Dynamic characterization and substrate binding of cis-2,3-dihydrobiphenyl-2,3-diol dehydrogenase, an enzyme used in bioremediation
Stefano Piccoli1, Francesco Musiani2,3,* and Alejandro Giorgetti1,*
1 Department of Biotechnology, University of Verona (Italy).
2 Scuola Internazionale Superiore di Studi Avanzati (SISSA/ISAS), Trieste (Italy).
3 Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna (Italy).
SUPPORTING IMFORMATION
S1. BPY conformational analysis
Density functional theory(DFT) computations have been carried out with the program ORCA 3.0.1 [1]using the B3LYP [2, 3] functional. All atoms have been described by the 6-31G(d) basis set[4]. Frequency computations have been performed to determine the nature of the various critical points.
The coordinates of BPY co-crystallized with biphenyl dehydrogenase (PDB accession code: 3ZV5) [5]was used as starting point for the calculations. The hydrogen atoms of the initial model were added using the program UCSF Chimera [6]. The geometry optimization at the B3LYP/6-31G(d) level resulted in a conformation characterized by one benzene ring twisted by 40 degrees in relation to the other, as a consequence of steric crowding of the hydrogen atoms (M1 in Fig. S1). The computation of the vibration frequencies identified this conformation as a minimum on the potential energy surface.It was also possible to identify a second conformational minimum corresponding to a benzene rings twisting angle of -40 degrees (M2 in Fig. S1). Conformations M1 and M2 have the same potential energy.The search for a conformational transition state along the reaction coordinate defined by the torsion of one benzene ring with respect to the other resulted in conformation TS1 (Fig. S1). The computation of the vibration frequencies confirmed that TS1 conformation is a saddle point of the first order (i.e. a transition state) on the potential energy surface. In the TS1 conformation the molecules is completely planar. The calculated conformational barrier between M1 (or M2) and TS1 is 17.6 kJ mol-1.
Figure S1.Energies of the biphenyl-2,3-diol conformations studied at the B3LYP/6-31G(d) level.
Table S1. Gibbs free enthalpies derived from the B3LYP/6-31G(d) calculations.
Point / Gibbs free enthalpy (Eh) / Relative Gibbs free enthalpy (kJ/mol)M1 / -613.5710 / 0.0
TS1 / -613.5643 / 17.6
M2 / -613.5710 / 0.0
S2. Comparison between the PpBphB crystal structures
The crystal structures of the the apo, binary and holo forms of PpBphB dimers (PDBid:2Y93, 2Y99, and 3ZV5, respectively) where superimposed using the program UCSF Chimera [6].
Table S2. Cα RMSD (nm) between the apo, binary, and holo forms of PpBphB dimers
RMSD (nm) / Apo / BinaryBinary / 0.05 / -
Holo / 0.12 / 0.12
S3. Molecular docking
Figure S2. Haddock score vs. interface-ligand-RMSD (i-l-RMSD) plot [7]. PpBphB-BPY complexes belonging to the same cluster are reported using dots of the same colour. Cluster average structures are highlighted using triangle of the same colour of the dots.
Figure S3. Comparison between the BPY binding pose found in the holoPpBphB crystal structure (PDBid: 3ZV5, grey) and the best docking pose obtained using Haddock (red). The experimental electron density of holoPpBphB is reported in cyan.
S4. Cluster analysis.
Clustering analysis has been performed using the g_cluster module of Gromacs[8], using the Gromos algorithm [9]. A 0.15 nm cut-off for the Cα RMSD was used to include structures in the same cluster. C-terminal residues 259-276 from each chainwere not considered in the RMSD calculation since they showed very high mobility during the MD simulations (see Fig. S9 below).
Figure S4. Cluster population (left plot) and cluster number of eachframe in the trajectory (right plot)for the apo form.
Figure S5.Cluster population (left plot) and cluster number of eachframe in the trajectory (right plot)for the binary form.
Figure S6.Cluster population (left plot) and cluster number of eachframe in the trajectory (right plot)for the holo form.
Figure S7. Structural alignment between the representative structure of the two most populated clusters (Fig. S4) in the trajectory of the holo form. The cartoons are reported in green and cyan for cluster #1 and #2, respectively.
S5. Essential dynamics
The essential vectors are determined by diagonalizing the covariance matrices (cov):
(Eq. 1)
where the sum goes over the M configurations or snapshots from the dynamics, corresponds to the ith Cartesian coordinate of the system in snapshot number k, and is the mean value. From the diagonalization of the covariance matrix, a set of eigenvalues () and eigenvectors () are obtained.Each obtained eigenvector corresponds to an essential mode (EM) of the protein. Together, all of the essential modes describe the motion of the protein along the MD run used to generate the computed matrix. The eigenvalues obtained represent the relative contribution of each EM to the overall dynamics. The EM are ranked according their eigenvalues and therefore their relative weight (), the first EM being the one with major contribution or larger eigenvalue.The weight of each EM in the trajectory is calculated by:
(Eq. 2)
C-terminal residues 259-276 from each chain were not considered in the RMSD calculation since they showed very high mobility during the MD simulations (see Fig. S9 below).
Figure S8.(A-C) Cartoon representation of the structural movement along the first EM eigenvector of apo, binary and holo forms of PpBphB (A, B, and C panel, respectively). Cartoons are colored with red and blue structure corresponding to the two extreme structures along the EM. (D) Schematic representation of the main movement along the first EM eigenvector in the three simulations.
S6. Root mean square fluctuations
Figure S9.Average CαRMSF values calculated for the apo (green line), binary (red line) and holo forms of PpBphB.
Table S3.Average CαRMSF (<RMSF>) of the residues in the central region of the substrate binding loop(residues 198-206).
Form / RMSF(Chain A) / RMSF(Chain B) / <RMSF> (A/B)Holo / 0.15 / 0.19 / 0.17
Binary / 0.14 / 0.25 / 0.19
Apo / 0.19 / 0.24 / 0.22
S7. Selected distances and H-bonds
Figure S10.Distance vs. time plot of BPY from selected residues in the binding pocket of the holo form. Distances betweenSer142(Oγ)-BPY(O2), Asn143(Oδ1)-BPY(O1), and Tyr155(Oη)-BPY(O2)are reported in the left, central and right panel, respectively.Grey lines represent the effective sampling of the distance during the simulation, black lines are obtained by applying a Fast Fourier Transform filter in order to cut-off noise.
Figure S11.Distance vs. time plot of NAD from selected residues in the binding pocket of the holo form. Distances between Tyr155(Oη)-NAD(O3D) and Lys159(Nζ)-NAD(O2D) are reported in the left and right panel, respectively.Grey lines represent the effective sampling of the distance during the simulation, black lines are obtained by applying a Fast Fourier Transform filter in order to cut-off noise.
Figure S12.Distance vs. time plot of the Ser142(Oγ)-Tyr155(Oη) distance in the apo, binary and holo form (left, central, and right panel, respectively).Grey lines represent the effective sampling of the distance during the simulation, black lines are obtained by applying a Fast Fourier Transform filter in order to cut-off noise.
Supplementary Information References
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