Viewpoint Cite This: Biochemistry XXXX, XXX, XXX−XXX
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Learning to Build a β‑Carboxysome Cecilia Blikstad, Avi I. Flamholz, Luke M. Oltrogge, and David F. Savage* Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, United States yanobacteria were the first lineage on Earth to fix CO2 via oxygen-producing photosynthesis, the same type found in contemporary land plants. Today, oxygenic photosynthesis is the ultimate source of nearly all carbon in the biosphere and cyanobacteria are responsible for roughly 10% of worldwide CO2 fixation. Rubisco, the primary CO2-fixing enzyme of the Calvin−Benson−Bassham cycle, is notorious, however, for being a relatively slow and nonspecific enzyme. Aside from carboxylation, it also catalyzes a reaction with O2 that results in a costly process called photorespiration. To overcome this problem, cyanobacteria, eukaryotic algae, and some plants have developed different types of CO2-concentrating mechanisms (CCMs), systems that increase CO2 levels near Rubisco to inhibit photorespiration and accelerate CO2 fixation. The key component of the cyanobacterial CCM is the carboxysome, a proteinaceous bacterial organelle found in all contemporary cyanobacteria.1 Carboxysomes range from 100 to 400 nm in diameter, have a protein shell, and encapsulate carbonic anhydrase together with Rubisco (Figure 1A). Two types of carboxysomes, α and β, appear to have evolved convergently. In either case, carboxysome genes are essential for growth in ambient CO2 concentrations (≈0.04%). Since CCMs accelerate Rubisco, there is a great interest in transplanting cyanobacterial CCMs into crop plants to increase photosynthetic efficiency. Exciting recent research has tested the feasibility of this approach by producing simplified carboxysome structures in tobacco chloroplasts.2 Still, the presence of carboxysomes in all cyanobacteria piques our curiosity: How exactly does the CCM work? Why do plants not express carboxysomes natively? How does this complex protein organelle self-assemble inside cells? In a recent paper, Hayer−Hartl, Price, and colleagues report findings that constitute a great leap forward in our understanding of β-carboxysome biogenesis.3 These findings shed light on how the major scaffolding protein of the βcarboxysome, CcmM, recruits Rubisco to the carboxysome. CcmM is found in a full-length and short form. Full-length CcmM consists of an N-terminal γ-carbonic anhydrase (γ-CA)like domain followed by a repeat of Rubisco small subunit-like (SSUL) domains (between 3 and 5 dependent on species) connected via flexible linkers. The short form (M35) contains only the SSUL domain repeats. The γ-CA domain interacts with CcmN, a scaffolding protein that anchors to the carboxysome shell, while the SSUL domains interact with Rubisco. Wang et al. used a combination of single-particle cryo-EM and crystallography to reconstitute the Rubisco− CcmM complex and solve the structure of the SSUL domain from the model cyanobacteria Synechococcus elongatus PCC 7942. Their results revealed several surprises, which, together with a complementary study by Ryan et al.,4 redraw our model of the biogenesis and internal organization of the βcarboxysome.
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Due to the predicted structural similarity to the small subunit of Rubisco (RbcS), it has long been thought that SSUL domains interact with Rubisco by displacing an RbcS. However, evidence from both research groups show that this is incorrect. Rather, the SSUL domains bind fully assembled heterohexadecameric Rubisco (L8S8, Figure 1B). In the reconstituted Rubisco−CcmM complex, the SSUL domains bind in a cleft between two dimers of the large subunit of Rubisco (RbcL). A short helical insertion, which is highly conserved in SSUL domains but absent in RbcS, makes critical salt bridges and van der Waals contacts with two RbcL subunits as well as one RbcS. This mode of interaction implies that CcmM only binds L8S8, ensuring that only fully assembled Rubisco is encapsulated in the carboxysome (Figure 1C). Wang et al. strengthen this hypothesis in an in vitro binding assay measuring the formation of Rubisco−CcmM aggregates. M35 was shown to bind L8S8, but not RbcL octamers, RbcS, or chaperone-bound intermediates. Since Rubisco (a heterohexadecamer) and CcmM (a repeat protein) are multivalent by nature, recent research argues for a liquid−liquid phase separation (LLPS) process generating the Rubisco−CcmM aggregate. Indeed, adding M35 to L8S8 Rubisco induced demixing into round droplet-like condensates containing both proteins (Figure 1C). Further assays confirmed the liquid-like characteristics of this condensate: droplets fuse over time and showed a quick