Crystal structure of CrSBPase. (A) Topology is displayed as cartoon with main chain colored from blue (amino-terminus) to red (carboxy-terminus). (B) Rotated view of A. by x-axis over -90°. (C) Close-up view of region A113SCAGTAC120 with disulfide bond shown in sticks. (D) Rotated view of C. by x-axis over -90°. (E) Putative active site residues inferred from alignment with FBPase bound to FBP (5l0a, human muscle fructose-1,6-bisphosphatase E69Q mutant in active R-state in complex with fructose-1,6-bisphosphate) are represented in sticks. Residue numbering is according to Chlamydomonas SBPase Uniprot entry P46284. (F) Surface representation in the B. orientation with putative active site residues colored in cyan. Water molecule 401 (W401) oxygen is represented as a red sphere. (G) Cysteines site chains are represented in spheres. (H) Threonines and serines side chains reported to be the target of phosphorylations are represented in spheres.

Functional characterization of CrSBPase in vitro. Reported enzymatic activity of CrSBPase assayed for (A) reduction by reduced dithiothreitol (DTTred) and 10 mM of MgSO4, (B) Magnesium sulfate (MgSO4) and 10 mM DTTred, and (C) recombinant thioredoxin f2 from Chlamydomonas reinhardtii (CrTRX-f2) and 10 mM MgSO4, 1 mM DTT.

Crystallographic structures of CrSBPase under reducing treatment: local disorder of the A113SCAGTAC120 loop. Main chain was traced according to crystallographic b-factor, with large orange sections representing high b-factor values and thin blue sections representing low b-factors. A-I. Aligned structures of CrSBPase protomers without redox treatment (A, 7b2o chain A) or in the presence of 10 mM TCEP reducing agent (B-I, 7zuv chains A-H).

Molecular dynamics simulation of CrSBPase after reduction. (A) Overlap of the crystallographic structure of oxidized CrSBPase and representative structures of equilibrated reduced CrSBPase during molecular dynamics simulation 2 (MD2). For structures extracted from MD, only residues 109 to 148 are displayed since most of the other residues are closely overlapping those of the crystallographic structure. (B) Overlap of the crystallographic structure of reduced CrSBPase and representative structures of equilibrated reduced CrSBPase during MD2. For structures extracted from MD, only residues 109 to 148 are displayed.

Oligomeric state of CrSBPase in the crystal. (A) Asymmetric unit dimer of untreated CrSBPase (7b2o). Chains are represented in cartoon and colored cyan (chain B) and salmon (chain E). (B) Asymmetric unit homotetramer under reducing treatment (7zuv). Chains A (cyan), C (magenta), F (white), and H (orange) belong to the same asymmetric unit. (C) Close-up view on figure A homodimer interface. Loop 113-ASCAGTAC-120 from chain B (in cyan) is in 5-Å distance of neighboring chain E (in salmon). C115 and C120 are bonded by a disulfide bridge.

Oligomeric state of CrSBPase in vitro. (A) Size-exclusion chromatograms of CrSBPase wild-type (blue), mutant C115S (orange), mutant C120S (gray) on Superdex 200 26/600 GL column. (B) Size-exclusion fractionation of Chlamydomonas cell extracts. Chlamydomonas cell culture was harvested, lysed and the soluble fraction of the lysate was loaded on Superose6 16/600 size-exclusion column. Chromatography fractions were analyzed by western blot with anti-CrSBPase primary antibodies. First membrane was loaded with fractions eluted from 40 to 80 mL. Second membrane was loaded with fractions eluted from 80 to 120 mL. M lane is loaded with molecular mass standards ladder. Recombinant CrSBPase was loaded was loaded on the last lane to the right..

Crystallographic data collections and models building statistics.

Primers used for point mutagenesis.