Supporting Information

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1 Supporting Information Shuffling Active Site Substate Populations Affects Catalytic Activity: The Case of Glucose Oxidase Dušan Petrović, 1 David Frank, 2, Shina Caroline Lynn Kamerlin, 3 Kurt Hoffmann, 2,* and Birgit Strodel 1,4,* 1 Institute of Complex Systems: Structural Biochemistry, Forschungszentrum Jülich, Jülich, Germany 2 Institute of Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, Aachen, Germany 3 Department of Cell and Molecular Biology, Uppsala University, BMC Box 596, S Uppsala, Sweden 4 Institute of Theoretical and Computational Chemistry, Heinrich Heine University Düsseldorf, Universitätsstraße 1, Düsseldorf, Germany Current address: aquila biolabs GmbH, Arnold-Sommerfeld-Ring 2, Baesweiler, Germany * Corresponding authors: kurt.hoffmann@rwth-aachen.de and b.strodel@fz-juelich.de S1

2 Table S1. Umbrella sampling conditions. Window Restraint Restraint Angle (kj mol -1 rad -1 ) Angle Window (kj mol -1 rad -1 ) (rad) (rad) WT A2 WT A S2

3 Figure S1. Backbone root-mean-square deviations (RMSD) over three independent 100 ns long MD simulations (red, green, and blue lines) of (a) WT GOx and its (b) P, (c) Pk, (d) Pv, (e) A2, and (f) F9 mutants. S3

4 Figure S2. (a) Root-mean-square fluctuation (RMSF) of the WT Cα-atoms, and (b f) the differential RMSF (ΔRMSF mutant = RMSF mutant RMSF wt ) for GOx mutants (b) P, (c) Pk, (d) Pv, (e) A2, (f) F9. Positive values denote increases and negative values denote decreases in flexibility. The positions of mutations in each variant are denoted by Ð, His516 by ê, and the first two shells around glucose are indicated in gray. S4

5 Figure S3. Dynamical cross-correlation maps (DCCMs) of (a) WT, (b) P, (c) Pk, (d) Pv, (e) A2, and (f) F9 GOx variants. Correlated and anticorrelated motions are shown in red and blue, respectively. The positions of His516 is denoted by ê, and mutations in each variant are denoted by +. The regions showing most significant changes in correlated and anticorrelated motions are framed with dashed and full lines, respectively. S5

6 Figure S4. Per-residue count of significantly correlated/anticorrelated motions (cutoff = ±0.3) for (a) WT, (b) P, (c) Pk, (d) Pv, (e) A2, and (f) F9 GOx variants. Correlated and anticorrelated motions are shown in red and blue, respectively. The positions of mutations in each variant are denoted by Ð, and His516 by ê. S6

7 Figure S5. The relationship between anticorrelated motions and the active site volume of (a) WT and (b) A2 GOx. Only residues performing highly anticorrelated motions in A2 GOx were selected for the analysis. For these residues in both variants, the pairwise distances between the Cα-atoms were calculated using the MDTraj library. 2 Principal component (PC) analysis was performed for the time evolution of these distances using the scikit-learn library in Python. The first two resulting PCs encompass around 60-70% of the total variance. Data points are plotted along the first two components, PC1 and PC2, and color-coded according to the active site volume at the corresponding time-frame (the average volumes are given in Table 3). For the A2 mutant, PC1 clearly separates two states that are characterized by different active site volumes: for PC the active site volume is large (> 300 Å 3 ), while the motion along this coordinate to PC1 < 5 reduces the active site volume to values of less than 200 Å 3. For the WT GOx, in which the anticorrelated motions are not as dominant, the separation between the states along the PC1 is not as clear, indicating the higher flexibility of the active site in the WT, as discussed in the main text. This figure shows that the WT GOx can easily switch between small and large active site volumes, which counteracts the stabilization of the substrate in the active site. S7

8 Figure S6. Glucose contact map for WT GOx: (a) all contacts within 6 Å and (b g) hydrogen bonds between glucose and the holoenzyme, using a 3.5 Å D A distance cutoff and 30 D H A angle. The hydrogen bond count was given for the whole glucose (b), as well as for the individual hydroxyl groups on atoms (c) C1, (d) C2, (e) C3, (f) C4, and (g) C6. Differential contact maps are given for all mutants (ΔFreq mutant = Freq mutant Freq wt ). S8

9 Figure S7. The active site geometries of WT GOx. (a) The catalytic conformation (g, Nt) of His516 shows binding similar to that determined by quantum mechanical calculations, 1 where the O1 atom of glucose is equidistant (2.8 Å) from the Nε and Nδ atoms of His516 and His559, respectively. The distance from the H1 atom of glucose to the N5 atom of FAD is 2.4 Å, as previously reported. 1 (b) In the non-catalytic conformation (g, Ng + ), His516 points towards His559 to form an H-bond, while simultaneously pushing glucose away, which is indicated by significantly longer distances (3.6 and 3.9 Å for O1 to His516 and His559, respectively, and 3.2 Å for H1 to FAD). S9

10 Figure S8. The quality of the active site electron density in different GOx crystals. The His516 electron density in wild-type (a d) and mutant (e f) GOx is shown in cyan. The positive and negative electron densities are shown in green and red, respectively. (a) 1GAL 2.3 Å resolution, (b) 1CF3 1.9 Å resolution, (c) 3QVP 1.2 Å resolution, (d) 3QVR 1.3 Å resolution, (e) 5NIW (F9) 1.8 Å resolution, and (f) 5NIT (A2) 1.9 Å resolution. S10

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12 Figure S9. The distribution of His516 side chain dihedral angles (χ 1 and χ 2 ) from classical MD simulations. Data points are color coded according to the simulation time. The blue star represents the initial conformation obtained after equilibration. Supporting references: (1) Meyer, M.; Wohlfahrt, G.; Knäblein, J.; Schomburg, D. J. Comput.-Aided Mol. Des. 1998, 12, (2) McGibbon, R. T.,; Beauchamp, K. A.; Harrigan, M. P.; Klein, C.; Swails, J. M.; Hernández, C. X.; Schwantes, C. R.; Wang, L.-P.; Lane, T. J.; Pande, V. S. Biophys. J. 2015, 109, S12