Synthetic Biology Parts, Devices and Applications

284 14 Sequestered: Design and Construction of Synthetic Organelles

Examples of these proteins are CcmK2 and CsoS1A from the CB, PduA from the

PDU, and EutM from the EUT [35]. There are roughly 4000–5000 copies of the

protein per shell. Additionally, there is a minor protein component of ~90 amino

acids that does not contain the canonical BMC domain and instead oligomerizes

into a pentamer (Pfam03319), as determined via X‐ray crystallography [36].

Cryo‐electron microscopy (EM) studies of purified BMCs suggest that the over­

all structure possesses a roughly icosahedral form [37, 38]. Topological con­

straints therefore dictate that the pentameric protein forms the vertices of the

icosahedron. There are 12 vertices in an icosahedron, placing the exact stoichi­

ometry of the monomer at 60 copies. Biochemical evidence also suggests the

pentamer is of very low abundance in purified BMCs. Examples of this family

include CcmL and CsoS4A from the CB, PduN in the PDU, and EutN from the

EUT. Intriguingly, although EutN crystallizes as a hexamer, protease cleavage

experiments suggest it is a pentamer in solution [36, 39]. This heterogeneity in

quaternary structure may explain the somewhat irregular form of isolated PDUs

and EUTs in comparison with the more icosahedral‐like CBs. The overall poly­

hedral structure therefore consists of (roughly) 20 triangular facets built from

thousands of shell proteins. Depending on the BMC and preparation protocol,

the overall structure is of size 80–400 nm. Finally, cargo proteins are targeted to

the lumen of the BMC through protein–protein interactions with the inside face

(a)

Lumen

Shell

Shell facet HCO

3 −

HCO 3 −

RUBP

RUBisCO

HCO 3 −

Lumen

Cytosol

3-Phosphoglycerate

3-Phosphoglycerate

1,5-bisphosphateRibulose

Ribulose 1,5-bisphosphate

(RBP)

CO 2

O 2

CO 2

CA

OOOOH

HOPOH OH

O

O

O

OH

OH

HO

HOP

OO

HOPOH

2 ×

75 °

Diameter ≈

100 nm

CA

~100 copies

CcmL/CsoS4

60 copies

RuBisCO

CcmK/CsoS 1 ~2000 copies

~5000 copies

(b)

(c)

Figure 14.3 The structure and function of a bacterial microcompartment, the carboxysome.

(a) Structural model showing some of the structural components and enzymatic cargo. (b)

Schematic of the carbon‐concentrating mechanism of carboxysome function. (c) Model of

shell permeability based on CcmK4 (PDB: 2A10).