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

Abstract

Protein compartments represent an important strategy for subcellular spatial control and compartmentalization. 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. This has hampered progress in understanding the distribution and functional diversity of encapsulins. 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 report the discovery and analysis of over 6,000 encapsulin-like systems in 31 bacterial and 4 archaeal phyla, including two novel encapsulin families as well as many new operon types that fall within the two already known families. We formulate hypotheses about the biological functions and biomedical relevance of newly identified operons which range from natural product biosynthesis and stress resistance to carbon metabolism and anaerobic hydrogen production. We conduct an evolutionary analysis of encapsulins and related HK97-type virus families and show that they share a common ancestor. We conclude that encapsulins likely evolved from HK97-type bacteriophages. Our study sheds new light on the evolutionary interplay of viruses and cellular organisms, the recruitment of protein folds for novel functions, and the functional diversity of microbial protein organelles.

Nanotechnological Applications Based on Bacterial Encapsulins

“…The encapsulins are a family of~30 kDa bacterial and archaeal proteins that selfassemble into icosahedral compartments which range in size from 20 to 42 nm. These nanocompartments are similar to virus capsids in the icosahedral architecture of the container as well as in the protomer fold, which is structurally similar to capsid proteins of the Hong Kong 97 (HK97)-like virus lineage that includes the Caudovirales bacteriophages [6,[29][30][31][32].…”

“…Cargo enzymes are targeted to the nanocompartment lumen via a cargo-loading peptide (CLP) or targeting peptide (TP) located at their termini, which interacts non-covalently with a binding pocket on the shell luminal surface [6,33,34]. Most archaeal systems have an even more compact configuration in which shell and cargo are fused, and a single gene encodes a completely functional nanocompartment [6,29,32,35,36] (Figure 1).…”

“…There has been a gradual improvement in the algorithms for sequence similarity database searches and a continuous increase in the size and diversity of sequenced genomes of bacteria and archaeal microorganisms; this is producing a progressively sharper picture of the true abundance and diversity of encapsulins in nature [6,29,31,35]. The most recent computational database search identified over 6000 encapsulin-like systems in 31 bacterial and four archaeal phyla, grouped in four different families based on particle structure [32]. Similar analysis of a small fraction of the environmental metagenomic data available uncovered new encapsulins [44], which suggests that other encapsulin systems await discovery, and that these systems constitute a widespread prokaryotic compartmentalization strategy.…”

“…The evolutionary origin of encapsulins remains elusive; this is due in part to the low sequence similarity between encapsulins and members of the HK97-like virus lineage, which implies very old origin, and in part to the few atomic structures available. Based on phylogenetic and structural analyses, encapsulins are proposed to have a viral origin, possibly via "domestication" events of prophage HK97-type capsid proteins [6,[29][30][31][32]. Eukaryotic cells show evidence of similar events involving the Ty3/gypsy superfamily of retrotransposons, ancient mobile elements that are widely distributed and often abundant in their genomes, considered ancestral to modern retroviruses [45].…”

Triggered reversible disassembly of an engineered protein nanocage

“…[8][9][10][11] Encapsulins are icosahedral protein nanocages found in bacteria and archaea with triangulation numbers of T=1 (24 nm), T=3 (32 nm) or T=4 (42 nm) containing sub-nanometer pores at the symmetry axes. 12 They self-assemble from a single HK97-fold capsid protein into 60mer (T=1), 180mer (T=3) or 240mer (T=4) protein cages and are involved in oxidative stress resistance, [13][14][15][16] iron mineralization and storage, 17,18 and sulfur metabolism. 19 Their defining feature is the ability to encapsulate dedicated cargo proteins via short C-terminal targeting peptides (TPs) found in cargo proteins which specifically interact with the interior of the protein shell during self-assembly.…”