The small RbcS-like domains of the β-carboxysome structural protein CcmM bind RubisCO at a site distinct from that binding the RbcS subunit

Abstract

Edited by Norma M. Allewell Carboxysomes are compartments in bacterial cells that promote efficient carbon fixation by sequestering RubisCO and carbonic anhydrase within a protein shell that impedes CO 2 escape. The key to assembling this protein complex is CcmM, a multidomain protein whose C-terminal region is required for RubisCO recruitment. This CcmM region is built as a series of copies (generally 3-5) of a small domain, CcmM S , joined by unstructured linkers. CcmM S domains have weak, but significant, sequence identity to RubisCO's small subunit, RbcS, suggesting that CcmM binds RubisCO by displacing RbcS. We report here the 1.35-Å structure of the first Thermosynechococcus elongatus CcmM S domain, revealing that it adopts a compact, well-defined structure that resembles that of RbcS. CcmM S , however, lacked key RbcS RubisCO-binding determinants, most notably an extended N-terminal loop. Nevertheless, individual CcmM S domains are able to bind RubisCO in vitro with 1.16 M affinity. Two or four linked CcmM S domains did not exhibit dramatic increases in this affinity, implying that short, disordered linkers may frustrate successive CcmM S domains attempting to simultaneously bind a single RubisCO oligomer. Size-exclusion chromatography-coupled right-angled light scattering (SEC-RALS) and native MS experiments indicated that multiple CcmM S domains can bind a single RubisCO holoenzyme and, moreover, that RbcS is not released from these complexes. CcmM S bound equally tightly to a RubisCO variant in which the ␣/␤ domain of RbcS was deleted, suggesting that CcmM S binds RubisCO independently of its RbcS subunit. We propose that, instead, the electropositive CcmM S may bind to an extended electronegative pocket between RbcL dimers.Cyanobacteria are oxygenic photosynthetic bacteria that, like higher plants, fix carbon dioxide using the Calvin cycle with ribulose-bisphosphate carboxylase/oxygenase (RubisCO; 2 EC 4.1.1.39) catalyzing the key inorganic carbon fixation reaction (1). RubisCO catalyzes a chemically challenging reaction made more difficult by modern low ambient CO 2 and high O 2 concentrations, the latter acting as a competing substrate that results in an unwanted side product that requires energy to recycle (2). Cyanobacteria enhance the efficiency of this reaction by expending energy to concentrate intracellular inorganic carbon using a varied set of CO 2 and HCO 3 Ϫ pumps (3). Because CO 2 is lipophilic and readily escapes through cellular membranes, cyanobacteria accumulate only HCO 3 Ϫ in the cytosol and encapsulate RubisCO behind a secondary, (relatively) CO 2impermeable protein barrier to form a carboxysome (5-7). Carbonic anhydrase (EC 4.2.1.1), the enzyme that interconverts CO 2 and HCO 3 Ϫ , is also encapsulated so that HCO 3 Ϫ pumped into the cell only evolves into CO 2 once within the carboxysome shell (8). Carboxysomes are polyphyletic with two deeply divergent lineages, termed ␣and ␤-carboxysomes. ␣-Carboxysomes, which contain form 1A RubisCO, likely originated in chemoautotrophic ␣-prot...

Single-Organelle Quantification Reveals Stoichiometric and Structural Variability of Carboxysomes Dependent on the Environment

“…Despite the potential effects caused by YFP fusion, it could suggest the presence of a "storage pool" of carboxysome proteins located in the cytoplasm, which are involved in the biogenesis, maturation and turnover of carboxysomes. The ratio of RbcL/S detected from cell lysates fraction is about 8:5.8 (n = 4) (Supplemental Table 2), in line with previous results (Long et al, 2011) but distinct from the in vitro reconstitution observations (Ryan et al, 2018;Wang et al, 2019).…”

“…Recent studies indicated that CcmM interacts with Rubisco (RbcL8S8) at distinct sites, without displacing RbcS (Ryan et al, 2018;Wang et al, 2019). Based on the L8S8 ratio and RbcS abundance per carboxysome determined, we estimated that there are approximately 853, 550, 367, and 1507 copies of Rubisco per β-carboxysome under Air/ML, CO2/ML, LL, and HL, respectively ( Figure 6A, Table 1).…”

Dual Role of a Rubisco Activase in Metabolic Repair and Carboxysome Organization

“…The SSUL domain shares 54-64 % sequence identity with the three SSUL modules of the CcmM protein (NosM58) and its shorter variant M35 (NosM35) (Long et al, 2010) (Figure 6A and S6A). The SSUL modules of CcmM function as scaffolding proteins to concentrate Rubisco into a dense condensate prior to encapsulation into the β-carboxysome (Ryan et al, 2019;Wang et al, 2019). Thus, it seemed plausible that the SSUL domains recruit NosRca into the Rubisco condensate (Lechno-Yossef et al, 2020).…”

An unexpected sticking point for carboxysome assembly

“…To test whether the small subunits of RubisCO are indeed displaced upon binding of CcmM, Ryan et al (8) used native MS and size exclusion chromatography methods to measure the size of the complex formed between isolated CcmM assembly domains and RubisCO. They showed that it was consistent with 1-5 CcmM assembly domains binding to each RubisCO hexadecamer, without the detection of any free RubisCO small subunits, strengthening the contention that CcmM can bind to RubisCO without displacing the small subunits ( Fig.…”

“…overall binding of the complete CcmM protein, which contains multiple assembly domains joined by short linker regions. To determine if this was indeed the case, Ryan et al (8) used surface plasmon resonance binding assays to measure the binding affinity of CcmM constructs containing either a single assembly domain, two assembly domains joined by a linker, or all four assembly domains. These assays showed that all CcmM constructs bound RubisCO with a similar affinity, in the low micromolar range, confirming that a single CcmM binding domain binds in a fashion similar to the full-length protein and providing one of the first direct measurements of the affinity of RubisCO for one of its binding partners.…”