Orange carotenoid proteins: structural understanding of evolution and function.

Cyanobacteria uniquely contain a primitive water-soluble carotenoprotein, the orange carotenoid protein (OCP). Nearly all extant cyanobacterial genomes contain genes for the OCP or its homologs, implying an evolutionary constraint for cyanobacteria to conserve its function. Genes encoding the OCP and its two constituent structural domains, the N-terminal domain, helical carotenoid proteins (HCPs), and its C-terminal domain, are found in the most basal lineages of extant cyanobacteria. These three carotenoproteins exemplify the importance of the protein for carotenoid properties, including protein dynamics, in response to environmental changes in facilitating a photoresponse and energy quenching. Here, we review new structural insights for these carotenoproteins and situate the role of the protein in what is currently understood about their functions.

Biogenesis of a bacterial organelle: the carboxysome assembly pathway.

The carboxysome is a protein-based organelle for carbon fixation in cyanobacteria, keystone organisms in the global carbon cycle. It is composed of thousands of subunits including hexameric and pentameric proteins that form a shell to encapsulate the enzymes ribulose 1,5-bisphosphate carboxylase/oxygenase and carbonic anhydrase. Here, we describe the stages of carboxysome assembly and the requisite gene products necessary for progression through each. Our results demonstrate that, unlike membrane-bound organelles of eukaryotes, in carboxysomes the interior of the compartment forms first, at a distinct site within the cell. Subsequently, shell proteins encapsulate this procarboxysome, inducing budding and distribution of functional organelles within the cell. We propose that the principles of carboxysome assembly that we have uncovered extend to diverse bacterial microcompartments.

Structures of a phycobilisome in light-harvesting and photoprotected states.

Phycobilisome (PBS) structures are elaborate antennae in cyanobacteria and red algae1,2. These large protein complexes capture incident sunlight and transfer the energy through a network of embedded pigment molecules called bilins to the photosynthetic reaction centres. However, light harvesting must also be balanced against the risks of photodamage. A known mode of photoprotection is mediated by orange carotenoid protein (OCP), which binds to PBS when light intensities are high to mediate photoprotective, non-photochemical quenching3-6. Here we use cryogenic electron microscopy to solve four structures of the 6.2 MDa PBS, with and without OCP bound, from the model cyanobacterium Synechocystis sp. PCC 6803. The structures contain a previously undescribed linker protein that binds to the membrane-facing side of PBS. For the unquenched PBS, the structures also reveal three different conformational states of the antenna, two previously unknown. The conformational states result from positional switching of two of the rods and may constitute a new mode of regulation of light harvesting. Only one of the three PBS conformations can bind to OCP, which suggests that not every PBS is equally susceptible to non-photochemical quenching. In the OCP-PBS complex, quenching is achieved through the binding of four 34 kDa OCPs organized as two dimers. The complex reveals the structure of the active form of OCP, in which an approximately 60 Å displacement of its regulatory carboxy terminal domain occurs. Finally, by combining our structure with spectroscopic properties7, we elucidate energy transfer pathways within PBS in both the quenched and light-harvesting states. Collectively, our results provide detailed insights into the biophysical underpinnings of the control of cyanobacterial light harvesting. The data also have implications for bioengineering PBS regulation in natural and artificial light-harvesting systems.

Phycobilisome protein ApcG interacts with PSII and regulates energy transfer in Synechocystis.

