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

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 glutamines to solubilize condensates. 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.

Cells contain many millions of protein molecules that must be in the right place at the right time to carry out their roles. A process called phase separation, in which a well-mixed solution separates into two phases ­ one concentrated and one dilute ­ is thought to help organize the contents of various cell types. The single-celled bacteria Synechococcus elongatus converts carbon dioxide from the air into sugars using internal reaction centers. This process depends on a protein called McdB which is crucial for spatially organizing these centers. McdB readily phase separates on its own in a test tube, raising the possibility that this phenomenon could be involved in the carbon dioxide-capturing process. To investigate, Basalla et al. identified the parts of McdB responsible for phase separation and modified them to make a version that was less able to separate. When viewed under the microscope, Synechococcus elongatus cells containing the altered McdB showed changes in the organization and structure of the reaction centers. This suggests that phase separation by McdB is required for optimal carbon capture by this bacterium. In the future, manipulation of McdB phase separation could be used to improve carbon capture technologies or enhance crop growth. Phase separation is also known to influence complex disease. Therefore, further understanding of the process could be important for developing new disease treatments.

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases.

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals and starch in plants. The crystal structures of glucan phosphatase with model glucan substrates reveal distinct glucan-binding interfaces made of DSP and carbohydrate-binding domains. However, quantitative measurements of glucan-glucan phosphatase interactions with physiologically relevant substrates are fundamental to the biological understanding of the glucan phosphatase family of enzymes and the regulation of energy metabolism. This manuscript reports a Concanavalin A (ConA)-based in vitro sedimentation assay designed to detect the substrate binding affinity of glucan phosphatases against different glucan substrates. As a proof of concept, the dissociation constant (KD) of glucan phosphatase Arabidopsis thaliana Starch Excess4 (SEX4) and amylopectin was determined. The characterization of SEX4 mutants and other members of the glucan phosphatase family of enzymes further demonstrates the utility of this assay to assess the differential binding of protein- carbohydrate interactions. These data demonstrate the suitability of this assay to characterize a wide range of starch and glycogen interacting proteins.

Cooperative Kinetics of the Glucan Phosphatase Starch Excess4.

Glucan phosphatases are members of a functionally diverse family of dual-specificity phosphatase (DSP) enzymes. The plant glucan phosphatase Starch Excess4 (SEX4) binds and dephosphorylates glucans, contributing to processive starch degradation in the chloroplast at night. Little is known about the complex kinetics of SEX4 when acting on its complex physiologically relevant glucan substrate. Therefore, we explored the kinetics of SEX4 against both insoluble starch and soluble amylopectin glucan substrates. SEX4 displays robust activity and a unique sigmoidal kinetic response to amylopectin, characterized by a Hill coefficient of 2.77 ± 0.63, a signature feature of cooperativity. We investigated the basis for this positive kinetic cooperativity and determined that the SEX4 carbohydrate-binding module (CBM) dramatically influences the binding cooperativity and substrate transformation rates. These findings provide insights into a previously unknown but important regulatory role for SEX4 in reversible starch phosphorylation and further advances our understanding of atypical kinetic mechanisms.