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

Hackenberg, Claudia; Hakanpää, Johanna; Cai, Fei; Antonyuk, S.V.; Eigner, Caroline; Meißner, Sven; Laitaoja, Mikko; Jänis, Janne; Kerfeld, Cheryl A.; Dittmann, Elke; Lamzin, Victor S.

doi:10.1073/pnas.1806668115

Cited by 21 publications

(28 citation statements)

References 49 publications

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“…It has a main-chain RMSD of 0.8 Å (Fig. 1D) to the conformation present in the GAPDH-CP12 crystal structures and is similar to that observed in the cyanobacterial CP12-CBS domain protein structure (9).…”

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confidence: 69%

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Structural basis of light-induced redox regulation in the Calvin cycle

McFarlane

Shah

Kabasakal

et al. 2018

Preprint

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In plants, carbon dioxide is fixed via the Calvin cycle in a tightly regulated process. Key to this regulation is the conditionally disordered protein CP12. CP12 forms a complex with two Calvin cycle enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), inhibiting their activities. The mode of CP12 action was unknown. By solving crystal structures of CP12 bound to GAPDH, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide pre-orders CP12 prior to binding the PRK active site. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our model explains how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation. One Sentence Summary:How plants turn off carbon fixation in the dark.The Calvin cycle fixes organic carbon to provide fuel for plants, a process tightly controlled by light-induced redox changes in regulated enzymes (1). Phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are essential enzymes in the photosynthetic dark reactions that control availability of substrate for the carboxylation enzyme Rubisco. PRK consumes ATP to produce the Rubisco substrate ribulose bisphosphate (RuBP), while GAPDH catalyses the reduction step of the Calvin cycle with NADPH to produce the sugar, glyceraldehyde 3-phosphate (GAP), which is used for regeneration of RuBP and is the main exit point of the cycle (2). GAPDH and PRK are coregulated in response to the light reactions, which produce ATP and NADPH. In the light, NADPH and reduced ferredoxin are produced in the chloroplast stroma and the two disulfides of PRK are reduced to switch on PRK activity (3). GAPDH has no disulfides, instead redox regulation of GAPDH is driven by two disulfide bonds in the small regulatory inhibitor protein CP12 (4, 5), which is disordered under reducing conditions (6). In the dark, the stroma becomes oxidizing and intramolecular disulfide bonds within PRK and CP12 are formed. CP12 binds and inhibits GAPDH and then PRK activity in an obligate sequential reaction (7). CP12 is present in oxygenic phototrophs from cyanobacteria to plants (8). Previous structural studies of the GAPDH-CP12 complex resolved only a C-terminal fragment of CP12, so it remained unclear how CP12 could regulate both enzymes. A recent structure of a cyanobacterial CP12-CBS protein resolved an N-terminal CP12-like region, however the protein does not form a ternary complex with GAPDH and PRK (9). The lack of

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confidence: 69%

“…Cys19 is in the middle of the ATPbinding Walker A motif (P-loop, residues 15-24, fig S8) and forms a disulfide with Cys41. When this disulfide bond is oxidized in free PRK, up to 20% activity is retained (9,20,21). The obligate sequential assembly of the inhibited ternary complex is driven by the relative strengths of interaction interfaces within the complex.…”

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confidence: 99%

Structural basis of light-induced redox regulation in the Calvin cycle

McFarlane

Shah

Kabasakal

et al. 2018

Preprint

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“…). This may contribute to the observed inhibitory effect of H 2 O 2 on CO 2 fixation, in addition to the dependence of several enzymes engaged in the CBB pathway and of protein CP12 on the redox status of the cells (Hackenberg et al ., ; Launay et al ., ).…”

Section: Resultsmentioning

confidence: 98%

The response of Microcystis aeruginosa strain MGK to a single or two consecutive H₂O₂ applications

Daniel

Weiss

Murik

et al. 2019

Environ Microbiol Rep

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Summary Various approaches have been proposed to control/eliminate toxic Microcystis sp. blooms including H2O2 treatments. Earlier studies showed that pre‐exposure of various algae to oxidative stress induced massive cell death when cultures were exposed to an additional H2O2 treatment. We examined the vulnerability of exponential and stationary‐phase Microcystis sp. strain MGK cultures to single and double H2O2 applications. Stationary cultures show a much higher ability to decompose H2O2 than younger cultures. Nevertheless, they are more sensitive to an additional H2O2 dose given 1–6 h after the first one. Transcript analyses following H2O2 application showed a fast rise in glutathione peroxidase abundance (227‐fold within an hour) followed by a steep decline thereafter. Other genes potentially engaged in oxidative stress were far less affected. Metabolic‐related genes were downregulated after H2O2 treatments. Among those examined, the transcript level of prk (encoding phosphoribulose kinase) was the slowest to recover in agreement with the decline in photosynthetic rate revealed by fluorescence measurements. Our findings shed light on the response of Microcystis MGK to oxidative stress suggesting that two consecutive H2O2 applications of low concentrations are far more effective in controlling Microcystis sp. population than a single dose of a higher concentration.