Photosynthetic organisms harvest light using pigment-protein complexes. In cyanobacteria, these are water-soluble antennae known as phycobilisomes (PBSs). The light absorbed by PBS is transferred to the photosystems in the thylakoid membrane to drive photosynthesis. The energy transfer between these complexes implies that protein-protein interactions allow the association of PBS with the photosystems. However, the specific proteins involved in the interaction of PBS with the photosystems are not fully characterized. Here, we show in Synechocystis sp. PCC 6803 that the recently discovered PBS linker protein ApcG (sll1873) interacts specifically with PSII through its N-terminal region. Growth of cyanobacteria is impaired in apcG deletion strains under light-limiting conditions. Furthermore, complementation of these strains using a phospho-mimicking version of ApcG causes reduced growth under normal growth conditions. Interestingly, the interaction of ApcG with PSII is affected when a phospho-mimicking version of ApcG is used, targeting the positively charged residues interacting with the thylakoid membrane, suggesting a regulatory role mediated by phosphorylation of ApcG. Low-temperature fluorescence measurements showed decreased PSI fluorescence in apcG deletion and complementation strains. The PSI fluorescence was the lowest in the phospho-mimicking complementation strain, while the pull-down experiment showed no interaction of ApcG with PSI under any tested condition. Our results highlight the importance of ApcG for selectively directing energy harvested by the PBS and imply that the phosphorylation status of ApcG plays a role in regulating energy transfer from PSII to PSI.

Toward a glycyl radical enzyme containing synthetic bacterial microcompartment to produce pyruvate from formate and acetate.

Formate has great potential to function as a feedstock for biorefineries because it can be sustainably produced by a variety of processes that don't compete with agricultural production. However, naturally formatotrophic organisms are unsuitable for large-scale cultivation, difficult to engineer, or have inefficient native formate assimilation pathways. Thus, metabolic engineering needs to be developed for model industrial organisms to enable efficient formatotrophic growth. Here, we build a prototype synthetic formate utilizing bacterial microcompartment (sFUT) encapsulating the oxygen-sensitive glycyl radical enzyme pyruvate formate lyase and a phosphate acyltransferase to convert formate and acetyl-phosphate into the central biosynthetic intermediate pyruvate. This metabolic module offers a defined environment with a private cofactor coenzyme A that can cycle efficiently between the encapsulated enzymes. To facilitate initial design-build-test-refine cycles to construct an active metabolic core, we used a "wiffleball" architecture, defined as an icosahedral bacterial microcompartment (BMC) shell with unoccupied pentameric vertices to freely permit substrate and product exchange. The resulting sFUT prototype wiffleball is an active multi enzyme synthetic BMC functioning as platform technology.

Arabidopsis stromal carbonic anhydrases exhibit non-overlapping roles in photosynthetic efficiency and development.

Carbonic anhydrases (CAs) are ubiquitous enzymes that accelerate the reversible conversion of CO2 to HCO3 - . The Arabidopsis genome encodes members of the α-, ß- and γ-CA families, and it has been hypothesized that ßCA activity has a role in photosynthesis. In this work, we tested this hypothesis by characterizing the two plastidial ßCAs, ßCA1 and ßCA5, in physiological conditions of growth. We conclusively established that both proteins are localized in the chloroplast stroma and that the loss of ßCA5 induced the expression of ßCA1, supporting the existence of regulatory mechanisms to control the expression of stromal ßCAs. We also established that ßCA1 and ßCA5 have markedly different enzymatic kinetics and physiological relevance. Specifically, we found that ßCA5 had a first-order rate constant ~10-fold lower than ßCA1, and that the loss of ßCA5 is detrimental to growth and could be rescued by high CO2 . Furthermore, we established that, while a ßCA1 mutation showed near wild-type growth and no significant impact on photosynthetic efficiency, the loss of ßCA5 markedly disrupted photosynthetic efficiency and light-harvesting capacity at ambient CO2 . Therefore, we conclude that in physiological autotrophic growth, the loss of the more highly expressed ßCA1 does not compensate for the loss of a less active ßCA5, which in turn is involved in growth and photosynthesis at ambient CO2 levels. These results lend support to the hypothesis that, in Arabidopsis,ßCAs have non-overlapping roles in photosynthesis and identify a critical activity of stromal ßCA5 and a dispensable role for ßCA1.

Interfacial Assembly of Bacterial Microcompartment Shell Proteins in Aqueous Multiphase Systems.

