α-carboxysome formation is mediated by the multivalent and disordered protein CsoS2

Abstract

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

Discovery of a carbonic anhydrase-Rubisco complex within the alpha-carboxysome

“…An emerging theme of CCM self-assembly is that Rubisco interacts with various CCM proteins via Short Linear Motifs (SLiMs) found in intrinsically disordered proteins/regions (IDP/Rs) (27)(28)(29). Since CsoSCA's N48-peptide appears to bind Rubisco (Fig.…”

The McdAB Carboxysome Positioning System is Widespread Among β-cyanobacteria

“…We purified S. elongatus McdB and found that it forms phase-separated droplets across a physiological pH-range. To our knowledge, this is the first experimental demonstration of LLPS behavior in a ParA-type ATPase partner protein and is an interesting finding given that carboxysome formation has recently been shown to involve LLPS (Oltrogge et al, 2019; Wang et al, 2019) . Collectively, these results have broad implications for understanding carboxysome formation, homeostasis, positioning, and function.…”

“…Our finding that S. elongatus McdB undergoes phase separation in vitro is intriguing on multiple fronts. First, two recent studies have demonstrated that α- and β-carboxysome formation involves LLPS (Oltrogge et al, 2019; Wang et al, 2019) . With β-carboxysomes, the protein CcmM aggregates RuBisCO to form a procarboxysome (Cameron et al, 2013) .…”

“…Binding of SSLD regions of CcmM between RbcL dimers scaffolds RuBisCO molecules and induces LLPS (Wang et al, 2019) . Likewise, in α-carboxysomes of the chemoautotrophic proteobacterium Halothiobacillus neapolitanus , N-terminal repeated motifs within the intrinsically disordered protein CsoS2 mediates interaction with RuBisCO and induces LLPS (Oltrogge et al, 2019) . The pyrenoid does not have a protein shell, but serves as the functional equivalent to carboxysomes in eukaryotic algae.…”

“…Comprised of thousands of proteins (Sun et al, 2019) , carboxysomes were once thought to be completely paracrystalline (Kaneko et al, 2006) . However, recent reports have challenged this idea by showing that carboxysome formation involves the process of Liquid-Liquid Phase Separation (LLPS) (Wang et al, 2019; Oltrogge et al, 2019) ; a process that describes the ability of proteins to spontaneously demix into dilute and dense phases that resemble liquid droplets (Reviewed in: Alberti et al, 2019) . Through these mechanisms, carboxysomes contribute to greater than 25% of global carbon-fixation through atmospheric CO 2 assimilation (Rae et al, 2013) .…”