ABSTRACT
Carbon assimilation in plants is regulated by the reduction of specific protein disulfides by light and their re-oxidation in the dark. The redox switch CP12 is an intrinsically disordered protein that can form two disulfide bridges. In the dark oxidized CP12 forms an inactive supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase, two enzymes of the carbon assimilation cycle. Here we show that binding of CP12 to GAPDH, the first step of ternary complex formation, follows an integrated mechanism that combines conformational selection with induced folding steps. Initially, a CP12 conformation characterized by a circular structural motif including the C-terminal disulfide is selected by GAPDH. Subsequently, the induced folding of the flexible C-terminal tail of CP12 in the active site of GAPDH stabilizes the binary complex. Formation of several hydrogen bonds compensates the entropic cost of CP12 fixation and terminates the interaction mechanism that contributes to carbon assimilation control.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Carrier Proteins/metabolism , Photosynthesis/physiology , Protein Folding , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Carrier Proteins/genetics , Disulfides/metabolism , Enzyme Stability/physiology , Glyceraldehyde-3-Phosphate Dehydrogenase (NADP+)(Phosphorylating)/genetics , Glyceraldehyde-3-Phosphate Dehydrogenase (NADP+)(Phosphorylating)/metabolism , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolismABSTRACT
The crystal structure of the A(4) isoform of photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Arabidopsis thaliana, expressed in recombinant form and complexed with NAD, is reported. The crystals, which were grown in 2.4 M ammonium sulfate and 0.1 M sodium citrate, belonged to space group I222. The asymmetric unit includes ten subunits, i.e. two independent tetramers plus a dimer that generates a third tetramer by a crystallographic symmetry operation. The crystal structure was solved by molecular replacement and refined to an R factor of 23.7% and an R(free) factor of 28.9% at 2.6 A resolution. In the final model, each subunit binds one NAD(+) molecule and two sulfates, which occupy the P(s) and the P(i) anion-binding sites. Detailed knowledge of this structure is instrumental for structural investigation of supramolecular complexes of A(4)-GAPDH, phosphoribulokinase and CP12, which are involved in the regulation of photosynthesis in the model plant A. thaliana.
