NB-DNJ/GCase-pH 7.4 NB-DNJ+/GCase-pH 7.4 NB-DNJ+/GCase-pH 4.5

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1 SUPPLEMENTARY TABLES Suppl. Table 1. Protonation states at ph 7.4 and 4.5. Protonation states of titratable residues in GCase at ph 7.4 and 4.5. Histidine: HID, H at δ-nitrogen; HIE, H at ε-nitrogen; HIP, H at both nitrogens. ASH, protonation of aspartic acid; GLH, protonation of glutamic acid. Residue Residue number name ph 7.4 ph HIS HID HIP 140 ASP ASP ASH 145 HIS HID HIP 162 HIS HID HIP 223 HIS HID HIP 233 GLU GLH GLH 273 HIS HID HIP 274 HIS HID HIP 290 HIS HID HIP 300 GLU GLU GLH 306 HIS HIE HIP 315 ASP ASP ASH 328 HIS HID HIP 365 HIS HID HIP 380 ASP ASP ASH 388 GLU GLU GLH 419 HIS HIE HIP 451 HIS HIE HIP 481 GLU GLU GLH 495 HIS HID HIP Suppl. Table 2. Overall significance comparisons. The number of stable, unstable (where NB-DNJ changes orientation) and dissociations (where NB-DNJ leaves the active site) were determined in 5 separate MD simulations. In two of the WT simulations, and in one N370S simulation, NB-DNJ dissociated from the enzyme towards the end of the simulation. NB-DNJ+ at ph 7.4 binds equally well to WT and N370S, although NB-DNJ+ changes its orientation slightly. At ph 4.5, the interaction between NB-DNJ+ and both WT and N370S is most stable, showing no reorientation. NB-DNJ/GCase-pH 7.4 NB-DNJ+/GCase-pH 7.4 NB-DNJ+/GCase-pH 4.5 WT N370S WT N370S WT N370S Stable Unstable Dissociation

2 Suppl. Table 3. Statistical significance for differences observed in the MD simulations between WT and N370S. Significant differences (p < 0.01) between WT and N370S are indicated by an asterisk. Significant scores at p < 0.05 are shown in parentheses. ph 7.4 ph 4.5 GCase NB- DNJ/GCase NB-DNJ+/GCase NB-DNJ+/GCase GCase Whole protein 0.88 * (1.00 * ) 0.25 (0.31) 0.05 (0.38) 0.35 (0.50) 0.13 (0.33) Loop * (0.86 * ) 0.00 (0.09) 0.18 (0.06) 0.10 (0.67 * ) 0.02 (0.00) Loop (0.38) 0.00 (0.42) 0.24 (0.83 * ) 0.29 (0.64) 0.11 (0.25) Loop * (0.44) 0.00 (0.00) 0.71 * (1.00 * ) 0.22 (0.20) 0.19 (0.18)

3 SUPPLEMENTARY FIGURES Suppl. Fig. 1. RMSF values for WT and GD mutants. RMSF averages are shown ± s.e.m. A dashed line indicates an unstable structure. For WT and N370S, n = 5. For F213I, D409H, L444P and R496H, n = 3. * indicates significant differences. For N370S, t-test results are given in Suppl. Table 3.

4 Suppl. Fig. 2. Comparison of predicted and experimental GCase stability/flexibility. RMSF values for the WT, with and without NB-DNJ+ bound (top) are compared to experimentally derived melting temperatures (bottom). Taken from Ref. (1) a. a Lieberman, R. L., D'Aquino J, A., Ringe, D., and Petsko, G. A. (2009) Biochemistry 48,

5 Suppl. Fig. 3. RMSF values for WT and N370S. n = 5. ** (p < 0.01), * (p < 0.05) indicates statistical significance; t-test results are given in Suppl. Table 3.

