Allosteric dynamic control of binding

Fidan Sumbul 1,2, Saliha Ece Acuner-Ozbabacan 1,2, Turkan Haliloglu1,*

1Department of Chemical Engineering and Polymer Research Center, Bogazici University,

Istanbul, Turkey 2These authors contributed equally to this work

Correspondence:

* Turkan Haliloglu (halilogt@boun.edu.tr)

SUPPORTING MATERIAL

MATERIALS AND METHODS

SKEMPI dataset analysis

The identity of the mutant residue is immaterial on the decision of whether a mutation is at a

hinge region or not. We thus eliminate the redundant mutations in the SKEMPI database, where

a residue in a chain is mutated into several different residues. The dataset containing 2007

mutations in 108 chains of 81 PDB structures is split into two as non-redundant (907) and

redundant (1100) entries, and a representative mutation is chosen for latter. The first criterion for

a representative entry is whether the wild type residue is mutated into Alanine, regardless of the

amount of binding free energy change. If none of the redundant entries are mutated into Alanine,

then the mutation with the highest change in the binding free energy is chosen (among the

mutations of the wild type residue into chemically similar ones, if possible).

In the Fisher’s exact test, the contingency table is composed of four elements: the numbers of

mutations corresponding to “hinge” and “non-hinge” residues below and above the threshold.

The initial null hypothesis is a balanced “hinge”/”non-hinge” ratio below and above the

threshold. If a p-value is less than or equal to 0.05 (confidence level of 95%), then a statistically

significant difference in “hinge”/”non-hinge” preference of mutations below and above the

threshold is obtained rejecting the initial null hypothesis. In other words, the probability of

observing an imbalanced “hinge”/”non-hinge” preference of mutations by chance is less than

5%. Lastly, the preferences of significant cases are evaluated as either “hinge” or “non-hinge” by

comparing the number of occurrences in the contingency table with the total number of

mutations below and above that threshold.

Additionally, the statistical dataset analysis is applied to Alanine mutations as a separate case, to

eliminate any impact of the mutant residue type on the binding free energy change with respect

to the correspondence to hinge/nearby residues.

MD trajectory analyses

RMSD (Root Mean Square Deviation). Minimized structures are used as references and

RMSD of MD sampled conformations are calculated using VMD 1.9.1 (1). RMSD profiles are

used to for the convergence within the time window of MD simulations performed.

Dihedral angles. An in-house script in VMD 1.9.1 (1) is used to calculate backbone

rotational/dihedral angles. In order to determine the divergence of distributions of backbone

dihedral angles, multiple Gaussians (according to the number of clusters) are fitted to the data

and then Jensen-Shannon divergence between the fitted models are calculated (2).

Correlation between residue fluctuations. Δ𝐑i and Δ𝐑jare the positional fluctuations of

residues i and j (Cα). The correlation between residue fluctuations, cross correlations, Cij, are

calculated as

Cij =⟨Δ𝐑iΔ𝐑j⟩

⟨Δ𝐑i2⟩1 2⁄ ⟨Δ𝐑j

2⟩1 2⁄ (S1)

Normalized correlation values range from +1 to -1, referring to highly positively to negatively

correlated fluctuations. Correlation values around zero mean either uncorrelated fluctuations or

orthogonal motion.

PCA (Principal Component Analysis). The data dimension is reduced in the covariance

analysis by PCA. The contribution of individual PCs higher than the first three to the total

variance of fluctuations falls below 10% in both hGH and the PYD dimer, and thus only first

three principal components (PC-1, PC-2, and PC-3) are considered in the analyses (See the

Results and Discussion and Figs S5 and S11). The PCs are extracted from MD trajectories using

MATLAB R2015a.

The overlap between ANM modes and PCs can be determined from the correlation; cosine

𝑂𝑖𝑗 = 𝑷𝑪𝑖.𝒖𝒋, where 𝒖𝑗 is the eigenvector of 𝑗𝑡ℎ ANM mode (3). This overlap value is for the

qualification of the MD sampled space in terms of functional long-time dynamics (4).

