tobias

doi:10.1039/d3tb00288h

Cited by 21 publications

(11 citation statements)

References 87 publications

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“…The disordered N-terminal domains have recently been proposed to function as cargo loading domains (CLD) in Family 2B encapsulin systems 22 . Similar N-terminal CLDs are found in Family 2A cysteine desulfurase cargo proteins and have already been confirmed as essential for cargo encapsulation 22,23,39,43 .…”

Section: Resultssupporting

confidence: 67%

“…The CLDs of Family 2 encapsulins are extended and flexible domains rich in proline, glycine, and alanine 22,23,39,43 . Inspection of Sg 2-MIBS AlphaFold models suggests two CLD stretches with helical propensity which we used as markers to subdivide the Sg 2-MIBS CLD into five regions – CLD_A to CLD_E – for mutational and cargo-loading analysis (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Andreas,

Giessen

2024

Preprint

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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 bacteriumStreptomyces griseus. Here, we revise the function of EshA, now referred to asSgEnc, and show that it is a Family 2B encapsulin shell protein. Using cryo-electron microscopy, we find thatSgEnc forms an icosahedral protein shell and encapsulates 2-methylisoborneol synthase (2-MIBS) as a cargo protein.SgEnc 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 thatSgEnc 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 andSgEnc 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.

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Section: Resultssupporting

confidence: 67%

Section: Resultsmentioning

confidence: 99%

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

Andreas,

Giessen

2024

Preprint

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“…Encapsulins consist of self-assembling protein shells sequestering dedicated cargo enzymes and are among the most widespread protein compartments in prokaryotes ( 21 , 23 , 24 ). Cargo encapsulation is mediated by targeting sequences present at the N or C terminus of all cargo proteins ( 21 , 22 , 25 , 26 ). Encapsulin shells have icosahedral (I) symmetry with triangulation numbers of T = 1 (60 subunits, 24 nm), T = 3 (180 subunits, 32 nm), or T = 4 (240 subunits, 42 nm), and an evolutionary connection with viral capsids has been proposed ( 21 , 22 , 27 – 29 ).…”

Section: Introductionmentioning

confidence: 99%

A widespread bacterial protein compartment sequesters and stores elemental sulfur

Benisch,

Andreas,

Giessen

2024

Sci. Adv.

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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.

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“…In a recent study, we identified a putative single domain rhodanese (Ab-RLD) associated with an encapsulin nanocompartment operon found in Acinetobacter baumannii 14 . Encapsulin nanocompartments are self-assembling compartmentalization systems widespread in prokaryotes where cargo enzymes are specifically encapsulated inside 18 to 42 nm icosahedral protein shells 15,16–18 . Encapsulins have been shown to be involved in iron storage 19–21 , peroxide detoxification 22,23 , and sulfur metabolism 24 .…”

Section: Introductionmentioning

confidence: 99%

Structural and biochemical characterization of an encapsulin-associated rhodanese fromAcinetobacter baumannii

Benisch,

Giessen

2024

Preprint

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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 domainAcinetobacter baumanniiRLD (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 andin vitroassays, 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.

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Encapsulin cargo loading: progress and potential

Abstract: Encapsulins are a recently discovered class of prokaryotic self-assembling icosahedral protein nanocompartments measuring between 24 and 42 nm in diameter, capable of selectively encapsulating dedicated cargo proteins in vivo. They...

Cited by 21 publications

References 87 publications

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

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

A widespread bacterial protein compartment sequesters and stores elemental sulfur

Structural and biochemical characterization of an encapsulin-associated rhodanese fromAcinetobacter baumannii

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