ABSTRACT
Across bacteria, protein-based organelles called bacterial microcompartments (BMCs) encapsulate key enzymes to regulate their activities. The model BMC is the carboxysome that encapsulates enzymes for CO2 fixation to increase efficiency and is found in many autotrophic bacteria, such as cyanobacteria. Despite their importance in the global carbon cycle, little is known about how carboxysomes are spatially regulated. We recently identified the two-factor system required for the maintenance of carboxysome distribution (McdAB). McdA drives the equal spacing of carboxysomes via interactions with McdB, which associates with carboxysomes. McdA is a ParA/MinD ATPase, a protein family well-studied in positioning diverse cellular structures in bacteria. However, the adaptor proteins like McdB that connect these ATPases to their cargos are extremely diverse. In fact, McdB represents a completely unstudied class of proteins. Despite the diversity, many adaptor proteins undergo phase separation, but functional roles remain unclear. Here, we define the domain architecture of McdB from the model cyanobacterium Synechococcus elongatus PCC 7942, and dissect its mode of biomolecular condensate formation. We identify an N-terminal intrinsically disordered region (IDR) that modulates condensate solubility, a central coiled-coil dimerizing domain that drives condensate formation, and a C-terminal domain that trimerizes McdB dimers and provides increased valency for condensate formation. We then identify critical basic residues in the IDR, which we mutate to fine-tune condensate solubility. Finally, we find that a condensate-defective mutant of McdB has altered association with carboxysomes and influences carboxysome enzyme content. The results have broad implications for understanding spatial organization of BMCs and the molecular grammar of protein condensates.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Fig 2C-D Updated: SEC-MALS data now includes full-length McdB and all truncation constructs. Fig 3 New: in vitro FRAP, droplet fusion imaging, csat determination, and DLS showing PSCP of McdB. Fig 4C-E New: in vitro sedimentation, FRAP, droplet fusion imaging showing condensate formation and maturation of McdB truncations compared to wildtype. Fig 5C-D New: Mutational analysis of McdB CTD with SEC-MALS showing hexamer destabilization. Fig 8 New: In vivo imaging of McdB and carboxysomes in S. elongatus showing that the fluidized mutant of McdB has altered carboxysome association and influences Rubisco content in carboxysomes. Fig 9 New: Model figure connecting McdB domain structure, condensate formation, and the influence of McdB condensate formation in its association with carboxysomes. Fig S3ANew: CD of all McdB truncations compared to wildtype. Fig S4New: DIC imaging showing McdB forms similar condensates in PEG or Ficoll. Fig S6New: Alanine-scanning mutagenesis of the NTD and CTD of McdB showing Alanine artificially induces McdB aggregation in most constructs and that basic residues in the NTD, but not CTD, influence McdB solubility. Fig S7New: Mutational analysis of McdB CTD with SEC-MALS showing hexamer destabilization. Fig S9New: In vivo imaging quantification of carboxysome distribution in wt S. elongatus cells compared to strains encoding the fluidized mutant of McdB with or without McdA present.