H-bonds. The H-bond frequencies over all the snapshots of equilibrated MD trajectories are

calculated using Visual Molecular Dynamics (VMD) 1.9.1 (1) with default parameters

(maximum donor (D)-acceptor (A) distance is 3Å and the D-H-A angle less than 20˚). The H-

bonds observed in all parallel MD simulations are considered and the most conserved H-bonds

are determined by their frequencies. Cytoscape (5) is used to construct and visualize the H-bond

network.

SUPPORTING FIGURES AND TABLES

FIGURE S1 The significance of correspondence of Alanine mutations to hinge/nearby residues when the change in the binding free energy upon mutation (∆∆G) is below and equal to/above different thresholds. Four different cases are studied: mutations are mapped to (A) hinge residues with their neighbors whose alpha carbon distances are within 6Å in space, (B) hinge residues with their first two neighbors in sequence, (C) exact positions of hinge residues without nearby residues, and (D) hinge residues with their first three neighbors in sequence. See the caption for Fig. 1 for details and Table S3 for data calculation.

C

A B

D

FIGURE S2 Comparison of p-values resulting from the analysis of (A) randomly generated 1000 sets of hinges and (B) hinges predicted by the dynamic analysis. The labels 6Å, seq2 and seq3 refer to considering also nearby residues to hinges; 6Å: residues whose alpha carbon distances to hinge residues are within 6Å in space, seq2 and seq3: first two and three neighbors of hinge residues in sequence, respectively.

B A

FIGURE S3 (A) Secondary structural elements on hGH (PDB ID: 1HWG, the missing loop is modelled. See the Materials and Methods). Correlations between residue fluctuations –cross-correlation maps- in the slowest (B) and second slowest (C) modes of the wild type hGH. Blue and red refer to residues fluctuating in the same direction and hinge residues are shown in green. The mutation site (I58) is shown on the correlation map of the slowest mode.

A

B

N

C H1

H1’

H1’’

H1’’

H2 H3

H4

C I58

FIGURE S4 (A) Root mean square deviation (RMSD) profiles of MD simulations of the wild type and I58A hGH. Correlations between residue fluctuations averaged over six parallel wild type and I58A mutant hGH MD simulations ((B) & (C), respectively), using 3 PCs that contribute significantly to the variance of the data ((D) and (E), respectively); The contribution of PCs > 3 to variance fall below 10% in all MD simulations. The hGH structure in (Fig. 2 A, B and in Fig. 2 C, D) is colored based on the average correlation of F54-I58 (H3) and the binding site s1-c (H4) are marked on the correlation maps with dashed and solid rectangles, respectively.

C

D

A

B

E

F54-

I58

P61-

T67

F54-

I58

P61-

T67

FIGURE S5. The overlap between three principal modes (PCs) observed in MD simulations and ten slowest ANM modes calculated with the cumulative overlap between 1-3 PCs and 1-5 slowest ANM modes (on the right) of the wild type (A) and I58A (B) hGH. High cumulative overlap of PC1-PC3 and ANM1-ANM5 modes shows that the first three PCs adequately represent the predicted functional motion by ANM.

A B

FIGURE S6 Scatter plots of backbone dihedral angles phi (φ) versus psi (ψ) of hGH for residues with Jensen-Shannon divergence (JSD) (2) value ≥ 0.2. Blue and red data points represent the data for the wild type and I58A hGH, respectively, showing significant changes in the distribution of dihedral angles including some functional residues with the mutation. Scatter plot of L73 is given as a reference.

FIGURE S7 H-bonding network of hGH by MD simulations. Residues are represented as nodes and H-bonds that have frequencies ≥20% are represented as edges. The thickness of the edges reflects the frequencies of H-bonds and colored by blue and red for the wild type and I58A hGH, respectively. Nodes that have H-bonds only in the wild type and only in the I58A hGH are colored in blue and red, respectively. Green nodes are those that have H-bonds in both wild type and I58A mutant hGH.

