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

Abstract

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

Arabidopsis and Chlamydomonas phosphoribulokinase crystal structures complete the redox structural proteome of the Calvin–Benson cycle

“…All PRKs from bacteria (type I PRKs) (23), including phototrophic organisms, are octamers (∼32-kDa subunits) that are allosterically activated by NADH and inhibited by AMP (24). Archaeal, cyanobacterial, and eukaryotic (i.e., plant-type) PRKs are dimers (∼40-kDa subunits; type II PRKs) (23) with no allosteric regulation (15,25). While little is known about the regulation of archaeal PRKs, in oxygenic phototrophs from ancient cyanobacteria to modern flowering plants it is well-established that PRK forms a complex with A 4 -GAPDH and the regulatory protein CP12 at low NADP(H)/NAD(H) ratios (26,27).…”

“…S2), suggesting a less rigid structure. Since cyanobacterial and eukaryotic PRKs are found together with CP12, the greater flexibility of PRK could enable the formation of regulatory GAPDH-CP12-PRK complexes (9)(10)(11)(12)(13)(14)(15). Moreover, AtPRK and CrPRK are characterized by an increased number of exposed random-coiled regions ( Fig.…”

“…S1). In addition, PRK forms a regulatory complex with the redox-sensitive CP12 and the photosynthetic GAPDH (A 4 -GAPDH and AB-GAPDH) (9)(10)(11)(12)(13)(14)(15). The formation of this ternary complex occurs in both plants and algae, and causes an almost complete inhibition of PRK activity (11,16).…”

Introduction of Exogenous Glycolate Catabolic Pathway Can Strongly Enhances Photosynthesis and Biomass Yield of Cucumber Grown in a Low-CO2 Environment

“…Carbonic anhydrase (CA), which is responsible for transporting CO 2 diffused into plant cells to the carboxylation center of RuBisCO (DiMario et al, 2016). RuBisCO, the key enzyme for carbon fixation; RuBisCO activase (RCA), which regulates the activity of RuBisCO enzyme (Bi et al, 2017; Mcfarlane et al, 2018). Glycolate oxidase (GO), the key enzyme in the oxidation pathway of glycolic acid, which affects the rate of photorespiration (Long et al, 1994; Kang et al, 2018).…”

A new type of disordered CP12 protein in the marine diatom Thalassiosira pseudonana

“…plants, green and red algae, and cyanobacteria, it is associated with two enzymes, phosphoribulokinase (PRK) and glyceraldehyde 3 phosphate dehydrogenase (GAPDH) from the Calvin Benson Bassham cycle that is responsible for CO 2 assimilation [4]. This ternary complex has been well-studied and its structure has been recently solved using cryo-electron microscopy in the cyanobacterium Synechococcus elongatus [5] and using X-ray diffraction in the model higher plant Arabidopsis thaliana [6]. CP12 proteins from different organisms have some highly conserved regions such as a AWD_VEEL motif [2] and in most cases have a pair of cysteine residues at the C-terminus and/or a second pair at the N-terminus.…”