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.

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21Carboxysomes are bacterial microcompartments that function as the centerpiece of the 22 bacterial CO2-concentrating mechanism, feeding high concentrations of CO2 to the enzyme 23Rubisco for fixation. The carboxysome self-assembles from thousands of individual proteins into 24 icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. 25In the case of the α-carboxysome, there is little molecular insight into protein-protein interactions 26 which drive the assembly process. Here we show that the N-terminus of CsoS2, an intrinsically 27 disordered protein found in the α-carboxysome, possesses a repeated peptide sequence that 28 binds Rubisco. X-ray structural analysis of the peptide bound to Rubisco reveals a series of 29 conserved electrostatic interactions that are only made with properly assembled hexadecameric 30Rubisco. Although biophysical measurements indicate this single interaction is weak, its implicit 31 multivalency induces high-affinity binding through avidity. Taken together, our results indicate 32 CsoS2 acts as an interaction hub to condense Rubisco and enable efficient α-carboxysome 33 formation. 34 35 2 Introduction: 36

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15 Many bacterial autotrophs rely on CO 2 concentrating mechanisms (CCMs) to assimilate carbon. Although 16 many CCM proteins have been identified, including a 200+ MDa protein organelle called the carboxysome, 17 a systematic screen of CCM components has not been carried out.