Heterohexamers Formed by CcmK3 and CcmK4 Increase the Complexity of Beta Carboxysome Shells

Abstract

Bacterial microcompartments (BMCs) encapsulate enzymes within a selectively permeable, proteinaceous shell. Carboxysomes are BMCs containing ribulose-1,5-bisphosphate carboxylase oxygenase and carbonic anhydrase that enhance carbon dioxide fixation. The carboxysome shell consists of three structurally characterized protein types, each named after the oligomer they form: BMC-H (hexamer), BMC-P (pentamer), and BMC-T (trimer). These three protein types form cyclic homooligomers with pores at the center of symmetry that enable metabolite transport across the shell. Carboxysome shells contain multiple BMC-H paralogs, each with distinctly conserved residues surrounding the pore, which are assumed to be associated with specific metabolites. We studied the regulation of b-carboxysome shell composition by investigating the BMC-H genes ccmK3 and ccmK4 situated in a locus remote from other carboxysome genes. We made single and double deletion mutants of ccmK3 and ccmK4 in Synechococcus elongatus PCC7942 and show that, unlike CcmK3, CcmK4 is necessary for optimal growth. In contrast to other CcmK proteins, CcmK3 does not form homohexamers; instead CcmK3 forms heterohexamers with CcmK4 with a 1:2 stoichiometry. The CcmK3-CcmK4 heterohexamers form stacked dodecamers in a pH-dependent manner. Our results indicate that CcmK3-CcmK4 heterohexamers potentially expand the range of permeability properties of metabolite channels in carboxysome shells. Moreover, the observed facultative formation of dodecamers in solution suggests that carboxysome shell permeability may be dynamically attenuated by "capping" facet-embedded hexamers with a second hexamer. Because b-carboxysomes are obligately expressed, heterohexamer formation and capping could provide a rapid and reversible means to alter metabolite flux across the shell in response to environmental/growth conditions.

Occurrence and stability of hetero-hexamer associations formed by β-carboxysome CcmK shell components

“…paralogs could be demonstrated. The formation of hetero-hexamers between CcmK3 and CcmK4 from Synechococcus elongatus PCC7942 (Syn7942) could be shown too, thus confirming and complementing results obtained on the same proteins and on paralogs from Halothece PCC 7418 (Hal7418) [34]. Such observations were reasoned on the basis of sequence identities and compensatory effects for residues implicated in inter-subunit contacts, also by means of dynamic simulations using homology models.…”

“…Despite well expressed, the low solubility of Syn6803 K3 precluded the investigation of its solution behavior or attempts to elucidate its crystal structure. To understand whether this behavior could be linked to an incorrect folding of the K3 monomer or to the presence of residues incompatible with oligomerization, the amino acid sequence was first aligned with those of other Syn6803 paralogs or of CcmK from Syn7942 and Hal7418 species recently studied by Kerfeld and coworkers [34] (Fig. 4A).…”

“…Controls were set up to quantify signals obtained when the same paralog (CcmK4) was co-expressed tagged with both His6 and FLAG tags. By the time of realization of these experiments, Kerfeld and colleagues published evidences on the formation of hetero-hexamers between Syn7942 K3 and K4 [34]. We decided nevertheless to complete this portion of our study, especially because differences between the two experimental designs might permit to obtain complementary information.…”

Single-Organelle Quantification Reveals Stoichiometric and Structural Variability of Carboxysomes Dependent on the Environment

“…While the abundance of most of the structural components varies, the ratio of CcmK4 and CcmK3 is relatively unaffected (ranging from 3.6 to 4.1, Supplemental Table 5) under the tested growth conditions, implying their spatial colocalization within the carboxysome shell ( Figure 6A). It is reminiscent of the recent observation that CcmK3 and CcmK4 can form a heterohexameric complex with a 1:2 stoichiometry and further form dodecamers in a pH-dependent manner (Sommer et al, 2019). The ccmK3 and ccmK4 genes are located in the same operon that is distant from the ccm operon and they may have different expression regulation compared with other carboxysome components (Rae et al, 2012;Sommer et al, 2017).…”

An Envelope Protein of a Prokaryotic Organelle Function towards Conserving Catalytic Activity of the Native Enzyme at Higher Temperatures

“…A question that still remains regarding the PduBB' is that whether the combination is a heterotrimer or a dimer of trimers of PduB and PduB'. Recently, Sommer et al 2019 have shown that in betacarboxysomes, shell proteins Ccmk3 and Ccmk4 exist as heterotrohexamers and alter the permeability properties of carboxysome shell (39). This suggests that shell protein combination can have a significant role in the functioning of MCPs.…”