Encapsulation mechanisms and structural studies of GRM2 bacterial microcompartment particles - PubMed (original) (raw)

Encapsulation mechanisms and structural studies of GRM2 bacterial microcompartment particles

Gints Kalnins et al. Nat Commun. 2020.

Abstract

Bacterial microcompartments (BMCs) are prokaryotic organelles consisting of a protein shell and an encapsulated enzymatic core. BMCs are involved in several biochemical processes, such as choline, glycerol and ethanolamine degradation and carbon fixation. Since non-native enzymes can also be encapsulated in BMCs, an improved understanding of BMC shell assembly and encapsulation processes could be useful for synthetic biology applications. Here we report the isolation and recombinant expression of BMC structural genes from the Klebsiella pneumoniae GRM2 locus, the investigation of mechanisms behind encapsulation of the core enzymes, and the characterization of shell particles by cryo-EM. We conclude that the enzymatic core is encapsulated in a hierarchical manner and that the CutC choline lyase may play a secondary role as an adaptor protein. We also present a cryo-EM structure of a pT = 4 quasi-symmetric icosahedral shell particle at 3.3 Å resolution, and demonstrate variability among the minor shell forms.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1

Fig. 1. Klebsiella pneumoniae GRM2 locus and variants of cmcC.

a Klebsiella pneumonia GRM2 locus. Structural shell BMC-H proteins cmcA, cmcB, cmcC, and cmcE are colored in green, and BMC-P protein cmcD is colored in yellow. Core enzymes CutF (aldehyde dehydrogenase), CutO (alcohol dehydrogenase), CutC (choline lyase), CutD (glycyl-radical activating enzyme), and CutH (phosphotransacylase) are colored in blue. Regulatory and transporter genes are colored in gray. The genes have been named according to previous research. b C-terminal amino acid sequences of three cmcC variants—cmcC (native), cmcC′ (mutated), and cmcCtrunc (truncated).

Fig. 2

Fig. 2. Characterization of cmcABC′ + cmcD BDPs.

a Gel filtration of sedimented BDPs and SDS-PAGE analysis of the fractions. Two noticeable BDP peaks were formed: one immediately after the empty volume of 60 ml (large type particle zone) and one at approximately 90–100 ml (small type particle zone). An intermediary zone with smaller BDP protein amounts was formed between these two zones. b Examples of TEM analysis of the BDP samples in the large particle zone (66 ml), intermediary zone (76 and 84 ml), and small particle zone (96 ml). Scale bar: 200 nm.

Fig. 3

Fig. 3. Proposed enzymatic core encapsulation mechanism of CutC, CutF, and CutO in GRM2 BDPs.

CutC is serving as an adaptor for the encapsulation of other enzymes. CutC C-terminal domain is responsible for encapsulation and also for interaction with CutO. The CutC N-terminal domain is responsible for CutC interaction with CutF. CutF together with CutC N-terminal domain crosslinks the enzymatic core and increases its size.

Fig. 4

Fig. 4. Cryo-EM structure of pT=4 quasi-icosahedral BDP and its penatameric and hexameric components.

a Surface model of pT = 4 quasi-icosahedral BDP particle, displayed on the left side. A ribbon model of a cmcD pentamer and three cmcC′ hexamers is displayed on the right side. Pentameric cmcD protein is colored in yellow and hexameric cmcC′ is colored in green. Note that the fivefold symmetry axis is located at the center of cmcD pentamer and threefold axis is located in the middle between three cmcC′ hexamers. b Electrostatic surface potential of pentameric cmcD and hexameric cmcC′. Note the pores in the centers of pentamers and hexamers. The surface contour levels were set to −1 kT/e (red) and +1 kT/e (blue).

Fig. 5

Fig. 5. Cryo-EM classes of BDP subtypes.

a 3D class at 3.3 Å resolution and atomic model of whole intact pT = 4 quasi-symmetric icosahedral particles, in total 69%. b 8.8 Å resolution 3D class of pT = 4 derived particles, with at least one missing pentameric unit, in total 23%. c 9.6 Å resolution 3D class of pT = 4, Q = 6 quasi-symmetric particles, in total 6%. d 2D class of triangular-looking particles, judging from the size probably derived from three fused pT = 4 particles, in total 0.7%. e 2D class of pT = 4, Q = 8 particles, in total 0.3%. f 2D class of pT = 7 or pT = 9 quasi-icosahedral particles, in total 0.2%. g Separate micrographs of tubular particles elongated with more than two hexameric rings, in total less than 0.1%. Scale bar: 60 nm.

Fig. 6

Fig. 6. Detailed view of hexameric–hexameric and pentameric–hexameric interfaces.

a Hexameric–hexameric cmcC′–cmcC′ interface; K–R–X (in our case K–R–A) triad and salt bridge networks are viewed as stick models, distances are measured in angstroms. b Hexameric–pentameric cmcC′–cmcD interface; amino acids involved in contacts are viewed as stick models. Distances are measured in angstroms.

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