fluorescence recovery after photobleaching. The dynamic nature of the condensate implies the interior of a β-carboxysome is not crystalline, as it is often depicted in pictorial models. Moreover, the demonstration of LLPS as a component of biogenesis is intriguing because carboxysomes, unlike many other biological structures formed by LLPS, have a protein shell and display a tight size distribution (150−250 nm for Synechococcus) much smaller than in vitro aggregates. The LLPS theory predicts a single large droplet at equilibrium, which leads us to wonder: how do carboxysomes robustly attain a defined size? Perhaps the kinetics of shell and cargo assembly cooperatively determines the β-carboxysome size.5 The researchers present intriguing results that point to cargo−shell cooperation during carboxysome biogenesis. The SSUL domain structure revealed a conserved pair of cysteines that form a disulfide bond located in close proximity to the RbcL binding interface. In Synechococcus, this cysteine pair is present in two of three SSUL domains. Disruption of both disulfide bonds in vivo resulted in a 4-fold slower growth and carboxysomes with abnormal elongated shapes. Moreover, in vitro Rubisco−M35 aggregates formed more slowly in oxidizing conditions than reducing conditions. Since the Received: March 8, 2019
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DOI: 10.1021/acs.biochem.9b00199 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry
Figure 1. (A) Schematic of the carboxysome. HCO3− concentrated in the cytosol enters the carboxysome and is converted to CO2 by the encapsulated carbonic anhydrase. Elevated CO2 near Rubisco increases the carboxylation rate and inhibits oxygenation. (B) Structure of the reconstructed Rubisco−CcmM complex. The small subunit-like (SSUL) domain of CcmM binds Rubisco at an interface between two large subunits (RbcL) and one small subunit (RbcS). (C) This mode of interaction requires both RbcL and RbcS and so specifically recruits the fully assembled L8S8 Rubisco. Assembly intermediates are not recruited. The multivalent interaction between Rubisco (a multimeric complex) and CcmM (contains a chain of three SSUL domains) generates a protein liquid-like aggregate in vitro. (3) Wang, H., Yan, X., Aigner, H., Bracher, A., Nguyen, N. D., Hee, W. Y., Long, B. M., Price, G. D., Hartl, F. U., and Hayer-Hartl, M. (2019) Rubisco condensate formation by CcmM in β-carboxysome biogenesis. Nature 566, 131−135. (4) Ryan, P., Forrester, T. J. B., Wroblewski, C., Kenney, T. M. G., Kitova, E. N., Klassen, J. S., and Kimber, M. S. (2019) The small RbcS-like domains of the β-carboxysome structural protein CcmM bind RubisCO at a site distinct from that binding the RbcS subunit. J. Biol. Chem. 294, 2593−2603. (5) Rotskoff, G. M., and Geissler, P. L. (2018) Robust nonequilibrium pathways to microcompartment assembly. Proc. Natl. Acad. Sci. U. S. A. 115, 6341−6346.
carboxysome shell should block cellular reducing agents, the carboxysome is presumed to be oxidizing even though the cytosol is reducing. As such, the Rubisco−CcmM interaction is likely weaker within the fully assembled and closed carboxysome. Though the purpose of weakening the Rubisco−CcmM interaction remains unclear, it is apparent that attenuation of affinity upon oxidation is important for the production of functional β-carboxysomes in vivo. Altogether, Wang et al. and Ryan et al. reframe our understanding of carboxysome biogenesis, forcing us to consider the thermodynamics and kinetics of aggregate formation, shell association, and closure as we attempt to understand how such remarkable structures are formed in cells. We hope that future studies will further delineate the pathway of β-carboxysome biogenesis, interrogate the role of redox biochemistry in bacterial compartmentalization, and clarify whether α-carboxysomes are formed by similar means and principles.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
David F. Savage: 0000-0003-0042-2257 Funding
This work was supported by a grant (R01GM129241) from the National Institute of General Medical Sciences. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We would like to thank Julia Borden for helpful comments on the text.
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REFERENCES
(1) Kerfeld, C. A., and Melnicki, M. R. (2016) Assembly, function and evolution of cyanobacterial carboxysomes. Curr. Opin. Plant Biol. 31, 66−75. (2) Long, B. M., Hee, W. Y., Sharwood, R. E., Rae, B. D., Kaines, S., Lim, Y.-L., Nguyen, N. D., Massey, B., Bala, S., von Caemmerer, S., Badger, M. R., and Price, G. D. (2018) Carboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts. Nat. Commun. 9, 3570. B
DOI: 10.1021/acs.biochem.9b00199 Biochemistry XXXX, XXX, XXX−XXX
