Molecular Docking of 1,2,3-trichloropropane (TCP) to Free DhaA and 2,3-dichloro-1-propanol (DCL) to DhaA.Cl- Complex by AUTODOCK

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Date: 28.09.04 - 08.10.04
Author: Klvana, M.

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Motivation: 

•	Preparation of DhaA.Cl-.DCL complex since an X-ray structure of DhaA with either DCL or any alcohol has not been available. Merely free DhaA and DhaA.I- X-ray structures are currently in Protein Data Bank. DhaA.Cl-.DCL complex will be used for random expulsion molecular dynamics simulation.

•	Preparation of DhaA.TCP complex since an X-ray structure of DhaA with either TCP or any other substrate has not been available.

•	Comparison of docking results using DhaA from Rhodococcus rhodochrous NCIMB13064 (used in experiments) and Rhodococcus sp. TDTM0003 (X-ray structure). The two DhaA (strain NCIMB13064 vs strain TDTM0003) differ in three positions: 172 (A vs V), 209 (L vs I) and 292 (A vs G).

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Methods:

•	List of Programmes and Scripts: PYMOL 0.97 (DeLano Scientific, San Diego, USA), WHATIF 5.0 (Vriend, G., 1990), AUTODOCK 3.05 (Morris et al., 1998), q.kollua and addsol;

•	Procedure: Rotatable bonds and small molecular centre were assigned for TCP and DCL using Autotors. TCP contains two rotatable bonds (C1-C2 and C2-C3); DCL has three such bonds (C1-O, C1-C2 and C2-C3). DhaA (PDB-ID 1CQW from Rhodococcus sp. TDTM0003) previously modified by adding three missing C-terminal  residues and called 1CQWA was mutated (V172A + I209L + G292A) using PYMOL. Resulting structure was called 1CQWA_ncimb. Both DhaA structures were subsequently modified by addition of polar hydrogens by WHATIF, partial atomic charges using the script q.kollua and solvation parameters by the script addsol. In case of docking to DhaA.Cl- complex anion was taken from the crystal structure but changed from iodide to chloride with charge of -1. Since there is no chlorine atomic type for protein structure in AUTODOCK chloride was represented by X atom type with well depth and non-bonded parameters of chlorine. Electrostatic and atomic (C, O, H and Cl) maps (81x81x81 points and grid spacing of 0.25 Å) were calculated using Autogrid. Centre of each grid was set to Cg of H272. Fifty docking runs to DhaA and DhaA.Cl- were performed for TCP and DCL. A Lamarckian genetic algorithm was used with an initial population size of 50 individuals. A maximum of 1.5x106 number of energy evaluations and a maximum number of 27 000 generation served as termination criteria for each docking run. Elitism was set to 1, mutation rate to 0.02 and crossover rate to 0.8. A local search based on a pseudo-Solis and Wets algorithm (with a maximum of 300 iteration per local search) was applied with probability of 0.06 of performing the local search on an individual. Final orientations from every docking were clustered with a clustering tolerance for the root-mean-square positional deviation of 0.5 Å. 

Results and Discussion:

•	Docking of TCP to free DhaA 1CQWA resulted in three clusters of orientations (Table 1). Orientation 1 (TCP1a) and 2 (TCP1b) were favoured for nucleophilic attack by Od1 of D106 on C1 whereas orientation 3 (TCP1c; called bm1 by Banas et. al, unpublished) was favoured for attack on C2. Chlorine Cl2 was oriented toward L246 in TCP1a (bm3) and toward N41 in TCP1b (bm2).

Table 1. Docking of TCP to 1CQWA.
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   Clusters      Orientations      Docked Energy (A, B)
   - Name -       - Number -           - kcal/mol -    
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   TCP1a (bm3)        25           -3.83 (-5.88, +2.05)
   TCP1b (bm2)        14           -3.82 (-5.76, +1.94)
   TCP1c (bm1)        11           -3.82 (-5.70, +1.88)
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	A = intermolecular energy, B = internal energy of ligand

•	Docking of TCP to free DhaA 1CQWA_ncimb resulted in only two orientations (TCP2a and TCP2b; Table 2) which were almost identical with TCP1a and TCP1b. Both clusters were highly populated (28 and 22, respectively). No orientation corresponding to TCP1c  was obtained. This result is in agreement with experimental finding that 2,3-dichloro-1-propanol is the sole alcohol product of TCP dehalogenation by DhaA from Rhodococcus rhodochrous NCIMB13064.