Compartments are a fundamental feature of life, based variously on lipid membranes, protein shells, or biopolymer phase separation. Here, this combines self-assembling bacterial microcompartment (BMC) shell proteins and liquid-liquid phase separation (LLPS) to develop new forms of compartmentalization. It is found that BMC shell proteins assemble at the liquid-liquid interfaces between either 1) the dextran-rich droplets and PEG-rich continuous phase of a poly(ethyleneglycol)(PEG)/dextran aqueous two-phase system, or 2) the polypeptide-rich coacervate droplets and continuous dilute phase of a polylysine/polyaspartate complex coacervate system. Interfacial protein assemblies in the coacervate system are sensitive to the ratio of cationic to anionic polypeptides, consistent with electrostatically-driven assembly. In both systems, interfacial protein assembly competes with aggregation, with protein concentration and polycation availability impacting coating. These two LLPS systems are then combined to form a three-phase system wherein coacervate droplets are contained within dextran-rich phase droplets. Interfacial localization of BMC hexameric shell proteins is tunable in a three-phase system by changing the polyelectrolyte charge ratio. The tens-of-micron scale BMC shell protein-coated droplets introduced here can accommodate bioactive cargo such as enzymes or RNA and represent a new synthetic cell strategy for organizing biomimetic functionality.

Bacterial microcompartments as a next-generation metabolic engineering tool: utilizing nature's solution for confining challenging catabolic pathways.

Advancements in synthetic biology have facilitated the incorporation of heterologous metabolic pathways into various bacterial chassis, leading to the synthesis of targeted bioproducts. However, total output from heterologous production pathways can suffer from low flux, enzyme promiscuity, formation of toxic intermediates, or intermediate loss to competing reactions, which ultimately hinder their full potential. The self-assembling, easy-to-modify, protein-based bacterial microcompartments (BMCs) offer a sophisticated way to overcome these obstacles by acting as an autonomous catalytic module decoupled from the cell's regulatory and metabolic networks. More than a decade of fundamental research on various types of BMCs, particularly structural studies of shells and their self-assembly, the recruitment of enzymes to BMC shell scaffolds, and the involvement of ancillary proteins such as transporters, regulators, and activating enzymes in the integration of BMCs into the cell's metabolism, has significantly moved the field forward. These advances have enabled bioengineers to design synthetic multi-enzyme BMCs to promote ethanol or hydrogen production, increase cellular polyphosphate levels, and convert glycerol to propanediol or formate to pyruvate. These pioneering efforts demonstrate the enormous potential of synthetic BMCs to encapsulate non-native multi-enzyme biochemical pathways for the synthesis of high-value products.

Photoactivation of the orange carotenoid protein requires two light-driven reactions mediated by a metastable monomeric intermediate.

The orange carotenoid protein (OCP) functions as a sensor of the ambient light intensity and as a quencher of bilin excitons when it binds to the core of the cyanobacterial phycobilisome. We show herein that the photoactivation mechanism that converts the resting, orange-colored state, OCPO, to the active red-colored state, OCPR, requires a sequence of two reactions, each requiring absorption of a single photon by an intrinsic ketocarotenoid chromophore. Global analysis of absorption spectra recorded during continuous illumination of OCPO preparations from Synechocystis sp. PCC 6803 detects the reversible formation of a metastable intermediate, OCPI, in which the ketocarotenoid canthaxanthin exhibits an absorption spectrum with a partial red shift and a broadened vibronic structure compared to that of the OCPO state. While the dark recovery from OCPR to OCPI is a first-order, unimolecular reaction, the subsequent conversion of OCPI to the resting OCPO state is bimolecular, involving association of two OCPO monomers to form the dark-stable OCPO dimer aggregate. These results indicate that photodissociation of the OCPO dimer to form the monomeric OCPO intermediate is the first step in the photoactivation mechanism. Formation of the OCPO monomer from the dimer increases the mean value and broadens the distribution of the solvent-accessible surface area of the canthaxanthin chromophore measured in molecular dynamics trajectories at 300 K. The second step in the photoactivation mechanism is initiated by absorption of a second photon, by canthaxanthin in the OCPO monomer, which obtains the fully red-shifted and broadened absorption spectrum detected in the OCPR product state owing to displacement of the C-terminal domain and the translocation of canthaxanthin more than 12 Å into the N-terminal domain. Both steps in the photoactivation reaction of OCP are likely to involve changes in the structure of the C-terminal domain elicited by excited-state conformational motions of the ketocarotenoid.

