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. We recently identified the two-component system responsible for spatially regulating carboxysomes, consisting of the proteins McdA and McdB. McdA is a member of the ParA/MinD-family of ATPases, which position a variety of cellular cargos across bacteria. McdB, however, represents a widespread but unstudied class of proteins. We previously found that McdB forms a hexamer and undergoes robust Liquid-Liquid Phase Separation (LLPS) in vitro, but the sequence and structural determinants underlying these properties are unknown. Here we define the domain architecture for McdB from the model cyanobacterium S. elongatus PCC 7942 which we use to dissect McdB oligomerization and LLPS. We identify an N-terminal Intrinsically Disordered Region (IDR), a central Q-rich dimerizing domain, and a C-terminal domain that trimerizes McdB dimers. Intriguingly, all three domains contributed to McdB LLPS. The Q-rich domain drove LLPS, the IDR tuned solubility, and the C-terminal domain provided further oligomerization to achieve full-length LLPS activity. We also identified critical basic residues in the IDR that modulate McdB LLPS, which we mutate to fine-tune condensate solubility both in vitro and in vivo. Our findings show that IDRs are not always drivers of LLPS, but can play secondary roles in modulating condensate solubility. Finally, we provide in silico evidence suggesting the N-terminal IDR of McdB acts as a MoRF, folding upon interaction with McdA. The data advance our understanding and application of carboxysomes, their positioning system, and the molecular grammar governing protein phase separation.SIGNIFICANCEThe recently characterized Maintenance of Carboxysome Distribution (Mcd) system is responsible for spatially regulating carbon-fixing organelles in bacteria called carboxysomes. Although an understanding of the Mcd system would advance our application of carboxysomes to help engineer carbon-fixing organisms and combat the climate crisis, one of its two essential components, McdB, has only recently been identified and is poorly understood. Here, we provide a thorough biochemical characterization of McdB from the model cyanobacterium S. elognatus. We define a structural model for McdB and identify how specific domains and residues contribute to its oligomerization and phase separation. Notably, we saw that a disordered region of McdB regulates phase separation in response to pH; impactful to both carboxysome regulation and protein phase separation.

An experimental framework to assess biomolecular condensates in bacteria

“…McdB sol mutant, the net negative charge of its IDR was increased 25 ; therefore, we speculate that the localization pattern of the fluidized PAmCherry-McdB condensate is due to nucleoid exclusion by repulsive electrostatic interactions. Consistently, we found that mCherry-McdB sol foci expanded and encroached throughout the cytoplasm as the nucleoid was compacted via drug treatment (Supplementary Fig.…”

“…Based on the linker lengths, PopTag LL -mCherry condensates should be more fluid than PopTag SL -mCherry condensates, which we expect to be more viscous or in a gel-like state 26 . Additionally, we probed a solubilized McdB mutant (McdB sol ), previously shown to be abrogated in its phase separation activity both in vitro and in vivo 25 . Finally, mCherry alone was used as a control for a completely soluble protein.…”

“…We developed and adapted a suite of molecular and cell biology methodologies along with super-resolution imaging techniques in Escherichia coli to characterize the formation, solubility, and dynamic exchange of condensates. We examined the protein McdB, a component of the carboxysome positioning system that robustly forms phase-separated droplets in vitro [23][24][25] , alongside wellestablished control proteins that form condensates 26 and insoluble aggregates 27,28 . First, we qualitatively probed the liquid-like properties of McdB condensates using overexpression assays.…”

An experimental framework to assess biomolecular condensates in bacteria

“…Based on the linker lengths, PopTag LL -mCherry condensates should be more uid than PopTag SL -mCherry condensates, which we expect to be more viscous or in a gel-like state 26 . Additionally, we probed a solubilized McdB mutant (McdB sol ), previously shown to be abrogated in its phase separation activity both in vitro and in vivo 25 . Finally, mCherry alone was used as a control for a completely soluble protein.…”

“…We developed and adapted a suite of molecular and cell biology methodologies along with super-resolution imaging techniques in Escherichia coli to characterize the formation, solubility, and dynamic exchange of condensates. We examined the protein McdB, a component of the carboxysome positioning system that robustly forms phase-separated droplets in vitro [23][24][25] , alongside well-established control proteins that form condensates 26 and insoluble aggregates 27,28 . First, we qualitatively probed the liquid-like properties of McdB condensates using overexpression assays.…”