Table 2. Docking of TCP to 1CQWA_ncimb.Cl-.
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   Clusters      Orientations      Docked Energy (A, B)
   - Name -       - Number -          - kcal/mol -    
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  TCP2a (bm2)         28           -3.88 (-5.81, +1.93)
  TCP2b (bm3)         22           -3.85 (-5.90, +2.05)
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•	Difference in two active site residues caused different docking results. 1CQWA with I209 enables favourable but unproductive (Banas and Jerabek, unpublished) binding of TCP cross to the binding modes assumed to be productive. This could be caused by favourable van der Waals contact of TCP with the residue 209. I209L mutation makes steric hindrance for binding of docked TCP into the orientation for dehalogenation from C2.

•	DCL was docked to DhaA.Cl- complex in order to reconstruct complex of DhaA with products of TCP dehalogenation. Experimental observations (Bosma et al., 2002), computer modelling studies (Banas and Jerabek, unpublished) as well as docking of TCP to 1CQWA_ncimb strongly suggest that dehalogenation occurs purely on C1 of TCP. Therefore only 2,3-dichloro-1-propanol (DCL) was taken into the docking study.

•	Docking of DCL to 1CQWA.Cl- complex provided six clusters of orientations (DCL1a to DCL1f; Table 3). All DCL orientations had lower docked energy than TCP (from 1.03 to 1.57 kcal/mol). This was, however, caused by more convenient internal energy of DCL (from -0.17 to -0.03 kcal/mol) compared to TCP (from +1.88 to +2.05 kcal/mol). DCL was bound closely to chloride anion in all cases. DCL docked to 1CQWA.CL- preferred hydrogen bonding (HB) interaction of its hydroxyl with carbonyl oxygen of N41 main chain (orientations DCL1a to DCL1d). DCL1e formed HB with Cl- and DCL1f with Od1 of D106.

Table 3. Docking of DCL to 1CQWA.
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   Clusters      Orientations       Docked Energy (A, B)
   - Name -       - Number -           - kcal/mol -    
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    DCL1a             28            -5.14 (-5.11, -0.03)
    DCL1b             12            -5.05 (-4.98, -0.07)
    DCL1c              6            -4.96 (-4.80, -0.16)
    DCL1d              1            -4.96 (-4.90, -0.06)
    DCL1e              2            -4.89 (-4.87, -0.02)
    DCL1f              1            -4.86 (-4.70, -0.17)
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•	Only two clusters (DCL2a and DCL2b; Table 4) were obtained in case of 1CQWA_ncimb.Cl-. Both DCL2a and DCL2b were docked similarly to the orientation DCL1e. DCL2a (49 orientations in the cluster; Figure 1) interacts via HB with both carboxylic oxygen atoms of D106; DCL2b  (1 orientation in the cluster) only with Od2 of D106.

Table 4. Docking of DCL to 1CQWA_ncimb.Cl-.
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   Clusters      Orientations       Docked Energy (A, B)
   - Name -       - Number -           - kcal/mol -    
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    DCL2a             49            -5.39 (-5.26, -0.13)
    DCL2b              1            -5.17 (-5.04, -0.13)
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Figure 1. 1CQWA_ncimb.Cl-.DCL2a complex.

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Conclusions:

•	Following molecular dynamics study will be conducted with 1CQWA_ncimb.Cl-.DCL2a complex (Figure 1). 1CQWA_ncimb has been chosen in order to allow direct comparison of experimental and theoretical results. DCL2a has been selected according to the docking preference over DCL2b.

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Data:

•	Protein structures (1CQWA.pdb and 1CQWA_ncimb.pdb):
	martink@eldar.chemi.muni.cz:/home/data/martink/STR/PROT/DhaA
	(PROT = Protein)

•	Molecular docking of TCP to 1CQWA:
	martink@eldar.chemi.muni.cz:/home/data/martink/DOCK/DhaA/1CQWA/TCP

•	Molecular docking of DCL to 1CQWA.Cl-:
	martink@eldar.chemi.muni.cz:/home/data/martink/DOCK/DhaA/1CQWA.Cl/DCL

•	Molecular docking of TCP to 1CQWA_ncimb:
	martink@eldar.chemi.muni.cz:/home/data/martink/DOCK/DhaA/1CQWA_ncimb/TCP

•	Molecular docking of DCL to 1CQWA_ncimb.Cl-:
	martink@eldar.chemi.muni.cz:/home/data/martink/DOCK/DhaA/1CQWA_ncimb.Cl/
	/DCL

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References:

•	Bosma, T., Damborsky, J., Stucki, G., and Jensen, D. B. (2002) Applied and Environmental Microbiology 68, 3582-3587.

•	Morris, G.M., Goodsell, D. S., Huey, R., Hart, W. E., Halliday, R. S., Belew, R. K., and Olson, A. J. (1998), Journal of Computational Chemistry 19, 1639-1662.

•	Vriend, G. (1990) Journal of Molecular Graphics 8, 52-56.