Integrated Structural Studies for Elucidating Carotenoid-Protein Interactions.

Carotenoids are ancient pigment molecules that, when associated with proteins, have a tremendous range of functional properties. Unlike most protein prosthetic groups, there are no recognizable primary structure motifs that predict carotenoid binding, hence the structural details of their amino acid interactions in proteins must be worked out empirically. Here we describe our recent efforts to combine complementary biophysical methods to elucidate the precise details of protein-carotenoid interactions in the Orange Carotenoid Protein and its evolutionary antecedents, the Helical Carotenoid Proteins (HCPs), CTD-like carotenoid proteins (CCPs).

Clues to the function of bacterial microcompartments from ancillary genes.

Bacterial microcompartments (BMCs) are prokaryotic organelles. Their bounding membrane is a selectively permeable protein shell, encapsulating enzymes of specialized metabolic pathways. While the function of a BMC is dictated by the encapsulated enzymes which vary with the type of the BMC, the shell is formed by conserved protein building blocks. The genes necessary to form a BMC are typically organized in a locus; they encode the shell proteins, encapsulated enzymes as well as ancillary proteins that integrate the BMC function into the cell's metabolism. Among these are transcriptional regulators which usually found at the beginning or end of a locus, and transmembrane proteins that presumably function to conduct the BMC substrate into the cell. Here, we describe the types of transcriptional regulators and permeases found in association with BMC loci, using a recently collected data set of more than 7000 BMC loci distributed over 45 bacterial phyla, including newly discovered BMC loci. We summarize the known BMC regulation mechanisms, and highlight how much remains to be uncovered. We also show how analysis of these ancillary proteins can inform hypotheses about BMC function; by examining the ligand-binding domain of the regulator and the transporter, we propose that nucleotides are the likely substrate for an enigmatic uncharacterized BMC of unknown function.

Structural and functional insights into the unique CBS-CP12 fusion protein family in cyanobacteria.

Cyanobacteria are important photosynthetic organisms inhabiting a range of dynamic environments. This phylum is distinctive among photosynthetic organisms in containing genes encoding uncharacterized cystathionine ß-synthase (CBS)-chloroplast protein (CP12) fusion proteins. These consist of two domains, each recognized as stand-alone photosynthetic regulators with different functions described in cyanobacteria (CP12) and plants (CP12 and CBSX). Here we show that CBS-CP12 fusion proteins are encoded in distinct gene neighborhoods, several unrelated to photosynthesis. Most frequently, CBS-CP12 genes are in a gene cluster with thioredoxin A (TrxA), which is prevalent in bloom-forming, marine symbiotic, and benthic mat cyanobacteria. Focusing on a CBS-CP12 from Microcystis aeruginosa PCC 7806 encoded in a gene cluster with TrxA, we reveal that the domain fusion led to the formation of a hexameric protein. We show that the CP12 domain is essential for hexamerization and contains an ordered, previously structurally uncharacterized N-terminal region. We provide evidence that CBS-CP12, while combining properties of both regulatory domains, behaves different from CP12 and plant CBSX. It does not form a ternary complex with phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase. Instead, CBS-CP12 decreases the activity of PRK in an AMP-dependent manner. We propose that the novel domain architecture and oligomeric state of CBS-CP12 expand its regulatory function beyond those of CP12 in cyanobacteria.

Visualizing in Vivo Dynamics of Designer Nanoscaffolds.

Enzymes of natural biochemical pathways are routinely subcellularly organized in space and time in order to improve pathway efficacy and control. Designer scaffolding platforms are under development to confer similar benefits upon engineered pathways. Herein, we evaluate bacterial microcompartment shell (pfam0936-domain) proteins as modules for constructing well-defined nanometer scale scaffolds in vivo. We use a suite of visualization techniques to evaluate scaffold assembly and dynamics. We demonstrate recruitment of target cargo molecules onto assembled scaffolds by appending reciprocally interacting adaptor domains. These interactions can be refined by fine-tuning the scaffold expression level. Real-time observation of this system reveals a nucleation-limited step where multiple scaffolds initially form within a cell. Over time, nucleated scaffolds reorganize into a single intracellular assembly, likely due to interscaffold competition for protein subunits. Our results suggest design considerations for using self-assembling proteins as building blocks to construct nanoscaffolds, while also providing a platform to visualize scaffold-cargo dynamics in vivo.

