Heterologous Assembly of Pleomorphic Bacterial Microcompartment Shell Architectures Spanning the Nano- to Microscale

Abstract

Many bacteria use protein-based organelles known as bacterial microcompartments (BMCs) to organize and sequester sequential enzymatic reactions. Regardless of their specialized metabolic function, all BMCs are delimited by a shell made of multiple structurally redundant, yet functionally diverse, hexameric (BMC-H), pseudohexameric/trimeric (BMC-T), or pentameric (BMC-P) shell protein paralogs. When expressed without their native cargo, shell proteins have been shown to self-assemble into 2D sheets, open-ended nanotubes, and closed shells of ≈40 nm diameter that are being developed as scaffolds and nanocontainers for applications in biotechnology. Here, by leveraging a strategy for affinity-based purification, it is demonstrated that a wide range of empty synthetic shells, many differing in end-cap structures, can be derived from a glycyl radical enzyme-associated microcompartment. The range of pleomorphic shells observed, which span ≈2 orders of magnitude in size from ≈25 nm to ≈1.8 µm, reveal the remarkable plasticity of BMC-based biomaterials. In addition, new capped nanotube and nanocone morphologies are observed that are consistent with a multicomponent geometric model in which architectural principles are shared among asymmetric carbon, viral protein, and BMC-based structures.

Keywords: bacterial microcompartments; fullerenes; nanocones; nanotubes; self-assembly; synthetic biology.

Figure 1.. GRM3C shell proteins and design of the synthetic operon.

All six genes encoding shell proteins in the GRM3C locus from R. palustris BisB18 are expressed in E. coli under the control of a T7 promoter. The Ribosome binding site (RBS) preceding BMC-H₁ is derived from pET29b . Native RBS sequences were used for BMC-T, BMC-H₂, and BMC-H₃ by including the intergenic sequences upstream of each gene in the R. palustris BisB18 genome. A synthetic RBS and intergenic region from Synechocystis sp. PCC 6803 were used preceding BMC-H₄ and BMC-P_strep, respectively. Due to a ~100 amino acid C-terminal extension, BMC-H₂ is almost twice the length as BMC-H_1-3. We modified the native BMC-P sequence to include a C-terminal Strep Tag II sequence for purification (BMC-P_strep). The shell proteins can be further classified into clades relative to all identified BMC shell protein sequences: BMC-H₁: H_robinEggBlue, BMC-H₂: H_pumpkin, BMC-H₃: H_tan, BMC-H₄: H_p_GrpU_ickyGreen, BMC-T: Tsp_turquoiseBlue, BMC-P: P_stone according to the classification of Sutter et al. and Melnicki et al. .

Figure 2.. Composition, size, and morphology of synthetic GRM3C shells.

A. Coomassie blue-stained SDS-polyacrylamide gel of concentrated StrepTrap eluate containing GRM3C shell proteins expressed in E. coli BL21(DE3). Numbered bands were analyzed by mass spectrometry. B. Hydrodynamic diameter of purified GRM3C particles measured by Dynamic Light Scattering (DLS). Data represent the average and standard deviation of three measurements. C/D. Negative-stained (C) and cryo electron micrographs (D) of GRM3C shells. Arrows indicate examples of different shell morphologies: small spheres (black), irregular polyhedra (yellow), nanotubes (cyan), ovoids (magenta), and nested shells (white). Scale bars are 200 nm.

Figure 3.. Negative-stained EM characterization of capped GRM3C nanotubes.

A. Frequency of observed nanotube lengths and B. widths measured from negative-stained EM images; n = 489. Widths represent the average of 2-5 measurements per nanotube. Round brackets are exclusive and square brackets are inclusive. C. Negative-stained EM images showing GRM3C nanotube diversity. Scale bars are 50 nm.

Figure 4.. Cryo-EM characterization and geometric model for GRM3C nanotubes.

A. Representative cryo-electron micrograph of purified synthetic GRM3C shells. Arrows indicate examples of different shell morphologies: small spheres (black), irregular polyhedra (yellow), nanotubes (cyan), and ovoids (magenta). Scale bar is 100 nm. B. Examples of nanotube heterogeneity observed by cryo-EM; the top panel, an enlargement from A, shows a nanotube ~135 nm long, with a diameter of ~36 nm. Scale bars are 50 nm. C. Four class averages of cylindrical nanotube cross sections 27 nm, 31 nm, 33 nm, and 36 nm in diameter. For additional class averages see Figure S4. D. End-on view of computational models of two example fullerene caps. The distribution of their six respective pentagons (yellow, numbered 1-6 and 7-12) are defined according to the nomenclature of Lair et al. : 5-2d, 3a (Cap 1, cyan) and 6-0a, 2e (Cap 2, magenta). E. Two orientations of a fullerene-based model of a GRM3C nanotube incorporating the two caps in panel D. The zig-zag (16,0) nanotube cylinder is 16 hexagons in circumference and 20 hexagons long, corresponding to a diameter of ~36 nm and length of ~140 nm if constructed from hexagonal BMC-H shell proteins with a side length of 4 nm.

Figure 5.. EM characterization and geometric model of GRM3C nanocones.

A. Examples of nanocones from purified GRM3C samples imaged by negative-stained (top) or cryo-EM (bottom). Scale bars are 50 nm. B. Distribution of nanocone lengths measured from negative-stained EM images (n = 216). Round brackets are exclusive and square brackets are inclusive. C. A geometric model of a fullerene cone derived from the atomic-level structure of a mature HIV-1 capsid (PDB ID: 3J3Q) comprised of 216 hexagons and 12 pentagons (yellow, numbered). When built with BMC shell proteins, this model corresponds to a nanocone of about 75 nm in length.