S1-a S1-c S1-b S1-d S2-a S2-b S2-c

FIGURE S8 (A) Three PYD monomers interacting via Type I interaction mode surfaces in the cryo-EM structure of the PYD filament (PDB ID: 3J63), colored according to the correlation of H1 with the rest of the monomer of the wild type and D48A PYD. (B) Three PYD monomers interacting via Type II interaction mode surfaces colored by the correlation of the H4&H5 loop with the rest of the monomer of the wild type and D48A PYD. The monomer dynamics visualized in the cryo-EM structure here serves as a reference for the comparison of the PYD dimer dynamics presented in Fig. 5 A-F

Type I Type I

A

B

Side view Top view Side view Top view

D48A Mutant Wild Type

PYD

Type II

H4&H5 Loop

H5&H6 Loop

Type II

H4&H5 Loop

H5&H6 Loop

FIGURE S9 Correlations between residue fluctuations in the slowest (A) and second slowest (B) modes of the wild type PYD dimer by GNM. Blue and red refer to the residues fluctuating in the same direction and hinge residues are shown in green. D48 is located on the correlation map of the second slowest mode.

A B

Chain 1 Chain 2

D48

Chain 1 Chain 2

D48

FIGURE S10 RMSD profiles of MD simulations of the PYD dimer (Extracted from cryo-EM structure, PDB ID: 3J63) (A) Correlations between residue fluctuations averaged over parallel MD trajectories using first 3PC of both wild type and D48A mutant PYD dimer ((B) and (C), respectively); the contribution of PCs > 3 to variance falls below 10% in all MD simulations (D) and (E) Axes thick labels are set according to the secondary structural units. The structural positions considered in the coloring of the PYD dimer in Fig. 5 A-F labelled on the correlation maps.

C

D

A

B

E

Chain 1 Chain 2 Chain

Chain 2

H1

H1

H5&

H6

Loop

H4&

H5

Loop

H4&

H5

Loop

H5&

H6

Loop

H1&

H2

Loop

H1&

H2

Loop

H1

H1

H5&

H6

Loop

H4&

H5

Loop

H4&

H5

Loop

H5&

H6

Loop

H1&

H2

Loop

H1&

H2

Loop

FIGURE S11 The overlap between each of the first 3 PCs observed in MD simulations and ten slowest ANM modes with cumulative overlap between 1-3 PCs and 1-5 slowest ANM modes (on the right) of the wild type and D48A PYD dimer ((A) and (B), respectively). High cumulative overlap of PC1-PC3 and ANM1-ANM5 modes shows that the first three PCs of the MD fluctuations agree the predicted ANM global motion.

FIGURE S12 H-bonding network obtained in parallel MD simulations of the wild type and D48A PYD monomer calculated using default values in VMD. Residues are represented as nodes and H-bonds that have frequencies ≥ 20% are represented as edges. The thickness of the edges scales with the frequencies of H-bonds and are colored by blue and red for the wild type and D48A PYD, respectively. Nodes that have H-bonds only in the wild type and only in the D48A PYD are colored in blue and red, respectively. Green nodes are those that have H-bonds in both wild type and D48A PYD monomer.

A B

PYD

Mon

omer

P

YD D

imer

P1-

P2

FIGURE S13 Scatter plots of backbone dihedral angles phi (φ) versus psi (ψ) of the wild type and D48A PYD monomer (upper panel) for residues with Jensen-Shannon divergence (JSD) (2) value ≥ 0.2. Blue and red data points represent the data for the wild type and D48A, respectively, showing significant changes in the distribution of dihedral angles including some functional residues with the mutation. Distribution of dihedral angles for chain 1 (blue) and 2 (red) of the wild type PYD dimer is given in lower panel.

TABLE S1 Number and percentage of hinge sites on each non-redundant chain (108) in the SKEMPI dataset.

PDB ID_chain residue # hinge site #

hinge site %

PDB ID_chain residue # hinge site #

hinge site %

1HE8_A 749 28 3.7 1VFB_C 129 21 16.3 1MAH_A 533 45 8.4 3HFM_Y 129 20.5 15.9 1A4Y_A 460 25.5 5.5 1FFW_A 128 15.5 12.1 1Z7X_W 460 25.5 5.5 1H9D_B 125 13.5 10.8 3BP8_A 381 14 3.7 2J0T_D 124 15 12.1 2B42_A 364 30 8.2 1A4Y_B 123 14 11.4 2PCB_A 294 30 10.2 1NMB_H 122 19.5 16.0 2PCC_A 294 31 10.5 1DVF_D 121 17.5 14.5 2C0L_A 292 30.5 10.4 2HRK_B 121 17.5 14.5 2VIR_C 267 27 10.1 2I26_N 118 16 13.6