6 Suppl. Fig. 4. Surface RMSF changes at ph 7.4 and 4.5. (A) Surface depiction of GCase in which RMSF differences (ΔRMSF) between WT and N370S at ph 7.4 are color-coded. Blue indicates decreased flexibility and red indicates increased flexibility for N370S. The active site is in pink, and residue 370 in green. (B) RMSF differences for ph 4.5. The yellow circles highlight the area with the biggest change in ΔRMSF between ph 7.4 and 4.5. (C) The solvent accessibility surface area (SASA) is shown in green, the ΔRMSF for ph 7.4 in orange, and in blue at ph 4.5. Positive values indicate an increase in N370S flexibility; negative values a decrease. The yellow box indicates the values corresponding to the surface patches highlighted by a yellow circle in panels A and B.

7 Suppl. Fig. 5. Active site distance analysis of WT and N370S. Average distances and standard deviations for the most important interacting residues in and around the active site are shown. This data is complemented by the HB occupancy data in Table 2 of the main text.

8 Suppl. Fig. 6. Distribution of distance E235 E340. For the 10,000 snapshots of all simulation setups, the observed distances between E235 and E340 were counted and partitioned into 12 different distance ranges.

9 Suppl. Fig. 7. Secondary structure of loop 3. Secondary structure assignment is shown for loop 3 (residues ). At ph 7.4, only small differences between WT and N370S are seen in the secondary structure; however, at ph 4.5 there is a tendency for residues to change a helical turn into a more extended helical confirmation, indicating a marked rearrangement of loop 3. H/h = helix; t = turn; E = strand; C = coil.

10 Suppl. Fig. 8. Interaction of loops 1 and 3 upon binding of NB-DNJ. (A) The interaction strength between loops 1 and 3 is plotted for all snapshots of the MD simulations, starting at an open configuration, via an intermediate, towards a closed loop configuration. This information is given for all possible configurations with NB-DNJ+. (B) The overall difference for interactions between loops 1 and 3 is shown for all possible combinations of WT and N370S with NB-DNJ+, as a heat-plot. Different behavior is colored red (score = 1), similar behavior is colored dark-blue (score = 0).

11 Suppl. Fig. 9. Average affinities between loops 1 and 3 for WT and the five most common GD mutations. The average and standard deviation of the loop affinity is shown for WT and the five main GD mutants. A dashed line indicates unstable mutants.

12 Suppl. Fig. 10. Correct cavity geometry. The graphs show the percent of confirmations for WT (black) and N370S (red) with correct cavity geometry, similar to the measured geometry of a reference crystal structure set (< 20% deviation). A dashed line indicates unstable mutants.

13 Suppl. Movie 1. GCase-pH 7.4. WT (blue) and N370S (red) GCase structures (domain III) are superimposed. Only loop 1 (top), loop 3 (bottom left with cylinder) and the helix (bottom right) on which N370 is situated are shown in full color. Important side-chains are shown as sticks. The distances between D315 on loop 3, and S366 on the helix are shown as dashed lines. Residue N370 (S) is shown at the bottom end of the helix on the left side; the active site residues are depicted above loop 3 together with the distance between E235 and E340. During the simulation WT and N370S mostly show differences regarding the flexibility of loop 1, and to some extent loop 3, which nevertheless does not break away towards the helix on the right. Suppl. Movie 2. NB-DNJ+/GCase-pH 7.4. The representation is defined as for Suppl. Movie 1. Binding of NB-DNJ+ (faint colors) at ph 7.4 results in less flexibility of WT and N370S. In particular loop 3 is held in place. However, towards the end of the movie, NB-DNJ+ becomes very flexible for N370S and changes orientation. Suppl. Movie 3. NB-DNJ+/GCase-pH 4.5. The representation is defined as for Suppl. Movie 1. Binding of NB-DNJ+ (faint colors) at ph 7.4 results in less flexibility of WT and N370S. In particular, loop 3 is held in place. NB-DNJ+ shows less flexibility regarding its position in the active site cavity compared to NB-DNJ+ at ph 7.4. Suppl. Movie 4. GCase-pH 4.5. The representation is defined as for Suppl. Movie 1. Loop 3 is shifted towards the helix due to the bond between D315 and S366, which is shorter for N370S compared to WT.