The Structural Diversity of Encapsulin Protein Shells

Abstract

Subcellular compartmentalization is a universal feature of all cells. Spatially distinct compartments, be they lipid- or protein-based, enable cells to optimize local reaction environments, store nutrients, and sequester toxic processes. Prokaryotes generally lack intracellular membrane systems and usually rely on protein-based compartments and organelles to regulate and optimize their metabolism. Encapsulins are one of the most diverse and widespread classes of prokaryotic protein compartments. They self-assemble into icosahedral protein shells and are able to specifically internalize dedicated cargo enzymes. This review discusses the structural diversity of encapsulin protein shells, focusing on shell assembly, symmetry, and dynamics. The properties and functions of pores found within encapsulin shells will also be discussed. In addition, fusion and insertion domains embedded within encapsulin shell protomers will be highlighted. Finally, future research directions for basic encapsulin biology, with a focus on the structural understand of encapsulins, are briefly outlined.

Keywords: Encapsulin; HK97; Nanocompartment; Protein capsid; Protein shell.

Figure 1

Encapsulin classification and protomer structures. (A) Encapsulin classification scheme based on sequence and structural homology as well as genome neighborhood conservation. Accessory genes represent non‐cargo but conserved operon components. Question marks indicate lack of experimental evidence. Enc: encapsulin shell protein. (B) Structures of Family 1 encapsulin protomers. Examples of protomers found in T=1, T=3, and T=4 shells are shown. Core domains of the HK97 phage‐like fold (A‐domain, P‐domain, and E‐loop) are colored consistently throughout all panels. PDB IDs for all structures shown in parentheses. (C) Family 2 encapsulin protomers. A Family 2 A protomer (left) and a Family 2B protomer (right) are shown. Distinct domains are highlighted by color. (D) Alpha Fold (AF)‐predicted structure of a putative Family 3 encapsulin shell protein. A unique N‐terminal extension consisting of an N‐helix and a N‐β‐strand are highlighted in pink. UniProt ID shown above the protomer. (E) Family 4 encapsulin protomer structure.

Figure 2

The diversity of encapsulin assemblies. (A) Family 1 encapsulin shells. T=1, T=3, and T=4 shells are shown. Pentamers are colored in blue, hexamers in grey. The number of pentamers and hexamers necessary to form a shell of a given triangulation (T) number is shown. (B) The symmetry and organization of icosahedral encapsulin shells with different T numbers. Asymmetric units (ASUs) are shown in red. (C) T=3 (left) and T=4 (right) Family 1 hexamers are shown highlighting their ability to occupy both three‐fold (T=3) and two‐fold (T=4) symmetrical positions. (D) Family 2 A (left) and 2B (right) shells. External CBD/MBDs are shown in green. (E) Family 1 encapsulin shell with a bound flavin mononucleotide (FMN) cofactor. FMN binding sites are arranged around the three‐fold symmetry axis (left). FMN binding site (right). (F) Schematic of the complex assembly of mixed two‐component encapsulin shells. Numbers highlight unique interactions around the 2‐, 3‐, and 5‐fold axes (G) Structurally characterized dimeric assembly of a Family 4 encapsulin.

Figure 3

Pores in Family 1 encapsulins. (A) Identified pore positions in T=1, T=3, and T=4 encapsulin shells (left) and summary of all pores identified in structurally characterized Family 1 shells highlighting their pore diameters (right). PDB IDs shown on the x‐axis. 2‐fold_a: two‐fold adjacent; 5‐fold_a: five‐fold adjacent; PHH: pentamer‐hexamer‐hexamer interface. (B) Representative examples of exterior and interior surface views of all pore types identified in Family 1 encapsulins. Electrostatic surfaces are shown. (C) Five‐fold pore dynamics with discrete closed (left) and open (right) states identified in the Haliangium ochraceum T=1 encapsulin. (D) Pore dynamics of the Acidipropionibacterium acidipropionici T=1 shell.

Figure 4

Pores in Family 2 encapsulins. (A) Exterior and interior surface views of all pore types identified in the Acinetobacter baumannii Family 2 A encapsulin. Electrostatic surfaces are shown. (B) Five‐ and three‐fold pores found in the Streptomyces griseus Family 2B shell are shown as electrostatic surface. (C) Detailed exterior and interior view of a two‐fold pore found in a Family 2 A shell. The N‐arm and N‐helix are shown in red. E‐loops are shown in yellow. Both ribbon and surface representations are shown. (D) The open two‐fold pore of the S. griseus Family 2B encapsulin. The position of CBDs above the two‐fold pores is outlined in green. Besides their outline, CBDs are fully transparent. (E) The different two‐fold pore states observed in the Streptomyces lydicus two‐component Family 2B shell – open (left) and closed (right). CBDs omitted for clarity. The N‐arm and N‐helix are shown in red.

Figure 5

CBD and MBD insertions in Family 2B encapsulins. (A) Top and side views of externally displayed CBD/MBD E‐loop insertions arranged around the two‐fold axis of symmetry. Surface representation is shown. MBDs highlighted in pink, CBDs shown in green. MBD: metal‐binding domain. CBD: cyclic nucleotide‐binding fold domain. (B) A single CBD/MBD insertion shown in ribbon representation. The metal ion coordinated by the MBD is highlighted. (C) Close‐up view of the metal‐binding loop within the MBD highlighting metal coordination. Residues shown in stick representation. The sequence of the metal binding loop within the S. griseus MBD is shown (bottom) and residues involved in metal coordination are highlighted in green. (D) Comparison of the putative CBD ligand binding pocket and the cAMP binding site found in E. coli catabolite activator protein (CAP). cAMP (yellow) and key binding site residues are shown in stick representation.

Figure 6

Fusion encapsulin systems. (A) Domain organization of archaeal Family 1 Flp fusion encapsulins. Flp: ferritin‐like protein. (B) Alpha Fold prediction of the P. furiosus Flp fusion protomer. All predicted structures are colored based on pLDDT scores. (C) Homo‐decameric helical assembly of the excised P. furiosus Flp. (D) Schematic highlighting individual Flps (red dot) involved in the formation of decameric complexes located below the five‐fold axis in the context of an assembled T=3 shell. (E) Domain organization of Family 1 cEnc encapsulins found in anammox bacteria. (F) Predicted Alpha Fold structure of a cEnc protomer. (G) Alpha Fold prediction of the diheme cytochrome c fusion domain (Cytc) highlighting the two stacked heme groups in stick representation. (H) Domain organization of helical domain (HD)‐containing Family 3 encapsulins. (I) Alpha Fold prediction of an HD‐containing fusion protomer. (J) Alpha Fold‐predicted pentamer highlighting the formation of a helical bundle above the five‐fold pore.