Dissecting the phase separation and oligomerization activities of the carboxysome positioning protein McdB

ABSTRACT

Carboxysomes are protein-based organelles essential for efficient CO₂-fixation in cyanobacteria and some chemoautotrophic bacteria. Understanding carboxysome homeostasis has implications for both microbial physiology and engineering CO₂-fixing organisms. We recently identified the two-component system that spatially regulates carboxysomes, consisting of the proteins McdA and McdB. McdA is a member of the ParA/MinD-family of ATPases which position various structures across bacteria. McdB, however, represents a widespread but unstudied class of proteins. We previously found that McdB forms a hexamer and undergoes robust phase separation in vitro, but the sequence and structural determinants underlying these properties are unknown. Here, we define the domain architecture of McdB from the model cyanobacterium S. elongatus PCC 7942. We identify an N-terminal Intrinsically Disordered Region (IDR) that modulates condensate solubility, a central glutamine-rich dimerizing domain that drives phase separation, and a C-terminal domain that trimerizes McdB dimers. We also identify critical basic residues in the IDR which we mutate to fine-tune condensate solubility both in vitro and in vivo. Finally, we provide in silico evidence suggesting the N-terminus of McdB acts as a MoRF, folding upon interaction with McdA. The data advance our understanding and application of carboxysome homeostasis and the molecular grammar governing protein phase separation.