SUPPLEMENTARY INFORMATION

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1 doi: /nature10458 Active Site Remodeling in the Bifunctional Fructose-1,6- bisphosphate aldolase/phosphatase Juan Du, Rafael F. Say, Wei Lü, Georg Fuchs & Oliver Einsle SUPPLEMENTARY FIGURES Figure S1: Structure of T. neutrophilus FBPAP. a) The monomer folds into a compact domain with a C-terminal extension that wraps around a neighboring protomer of the octameric complex. Phosphatase lid is shown in blue, the aldolase loop in red and the anchor loop in yellow. b) The FBPAP octamer in surface representation, with a single monomer shown as cartoon to emphasize the position of the chain. 1

2 Figure S2: Stereo view of the active site of TnFBPAP in the native state with two bound Mg 2+ ions. Direct ligands of the two cations are D11, H18, D52, D53, D132 and D234. The water molecule coordinated to D11, D53 and Mg2 (marked H 2 O) is at the position from which the nucleophilic attack is carried out in the phosphatase reaction. This position was occupied with a water in all structures. When treated with 50 mm EDTA, Mg 1 is removed, but Mg 2 remains bound to the protein. 2

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4 Figure S3 (previous page): Top view of the active site in the four structures of TnFBPAP. A) Native state, B) DHAP-bound state with 2 molecules of DHAP as ligands, C) FBP complex and D) F6P product complex. For all soaked ligands, F o F c omit difference electron density maps were calculated after simulated annealing refinement without the adduct and contoured at the 3.5σ level. Figure S4: Stereo view of the active site of TnFBPAP in complex with FBP and four bound Mg 2+ ions (Fig. 1C, 2C). The phosphatase lid is in its closed conformation and the aldolase loop is in the locked conformation, with the side chain of the catalytic residue for the aldol condensation, K232, forming a hydrogen bond to the backbone carbonyl of P111 at the base of the phosphatase lid. Residue D233 that points away from the active site when the enzyme acts as an aldolase (in conformation of the aldolase loop) now coordinates both Mg3 and Mg4. E357 in the anchor loop contacts Mg4 through two well-defined water molecules and the substrate is fixed in place by Y91, D297 and Y

5 Figure S5: Mg 2+ binding to native TnFBPAP. Isothermal calorimetric titration of the enzyme at 50 C after EDTA-treatment shows entropy-driven binding to a single site with a K d = 27 µm. The structure of the EDTA-treated form revealed that Mg 2 is retained, so that the above event represents the binding of Mg 1. The experiment was carried out in the absence of DHAP, and no binding of a second Mg 2+ ion, Mg 3, is observed. Hollow squares indicate the reference curve that was subtracted from the experimental data. 5

6 Figure S6: TnFBPAP activities dependent on the concentration of Mg 2+ in the reaction buffer. a) aldolase activity and b) phosphatase activity differ in v max, but show very similar K M values. Under physiological conditions of > 10 mm Mg 2+ the availability of the cation should not be limiting for enzymatic activity. 6

7 Figure S7: Binding of the first substrate, DHAP. At 42 C the binding of substrate is an entropy-driven process with a dissociation constant of K d = 25.5 µm. The experiment was carried out in the presence of 10 mm MgCl 2 in the reaction buffer. Note that only a single binding event is observed. The second binding site for DHAP that was observed in the crystal structure will likely be only occupied when the substrate is added in large excess, as was the case during crystal soaking. 7

8 SUPPLEMENTARY METHODS Isothermal Titration Calorimetry Thermodynamic parameters for binding of Mg 2+ and the substrates G3P and DHAP to TnFBPAP were determined by isothermal calorimetric titrations on a Microcal VP-ITC calorimeter (GE Healthcare). Both protein and ligand solutions were prepared in the same buffer condition. As a control, a reference titration was performed by adding the ligand to buffer without protein, in order to correct for the heat of dilution of the titrant. ITC measurements of Mg 2+ binding to the enzyme were carried out at 30 C and 50 C in buffer containing 20 mm Tris-HCl (ph 8.0), 150 mm NaCl and 10% glycerol. Mg ions in the enzyme were removed by 50 mm EDTA. In this instance 122 µl Mg 2+ (3 mm MgCl 2, 20mM Tris/HCl at ph 8.0, 150 mm NaCl and 10% glycerol) was titrated to 1.4 ml TnFBPAP (0.08 mm) in 13 injections (1 2 µl µl). Due to the instability of the triose phosphates at high temperatures, the titrations of G3P and DHAP (1 mm and 1.2 mm in 20 mm Tris/HCl at ph 8.0, 150 mm NaCl, 10 mm MgCl 2 and 10% glycerol) to TnFBPAP (0.04 mm) were done at 42 C, with 13 injections (1 x 3 µl + 12 x 10 µl). Microcal Origin was used for processing and analysis of the calorimetric data. 8

9 SUPPLEMENTARY TABLES Table S1 Data collection and refinement statistics Ligand-free DHAP Fru-1,6-BP Fru-6-P EDTA Y229F Data collection Space group I422 I422 I422 I422 I422 I422 Cell dimensions a, b, c (Å) 112.5, 112.5, , 112.4, , 112.3, , 112.3, , 112,3, , 112.4, α, β, γ ( ) 90.0, 90.0, , 90.0, , 90.0, , 90.0, , 90.0, , 90.0, 90.0 Resolution (Å) ( ) * ( ) ( ) ( ) ( ) ( ) R sym or R merge (0.363) (0.373) (0.424) (0.371) (0.714) (0.599) I/σI 22.4 (6.0) 17.4 (4.9) 18.5 (5.1) 15.3 (5.2) 16.6 (2.5) 10.7 (3.0) Completeness (%) 99.9 (99.9) 99.8 (99.9) 99.9 (99.9) 99.8 (99.9) (100) 99.3 (99.6) Redundancy 10.0 (10.3) 7.9 (7.3) 9.2 (8.5) 8.0 (7.3) 5.8 (5.9) 6.4 (6.5) Refinement Resolution (Å) ( ) ( ) ( ) ( ) ( ) ( ) No. reflections 70,543 (5,416) 111,585 (8,613) 97,834 (7,534) 54,151 (4,060) 38,467 (3,111) 9,583 (680) R work/ R free 0.165/0.188 (0.236/0.259) 0.108/0.129 (0.167/0.186) 0.104/0.126 (0.160/0.204) 0.149/0.178 (0.210/0.231) 0.154/0.191 (0.217/0.274) 0.178/0.234 (0.198/0.288) No. atoms Protein Ligand/ion Water B-factors Protein Ligand/ion Water R.m.s deviations Bond lengths (Å) Bond angles (º) *Highest resolution shell is shown in parenthesis. 9

10 Table S2 Specific activities and substrate affinities for the FBP-Aldolase/Phosphatase from Cenarchaeum symbiosum (CS) and Thermoproteus neutrophilus (TN) and its variants. The specific activities correspond to the relative activities in Fig. 3. Enzyme variant Phosphatase reaction Specific activity (µmol min -1 mg -1 ) K M (mm) Aldolase reaction (catabolic*) Specific activity (µmol min -1 mg -1 ) K M (mm) CS wildtype ± ± ± ± 20 CS Y229F ± ± ± i.m. CS K232R ± ± < i.m. CS D233N ± i.m ± i.m. CS E347Q ± i.m ± ND CS Y348F ± i.m ± ND TN wildtype ± ND ± ND TN Y229F ± ND < i.m. TN D297N ± ND < i.m. ND = not determined i.m. = incapable of measurement * = measured with 100 mm FBP 10