2G2U_A 265 24 9.1 1DAN_U 116 9 7.8 2I9B_E 265 16.5 6.2 1DVF_B 116 15.5 13.4 1JTG_A 262 28.5 10.9 1VFB_B 116 16.5 14.2 1DAN_H 254 20.5 8.1 2WPT_B 113 13.5 11.9 1EAW_A 241 29 12.0 1NMB_L 109 18 16.5 3BN9_B 241 27 11.2 1BRS_A 108 13.5 12.5 3NPS_A 241 28 11.6 2PCC_B 108 12.5 11.6 2VLJ_E 240 14.5 6.0 1DVF_A 107 16 15.0 1JCK_B 239 25 10.5 1DVF_C 107 18.5 17.3 1SBB_B 239 22 9.2 1VFB_A 107 16 15.0 1NCA_H 221 18.5 8.4 2SIC_I 107 12.5 11.7 3TGK_E 217 27 12.4 1KTZ_B 106 12.5 11.8 1FY8_E 215 27.5 12.8 1JRH_I 95 16 16.8

3HFM_H 215 18 8.4 1BRS_D 87 12 13.8 1DQJ_A 214 16.5 7.7 1LFD_A 87 9 10.3 3HFM_L 214 19.5 9.1 1REW_C 86 11.5 13.4 1DQJ_B 210 19.5 9.3 2JEL_P 85 14 16.5 2A9K_B 207 21 10.1 1EMV_A 83 11 13.3

1AHW_C 200 17 8.5 1KTZ_A 82 11 13.4 2VLJ_D 199 17 8.5 2WPT_A 82 14 17.1 1A22_B 192 15 7.8 1DAN_T 75 11 14.7 1GRN_A 191 18 9.4 1XD3_B 75 12 16.0 2HLE_A 188 17 9.0 3BP8_C 75 10.5 14.0

1KAC_A 185 17 9.2 1GCQ_C 69 10 14.5 2J12_A 182 15 8.2 1FFW_B 68 10 14.7 2J1K_C 182 21.5 11.8 3BK3_C 67 5 7.5 1GC1_C 181 11.5 6.4 2GOX_B 65 7 10.8 1A22_A 180 15.5 8.6 1TM1_I 64 11.5 18.0 1JRH_H 180 17 9.4 1ACB_I 63 11 17.5 1E96_A 178 24.5 13.8 1CSE_I 63 10 15.9 2HRK_A 177 18 10.2 1CBW_I 58 12 20.7 2AJF_E 174 20.5 11.8 1EFN_A 57 11 19.3 1JRH_L 167 14 8.4 2FTL_I 57 7.5 13.2 1JTG_B 165 16.5 10.0 1FCC_C 56 7.5 13.4 2G2U_B 165 15.5 9.4 1PPF_I 56 11.5 20.5 1AK4_D 145 11.5 7.9 1CHO_I 53 10 18.9 2BTF_P 139 16.5 11.9 1R0R_I 51 10 19.6 1S1Q_A 137 14.5 10.6 3SGB_I 50 11.5 23.0 1N8O_E 136 12 8.8 2OOB_A 44 5 11.4 2O3B_B 135 15 11.1 1FC2_C 43 7.5 17.4 1EMV_B 131 12 9.2 4CPA_I 37 7 18.9 1DQJ_C 129 21 16.3 1GL1_I 34 6.5 19.1 1IAR_A 129 11.5 8.9 1F47_A 17 3 17.6 1UUZ_A 129 16 12.4 1SMF_I 9 2 22.2

TABLE S2 The number of mutations below (low) and above (high) each of the energy threshold values, the number of mutations corresponding to hinge/nearby residues and the logarithm of p-values of their correspondence.