X-ray radiolytic labeling reveals the molecular basis of orange carotenoid protein photoprotection and its interactions with fluorescence recovery protein.

In cyanobacterial photoprotection, the orange carotenoid protein (OCP) is photoactivated under excess light conditions and binds to the light-harvesting antenna, triggering the dissipation of captured light energy. In low light, the OCP relaxes to the native state, a process that is accelerated in the presence of fluorescence recovery protein (FRP). Despite the importance of the OCP in photoprotection, the precise mechanism of photoactivation by this protein is not well-understood. Using time-resolved X-ray-mediated in situ hydroxyl radical labeling, we probed real-time solvent accessibility (SA) changes at key OCP residues during photoactivation and relaxation. We observed a biphasic photoactivation process in which carotenoid migration preceded domain dissociation. We also observed a multiphasic relaxation process, with collapsed domain association preceding the final conformational rearrangement of the carotenoid. Using steady-state hydroxyl radical labeling, we identified sites of interaction between the FRP and OCP. In combination, the findings in this study provide molecular-level insights into the factors driving structural changes during OCP-mediated photoprotection in cyanobacteria, and furnish a basis for understanding the physiological relevance of the FRP-mediated relaxation process.

Cyanobacterial carboxysomes contain an unique rubisco-activase-like protein.

In plants, rubisco activase (Rca) regulates rubisco by removing inhibitory molecules such as ribulose-1,5-bisphosphate (RuBP). In cyanobacteria, a homologous protein (activase-like cyanobacterial protein, ALC), contains a distinctive C-terminal fusion resembling the small-subunit of rubisco. Although cyanobacterial rubisco is believed to be less sensitive to RuBP inhibition, the ALC is widely distributed among diverse cyanobacteria. Using microscopy, biochemistry and molecular biology, the cellular localization of the ALC, its effect on carboxysome/cell ultrastructure in Fremyella diplosiphon, and its function in vitro were studied. Bioinformatic analysis uncovered evolutionary relationships between the ALC and rubisco. ALC localizes to carboxysomes and exhibits ATPase activity. Furthermore, the ALC induces rubisco aggregation in a manner similar to that of another carboxysomal protein, M35, and this activity is affected by ATP. An alc deletion mutant showed modified cell morphology when grown under enriched CO2 and impaired regulation of carboxysome biogenesis, without affecting growth rate. Carbamylation of Fremyella recombinant rubisco was inhibited by RuBP, but this inhibition was not relieved by the ALC. The ALC does not appear to function like a canonical Rca; instead, it exerts an effect on the response to CO2 availability at the level of a metabolic module, the carboxysome, through rubisco network formation, and carboxysome organization.

Structure of a Synthetic ß-Carboxysome Shell.

Carboxysomes are capsid-like, CO2-fixing organelles that are present in all cyanobacteria and some chemoautotrophs and that substantially contribute to global primary production. They are composed of a selectively permeable protein shell that encapsulates Rubisco, the principal CO2-fixing enzyme, and carbonic anhydrase. As the centerpiece of the carbon-concentrating mechanism, by packaging enzymes that collectively enhance catalysis, the carboxysome shell enables the generation of a locally elevated concentration of substrate CO2 and the prevention of CO2 escape. A functional carboxysome consisting of an intact shell and cargo is essential for cyanobacterial growth under ambient CO2 concentrations. Using cryo-electron microscopy, we have determined the structure of a recombinantly produced simplified ß-carboxysome shell. The structure reveals the sidedness and the specific interactions between the carboxysome shell proteins. The model provides insight into the structural basis of selective permeability of the carboxysome shell and can be used to design modifications to investigate the mechanisms of cargo encapsulation and other physiochemical properties such as permeability. Notably, the permeability properties are of great interest for modeling and evaluating this carbon-concentrating mechanism in metabolic engineering. Moreover, we find striking similarity between the carboxysome shell and the structurally characterized, evolutionarily distant metabolosome shell, implying universal architectural principles for bacterial microcompartment shells.