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“…So far, no such putative CBS has been reported to bind with ATP, ADP, AMP and NADH [ 64 ]. The only CBS-CP12 so far characterized in cyanobacteria showed binding ability with AMP only and not with other adenosyl ligands [ 65 ]. Binding with different adenosyl ligands indicates the profound ability…”

Section: Discussionmentioning

confidence: 99%

Functional Characterization of Alr0765, A Hypothetical Protein from Anabaena PCC 7120 Involved in Cellular Energy Status Sensing, Iron Acquisition and Abiotic Stress Management in E. coli Using Molecular, Biochemical and Computational Approaches

Chatterjee

Singh

Rai

et al. 2020

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Background: Cyanobacteria are excellent model to understand the basic metabolic processes taking place in response to abiotic stress. The present study involves characterization of a hypothetical protein Alr0765 of Anabaena PCC7120 comprising CBS-CP12 domain and deciphering its role in abiotic stress tolerance. Methods: Molecular cloning, heterologous expression and protein purification using affinity chromatography was performed to obtain native purified protein Alr0765. Energy sensing property of Alr0765 was inferred from its binding affinity with different ligand molecules as analyzed by FTIR and TNP-ATP binding assay. AAS and real time-PCR were applied to evaluate the iron acquisition property and cyclic voltammetry was employed to check redox sensitivity of the target protein. Transcript level under different abiotic stresses as well as spot assay, CFU count, ROS level and cellular H2O2 level were used to show potential role of Alr0765 in abiotic stress tolerance. In-silico analysis of Alr0765 included molecular function probability analysis, multiple sequence analysis, protein domain and motif finding, secondary structure analysis, protein ligand interaction, homologous modeling, model refinement and verification and molecular docking was performed with COFACTOR, PROMALS-3D, InterProScan, MEME, TheaDomEx, COACH, Swiss modeller, Modrefiner, PROCHECK, ERRAT, MolProbity, ProSA, TM-align, and Discovery studio respectively. Results: Transcript levels of alr0765 significantly increased by 20, 13, 15, 14.8, 12, 7, 6 and 2.5 fold when Anabaena PCC7120 treated with LC50 dose of heat, arsenic, cadmium, butachlor, salt, mannitol (drought), UV-B, and methyl viologen respectively, with respect to control (untreated). Heterologous expression resulted in 23KDa protein observed on the SDS-PAGE. Immunoblotting and MALDI-TOF-MS/MS followed by MASCOT search analysis confirmed the identity of the protein and ESI/MS revealed the purified protein was a dimer. Binding possibility of Alr0765 with ATP was observed with almost 6-fold increment in relative fluorescence during TNP-ATP binding assay with a ƛ max of 538 nm. FTIR spectra revealed modification in protein confirmation upon binding of Alr0765 with ATP, ADP, AMP and NADH. A 10-fold higher accumulation of iron was observed in digests of E. coli with recombinant vector after induction as compared to control affirms the iron acquisition property of protein. Moreover, generation of redox potential of 146 mV by Alr0765 suggested its probable role in maintaining redox status of the cell under environmental constraints. As per CFU count recombinant E. coli BL21 cells showed about 14.7, 7.3, 6.9, 1.9, 3, 4.9 fold higher number of colonies under heat, cadmium (CdCl2), arsenic (Na3AsO4), salt (NaCl), UV-B and drought (mannitol) respectively compared to pET21a harboring E. coli BL21 cells. Deterioration in cellular ROS level and total cellular H2O2 concentration validated stress tolerance ability of Alr0765. In-silico analysis unraveled novel findings and attested experimental findings in determining the role of Alr0765. Conclusion: Alr0765 is a novel CBS-CP12 domain protein that maintains cellular energy level and iron homeostasis provide tolerance against multiple abiotic stresses.

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

Cited by 21 publications

References 49 publications

Structural basis of light-induced redox regulation in the Calvin cycle

Structural basis of light-induced redox regulation in the Calvin cycle

The response of Microcystis aeruginosa strain MGK to a single or two consecutive H₂O₂ applications

Functional Characterization of Alr0765, A Hypothetical Protein from Anabaena PCC 7120 Involved in Cellular Energy Status Sensing, Iron Acquisition and Abiotic Stress Management in E. coli Using Molecular, Biochemical and Computational Approaches

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

Cited by 21 publications

References 49 publications

Structural basis of light-induced redox regulation in the Calvin cycle

Structural basis of light-induced redox regulation in the Calvin cycle

The response of Microcystis aeruginosa strain MGK to a single or two consecutive H2O2 applications

Functional Characterization of Alr0765, A Hypothetical Protein from Anabaena PCC 7120 Involved in Cellular Energy Status Sensing, Iron Acquisition and Abiotic Stress Management in E. coli Using Molecular, Biochemical and Computational Approaches

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The response of Microcystis aeruginosa strain MGK to a single or two consecutive H₂O₂ applications