Energy Threshold (kcal/mol) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 low_allosteric_mutation 90 147 180 202 219 231 240 247 253 259 high_allosteric_mutation 193 136 103 81 64 52 43 36 30 24

low_allosteric_hinge 15 21 27 32 32 35 38 40 42 42 high_allosteric_hinge 37 31 25 20 20 17 14 12 10 10 log(hinge_p_value) -0.130 -1.170 -1.243 -1.041 -2.281 -2.281 -1.774 -1.698 -1.374 -2.324

6Å_ low_allosteric_hinge 43 71 83 93 101 106 112 115 118 120 6Å_ high_allosteric_hinge 97 69 57 47 39 34 28 25 22 20

log(6Å_hinge_p_value) -0.152 -0.142 -0.852 -1.060 -1.334 -1.861 -1.506 -1.913 -2.183 -3.309 seq2_ low_allosteric_hinge 48 71 85 96 104 109 114 116 119 120 seq2_ high_allosteric_hinge 94 71 57 46 38 33 28 26 23 22

log(seq2_hinge_p_value) -0.281 -0.258 -0.663 -0.724 -0.929 -1.343 -1.337 -2.167 -2.495 -4.765 seq3_ low_allosteric_hinge 55 93 111 124 133 140 147 150 153 154 seq3_ high_allosteric_hinge 122 84 66 53 44 37 30 27 24 23

log(seq3_hinge_p_value) -0.101 -0.093 -0.153 -0.231 -0.517 -0.690 -0.508 -0.856 -1.344 -3.641 Energy Threshold (kcal/mol) 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2

low_allosteric_mutation 262 266 269 271 274 277 279 279 280 280 high_allosteric_mutation 21 17 14 12 9 6 4 4 3 3

low_allosteric_hinge 42 43 45 46 48 50 51 51 51 51 high_allosteric_hinge 10 9 7 6 4 2 1 1 1 1 log(hinge_p_value) -2.869 -3.031 -2.234 -1.955 -1.204 -0.517 -0.253 -0.253 -0.340 -0.340

6Å_ low_allosteric_hinge 123 125 127 129 132 135 136 136 137 137 6Å_ high_allosteric_hinge 17 15 13 11 8 5 4 4 3 3

log(6Å_hinge_p_value) -2.544 -3.065 -3.171 -2.583 -1.731 -0.929 -1.232 -1.232 -0.922 -0.922 seq2_ low_allosteric_hinge 123 127 129 131 134 137 138 138 139 139 seq2_ high_allosteric_hinge 19 15 13 11 8 5 4 4 3 3

log(seq2_hinge_p_value) -3.882 -2.758 -2.844 -2.270 -1.442 -0.670 -0.912 -0.912 -0.607 -0.607 seq3_ low_allosteric_hinge 157 161 163 165 168 171 173 173 174 174 seq3_ high_allosteric_hinge 20 16 14 12 9 6 4 4 3 3

log(seq3_hinge_p_value) -3.151 -2.400 -2.867 -2.361 -1.538 -1.061 -0.522 -0.522 -0.531 -0.531 * Hinge residues are predicted for the first two global modes of motion by GNM. The labels 6Å, seq2 and seq3 stand for the method of finding nearby residues to hinge residues such that 6Å: residues whose alpha carbon distances to the hinge residues are smaller than 6Å, seq2 and seq3: the first two and three neighbors of hinge residues in sequence, respectively. p-values are calculated in Fisher’s exact test (see Fig. 1 for visualization of the data). Orange: false positives, where mutant residues significantly correspond to residues other than hinge residues both below and above the threshold, Green: true positives, where mutant residues significantly correspond to hinge/nearby residues above the threshold; and to residues other than hinge/nearby residues below the threshold.

TABLE S3 The number of mutations to Alanine below (low) and above (high) each of the energy threshold values, the number of mutations corresponding to hinge/nearby residues and the logarithm of p-values of their correspondence.

Energy Threshold (kcal/mol) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 low_allosteric 66 115 147 166 179 188 195 200 204 high_allosteric 158 109 77 58 45 36 29 24 20

low_allosteric_hinge 10 14 20 24 24 27 30 31 33 high_allosteric_hinge 30 26 20 16 16 13 10 9 7 log(hinge_p_value) -0.245 -1.614 -1.561 -1.529 -2.771 -2.428 -1.727 -1.710 -1.224

6Å_low_allosteric_hinge 28 50 62 71 76 80 85 87 90 6Å_high_allosteric_hinge 77 55 43 34 29 25 20 18 15 log(6Å_hinge_p_value) -0.334 -0.458 -1.176 -1.330 -1.932 -2.456 -1.805 -2.358 -2.015

seq2_low_allosteric_hinge 29 47 61 71 77 81 85 86 89 seq2_high_allosteric_hinge 75 57 43 33 27 23 19 18 15 log(seq2_hinge_p_value) -0.180 -0.968 -1.314 -1.165 -1.338 -1.550 -1.526 -2.380 -2.040

seq3_low_allosteric_hinge 32 63 81 93 99 104 109 111 114 seq3_high_allosteric_hinge 98 67 49 37 31 26 21 19 16

seq3_hinge_p_value 0.075 0.344 0.255 0.355 0.128 0.067 0.109 0.029 0.055 log(seq3_hinge_p_value) -1.126 -0.463 -0.594 -0.450 -0.893 -1.174 -0.964 -1.535 -1.257

Energy Threshold (kcal/mol) 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 low_allosteric 208 211 212 215 217 218 221 223 223 high_allosteric 16 13 12 9 7 6 3 1 1

low_allosteric_hinge 33 33 33 35 36 37 39 40 40 high_allosteric_hinge 7 7 7 5 4 3 1 0 0 log(hinge_p_value) -1.941 -2.577 -2.850 -1.982 -1.686 -1.145 -0.349 0.000 0.000

6Å_low_allosteric_hinge 92 95 95 97 99 100 103 104 104 6Å_high_allosteric_hinge 13 10 10 8 6 5 2 1 1 log(6Å_hinge_p_value) -2.130 -1.381 -1.843 -1.860 -1.276 -0.996 -0.221 -0.329 -0.329

seq2_low_allosteric_hinge 90 93 94 96 98 99 102 103 103 seq2_high_allosteric_hinge 14 11 10 8 6 5 2 1 1 log(seq2_hinge_p_value) -2.984 -2.128 -1.853 -1.877 -1.289 -1.005 -0.223 -0.333 -0.333

seq3_low_allosteric_hinge 115 118 119 121 123 124 127 129 129 seq3_high_allosteric_hinge 15 12 11 9 7 6 3 1 1 log(seq3_hinge_p_value) -2.576 -2.047 -1.804 -1.947 -1.644 -1.384 -0.575 0.000 0.000

* See the footnote for Table S2 for details and Fig. S1 for visualization of the data.

TABLE S4 MD Simulation Details.

System # of Amino

Acids Atoms

[#]* Total # of

Atoms Simulation Length [ns]

Equilibration Length [ns]

1HWG wt-1 190 3059 19842 100 45 1HWG wt-2 190 3059 19842 100 10 1HWG wt-3 190 3059 19842 100 40 1HWG wt-4 190 3059 19842 100 5 1HWG wt-5 190 3059 19842 100 5

1HWG wt-6 190 3059 19842 100 5 1HWG I58A-1 190 3050 19833 100 45 1HWG I58A-2 190 3050 19833 100 5 1HWG I58A-3 190 3050 19833 100 20 1HWG I58A-4 190 3050 19833 100 25 1HWG I58A-5 190 3050 19833 100 10 1HWG I58A-6 190 3050 19833 100 20 PYD-PYD wt-1 192 2872 19370 100 20 PYD-PYD wt-2 192 2872 19370 100 40 PYD-PYD wt-3 192 2872 19370 100 10 PYD-PYD wt-4 192 2872 19370 100 15 PYD-PYD wt-5 192 2872 19370 100 10 PYD-PYD wt-6 192 2872 19370 100 10

PYD-PYD D48A-1 192 2868 19366 100 25 PYD-PYD D48A-2 192 2868 19366 100 10 PYD-PYD D48A-3 192 2868 19366 100 45 PYD-PYD D48A-4 192 2868 19366 100 10 PYD-PYD D48A-5 192 2868 19366 100 10 PYD-PYD D48A-6 192 2868 19366 100 5

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