Multivalent interactions between CsoS2 and Rubisco mediate α-carboxysome formation

Abstract

Carboxysomes are bacterial microcompartments that function as the centerpiece of the bacterial CO2-concentrating mechanism by facilitating high CO2 concentrations near the carboxylase Rubisco. The carboxysome self-assembles from thousands of individual proteins into icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. In the case of the α-carboxysome, there is little molecular insight into protein-protein interactions that drive the assembly process. Here, studies on the α-carboxysome from Halothiobacillus neapolitanus demonstrate that Rubisco interacts with the N-terminus of CsoS2, a multivalent, intrinsically disordered protein. X-ray structural analysis of the CsoS2 interaction motif bound to Rubisco reveals a series of conserved electrostatic interactions that are only made with properly assembled hexadecameric Rubisco. Although biophysical measurements indicate this single interaction is weak, its implicit multivalency induces high-affinity binding through avidity. Taken together, our results indicate CsoS2 acts as an interaction hub to condense Rubisco and enable efficient α-carboxysome formation.

The emergence of phase separation as an organizing principle in bacteria

“…For a more in-depth review of BR-body function and phylogenetic distribution, see Muthunayake et al (4). (21) In vitro (18) In vitro (18) In vitro (18) In vitro (22) In vitro (22) In vitro (22) In vitro (47) In vitro (23) In vivo In vitro spherical (23,24,26) In situ icosahedral (95)(96)(97)(98) In situ (25,29) In vitro (23) In vivo (91) In vitro (26) In vitro (23,24,26) ND…”

“…In vivo (25,74) In vivo (25,27) Cluster size independent of concentration (27) ND In situ (61) In situ (61) ND ND ND…”

“…). Still, the N-terminal domain of CsoS2, a required protein for α-carboxysome assembly, can form phase-separated droplets with RuBisCO(25). Since CsoS2 is an intrinsically disordered protein (IDP) that is believed to act as a scaffold for RuBisCO coalescence (103), it is possible that like CcmM, CsoS2 might use LLPS to initiate carboxysome formation.Carboxysome structure, composition, function, and organization are all highly responsive and adaptable to environmental change including changes in growth temperature, light, and CO2 concentration(94,104,105).…”

The McdAB system positions α-carboxysomes in proteobacteria

“…Likewise, while sharing no identifiable characteristics with known cyanobacterial McdB sequences, we also tested whether the small coding sequence following the McdA-like sequence was a functional homolog to cyanobacterial McdB. To explore this, we used the sulfur-oxidizing chemoautotroph Halothiobacillus neapolitanus c2 (hereafter H. neapolitanus), which has been established as a model system for studying α-carboxysome biology in proteobacteria (Shively et al, 1973;Cannon and Shively, 1983;Holthuijzen et al, 1987;Schmid et al, 2006;Dou et al, 2008;Rae et al, 2013;Sutter et al, 2015;Oltrogge et al, 2020). First, in H. neapolitanus, we fused the native gene that encodes for the small subunit of RuBisCO (CbbS) with the fluorescent protein mTurquoise2 to form CbbS-mTQ (Goedhart et al, 2012).…”

Rubisco proton production can drive the elevation of CO2within condensates and carboxysomes

“…The microalgal CCM accumulates HCO 3 and converts it to CO 2 within the pyrenoid to maximize CO 2 -fixation. Common to both cyanobacterial and microalgal systems is the presence of unique Rubisco-binding proteins, enabling liquid-liquid phase separation (LLPS) of Rubisco from the bulk cytoplasm (17)(18)(19)(20)(21)(22)(23)(24). Condensation of proteins to form LLPS compartments within the cell is an increasingly apparent means by which cellular processes can be segregated and organized, across a broad range of biological systems (25)(26)(27).…”