Heterohexamers Formed by CcmK3 and CcmK4 Increase the Complexity of Beta Carboxysome Shells.

Bacterial microcompartments (BMCs) encapsulate enzymes within a selectively permeable, proteinaceous shell. Carboxysomes are BMCs containing ribulose-1,5-bisphosphate carboxylase oxygenase and carbonic anhydrase that enhance carbon dioxide fixation. The carboxysome shell consists of three structurally characterized protein types, each named after the oligomer they form: BMC-H (hexamer), BMC-P (pentamer), and BMC-T (trimer). These three protein types form cyclic homooligomers with pores at the center of symmetry that enable metabolite transport across the shell. Carboxysome shells contain multiple BMC-H paralogs, each with distinctly conserved residues surrounding the pore, which are assumed to be associated with specific metabolites. We studied the regulation of ß-carboxysome shell composition by investigating the BMC-H genes ccmK3 and ccmK4 situated in a locus remote from other carboxysome genes. We made single and double deletion mutants of ccmK3 and ccmK4 in Synechococcus elongatus PCC7942 and show that, unlike CcmK3, CcmK4 is necessary for optimal growth. In contrast to other CcmK proteins, CcmK3 does not form homohexamers; instead CcmK3 forms heterohexamers with CcmK4 with a 1:2 stoichiometry. The CcmK3-CcmK4 heterohexamers form stacked dodecamers in a pH-dependent manner. Our results indicate that CcmK3-CcmK4 heterohexamers potentially expand the range of permeability properties of metabolite channels in carboxysome shells. Moreover, the observed facultative formation of dodecamers in solution suggests that carboxysome shell permeability may be dynamically attenuated by "capping" facet-embedded hexamers with a second hexamer. Because ß-carboxysomes are obligately expressed, heterohexamer formation and capping could provide a rapid and reversible means to alter metabolite flux across the shell in response to environmental/growth conditions.

Bacterial microcompartments: catalysis-enhancing metabolic modules for next generation metabolic and biomedical engineering.

Bacterial cells have long been thought to be simple cells with little spatial organization, but recent research has shown that they exhibit a remarkable degree of subcellular differentiation. Indeed, bacteria even have organelles such as magnetosomes for sensing magnetic fields or gas vesicles controlling cell buoyancy. A functionally diverse group of bacterial organelles are the bacterial microcompartments (BMCs) that fulfill specialized metabolic needs. Modification and reengineering of these BMCs enable innovative approaches for metabolic engineering and nanomedicine.

A designed bacterial microcompartment shell with tunable composition and precision cargo loading.

Microbes often augment their metabolism by conditionally constructing proteinaceous organelles, known as bacterial microcompartments (BMCs), that encapsulate enzymes to degrade organic compounds or assimilate CO2. BMCs self-assemble and are spatially delimited by a semi-permeable shell made up of hexameric, trimeric, and pentameric shell proteins. Bioengineers aim to recapitulate the organization and efficiency of these complex biological architectures by redesigning the shell to incorporate non-native enzymes from biotechnologically relevant pathways. To meet this challenge, a diverse set of synthetic biology tools are required, including methods to manipulate the properties of the shell as well as target and organize cargo encapsulation. We designed and determined the crystal structure of a synthetic shell protein building block with an inverted sidedness of its N- and C-terminal residues relative to its natural counterpart; the inversion targets genetically fused protein cargo to the lumen of the shell. Moreover, the titer of fluorescent protein cargo encapsulated using this strategy is controllable using an inducible tetracycline promoter. These results expand the available set of building blocks for precision engineering of BMC-based nanoreactors and are compatible with orthogonal methods which will facilitate the installation and organization of multi-enzyme pathways.

The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle.

Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen.