Lin et al. 10.1073/pnas.0606006103. |
Fig. 3. Schematic representation of the QM/MM partition in the reactive state structure. (a) The system selected for the QM/MM calculation. QM atoms are in ball-and-stick format, dsDNA (stick), complete DNA polymerase b (cartoon and lines, hydrogen atoms not shown), counter ions (Na+ and Cl-, spheres), and selected waters (up to 15 Å from QM atoms) are also included. Only MM atoms within 10 Å of the QM atoms are allowed to move during the optimization. (b) Optimized active-site initial state structure. Key distances are listed with reference to the experimental values (in parentheses) in the gapped DNA-2'-deoxy-uridine-5'-(a,b)-imido triphosphate-pol b complex.
Fig. 4. Schematic minimum energy path along the potential surface (arbitrary reaction coordinate). Reaction state (RS), local minimum state after the proton transfer to Asp-256 (LM), TS, and product state (PS) are connected with a 0.1-Å step along O3'-P1A and P1A-O3A distances. Both open and filled circles are calculated points.
Fig. 5. Schematic of the TS structure of the active site. Residues with significant contribution to the electrostatic stabilization of TS are also included.
Table 1. Residue TS stabilizations (kcal/mol) based on electrostatic calculations and mutagenesis data
(Sub) Domain | Residues | Reference to GS† | Reference to LM‡ | Mutant | Catalytic activity (WT/mutant) | X-family conservation |
Lyase | Asp-17 | 1.41 | 1.08 |
|
|
|
| Lys-27 | -2.04 | -1.13 |
|
|
|
| Arg-40 | -2.79 | -1.78 |
|
|
|
| Lys-48 | -1.52 | -1.08 |
|
|
|
| Thr-79 | 0.00 | 0.00 | T79S | 0.9-10.6 (1) |
|
|
|
|
|
|
|
|
D | Glu-147 | 1.47 | 1.20 |
|
|
|
| Arg-149 | -5.03 | -4.64 | R149A | 6.0-22.3 (2) | Yes* |
|
|
|
|
|
|
|
C | Ser-180 | -1.40 | -1.39 | S180A | 287-439 (2) |
|
| Arg-182 | -1.63 | -1.27 | R182Q | 28.5 (3) | Yes |
Arg-183 | -8.01 | -7.06 | R183A | 15-450 (2) | Yes | |
43000 (3) | ||||||
|
|
|
| R183Q | 54.7 (3) |
|
| Ser-188 | -0.80 | -0.82 | S188A | 1.7-8.5 (2) |
|
| Lys-234 | -0.44 | 1.00 |
|
|
|
| Asp-246 | 0.33 | 0.30 | D246V | 0.5-6.5 (4) |
|
| Glu-249 | 0.35 | 0.36 | E249K | 0.4-2.4 (5) |
|
| Arg-253 | -1.10 | -1.15 | R253M | 2 (6) | Yes |
Arg-254 | 7.67 | 3.62 | R254A | 27.1 (7) | Yes | |
|
|
|
| R254K | 3.2 (7) |
|
| Arg-258 | -0.74 | 0.35 | R258A | 7.0 (7) |
|
Ile-260 | -0.05 | -0.02 | I260D,E,K,N, or R | No activity (8) | ||
I260M | 2.9-45.0 (9) | |||||
|
|
|
| I260Q | 4.7-8.0 (10) |
|
N | Tyr-265 | 0.05 | 0.02 | Y265F | 0.5-2.0 (11, 12) | |
Y265H | 0.02-12.2 (13) | |||||
Y265L | 1.1 (12) | |||||
Y265S | 5.5 (12) | |||||
|
|
|
| Y265W | 0.5-0.9 (11, 12) |
|
Tyr-271 | 0.04 | 0.08 | Y271A | 0.6-1.4 (14) | ||
1.0 (15) | ||||||
Y271H | 0.6-6.0 (14) | |||||
Y271F | 2 (15) | |||||
|
|
|
| Y271S | 2.4-10.9 (14) |
|
| Phe-272 | 1.24 | 1.32 | F272L | 1.1-2.3 (16) |
|
| Gly-274 | -0.38 | -0.25 | G274P | 10000 (17) |
|
Asp-276 | 4.06 | 2.77 | D276E | 0.04 (18) | ||
D276R | 8.5 (19) | |||||
|
|
|
| D276V | 0.2-0.4 (20) |
|
Asn-279 | -0.43 | -0.17 | N279A | 13.5-16.4 (14) | ||
5 (15) | ||||||
N279L | 14.0 (15) | |||||
|
|
|
| N279Q | 12-18.2 (14) |
|
Lys-280 | -2.26 | -1.29 | K280A | 2.1-3.3 (2) | Yes* | |
30 (21) | ||||||
K280R,K,M,I | ~1 (21) | |||||
K280L | 2 (21) | |||||
K280Q | 3 (21) | |||||
|
|
|
| K280G | 40 (21) |
|
| Met282 | -0.05 | -0.03 | M282L | 2 (22) |
|
Arg-283 | -2.06 | -0.89 | R283A | 5000 (15) | Yes | |
224-461 (23) | ||||||
R283K | 125 (15) | |||||
|
|
|
| R283L | 2500 (15) |
|
| Lys-289 | -0.83 | -0.53 | K289M | 5.8-130 (24) |
|
Asn-294 | -0.03 | 0.02 | N294A | 11-190 (2) | ||
|
|
|
| N294Q | 4.1-15.8 (2) |
|
| Glu-295 | 1.42 | 0.27 | E295A | 33-1300 (2) |
|
| Tyr-296 | -0.03 | -0.02 |
|
|
|
| Glu-316 | 1.34 | 0.93 |
|
|
|
| Arg-328 | -1.63 | -1.17 |
|
| Yes* |
| Lys-331 | -1.19 | -0.93 |
|
|
|
| Asp-332 | 1.39 | 1.06 |
|
|
|
| Arg-333 | -1.75 | -1.26 |
|
| Yes |
| Glu-335 | 1.85 | 1.31 |
|
|
|
|
|
|
|
|
|
|
Solvent | WAT#475 | -1.86 | -1.87 |
|
|
|
| WAT#402 | -1.61 | -1.54 |
|
|
|
Domain/subdomain nomenclature is according to Beard et al. (21): Lyase,domain (L) and the DNA binding (D), DNA synthesis (C), and dNTP selection (N) subdomains of the polymerase domain. * denotes that the residue is preserved in at least two X-family members (pol b, pol l, pol m, TdT).
†
Energy relative to the energy of ground state (GS).‡
Energy relative to the proton-transferred local minimum state (LM).References
1. Maitra, M., Gudzelak, A., Li, S. X., Matsumoto, Y., Eckert, K. A., Jager, J. & Sweasy, J. B. (2002) J. Biol. Chem. 277, 35550-35560.
2. Kraynov, V. S., Showalter, A. K., Liu, J., Zhong, X. J. & Tsai, M. D. (2000) Biochemistry 39, 16008-16015.
3. Date, T., Yamamoto, S., Tanihara, K., Nishimoto, Y., Liu, N. & Matsukage, A. (1990) Biochemistry 29, 5027-5034.
4. Dalal, S., Kosa, J. L. & Sweasy, J. B. (2004) J. Biol. Chem. 279, 577-584.
5. Kosa, J. L. & Sweasy, J. B. (1999) J. Biol. Chem. 274, 35866-35872.
6. Kosa, J. L. & Sweasy, J. B. (1999) J. Biol. Chem. 274, 3851-3858.
7. Menge, K. L., Hostomsky, Z., Nodes, B. R., Hudson, G. O., Rahmati, S., Moomaw, E. W., Almassy, R. J. & Hostomska, Z. (1995) Biochemistry 34, 15934-15942.
8. Starcevic, D., Dalal, S. & Sweasy, J. (2005) Biochemistry 44, 3775-3784.
9. Dalal, S., Hile, S., Eckert, K. A., Sun, K., Starcevic, D. & Sweasy, J. B. (2005) Biochemistry 44, 15664-15673.
10. Starcevic, D., Dalal, S., Jaeger, J. &, Sweasy, J. B. (2005) J. Biol. Chem. 280, 28388-28393.
11. Shah, A.M., Maitra, M. & Sweasy, J. B. (2003) Biochemistry 42, 10709-10717.
12. Opresko, P. L., Shiman, R. & Eckert, K. A. (2000) Biochemistry 39, 11399-11407.
13. Shah, A. M., Li, S. X., Anderson, K. S. & Sweasy, J. B. (2001) J. Biol. Chem. 276, 10824-10831.
14. Kraynov, V. S., Werneburg, B. G., Zhong, X. J., Lee, H., Ahn, J. W. & Tsai, M. D. (1997) Biochem. J. 323, 103-111.
15. Beard, W. A., Osheroff, W. P., Prasad, R., Sawaya, M. R., Jaju, M., Wood, T. G., Kraut, J., Kunkel, T. A. & Wilson, S. H. (1996) J. Biol. Chem. 271, 12141-12144.
16. Li, S. X., Vaccaro, J. A. & Sweasy, J. B. (1999) Biochemistry 38, 4800-4808
17. Beard, W. A., Shock, D. D., Vande Berg, B. J. & Wilson, S. H. (2002) J. Biol. Chem. 277, 47393-47398.
18. Skandalis, A. & Loeb, L. A. (2001) Nucleic Acids Res. 29, 2418-2426.
19. Liu, J. & Tsai, M. D. (2001) Biochemistry 40, 9014-9022.
20. Vande Berg, B. J., Beard, W. A. & Wilson, S.H. (2001) J. Biol. Chem. 276, 3408-3416.
21. Beard, W. A., Shock, D. D., Yang, X. P., DeLauder, S. F. & Wilson, S. H. (2002) J. Biol. Chem. 277, 8235-8242.
22. Shah, A. M., Conn, D. A., Li, S. X., Capaldi, A., Jager, J. & Sweasy, J. B. (2001) Biochemistry 40, 11372-11381.
23. Ahn, J., Werneburg, B. G. & Tsai, M. D. (1997) Biochemistry 36, 1100-1107.
24. Lang, T. M., Maitra, M., Starcevic, D., Li, S. X. & Sweasy, J. B. (2004) Proc. Natl. Acad. Sci. USA 101, 6074-6079.
Atom number | Atom name | Atom type | RESP charges |
1 | O1G | O2 | -0.9400 |
2 | PG | P | 1.5440 |
3 | O2G | OH | -0.7010 |
4 | H2G | HO | 0.3930 |
5 | O3G | O2 | -0.9400 |
6 | O3B | OS | -0.7410 |
7 | PB | P | 1.5510 |
8 | O1B | O2 | -0.9290 |
9 | O2B | O2 | -0.9290 |
10 | O3A | OS | -0.4940 |
11 | PA | P | 1.0150 |
12 | O1A | O2 | -0.6870 |
13 | O2A | O2 | -0.6870 |
14 | O5' | OS | -0.6446 |
15 | C5' | CT | -0.0069 |
16 | H5'1 | H1 | 0.0754 |
17 | H5'2 | H1 | 0.0754 |
18 | C4' | CT | 0.1629 |
19 | H4' | H1 | 0.1176 |
20 | O4' | OS | -0.3691 |
21 | C1' | CT | 0.0680 |
22 | H1' | H2 | 0.1804 |
23 | N1 | N* | -0.0239 |
24 | C6 | CM | -0.2209 |
25 | H6 | H4 | 0.2607 |
26 | C5 | CM | 0.0025 |
27 | C7 | CT | -0.2269 |
28 | H71 | HC | 0.0770 |
29 | H72 | HC | 0.0770 |
30 | H73 | HC | 0.0770 |
31 | C4 | C | 0.5194 |
32 | O4 | O | -0.5563 |
33 | N3 | NA | -0.4340 |
34 | H3 | H | 0.3420 |
35 | C2 | C | 0.5677 |
36 | O2 | O | -0.5881 |
37 | C3' | CT | 0.0713 |
38 | H3' | H1 | 0.0985 |
39 | C2' | CT | -0.0854 |
40 | H2'1 | HC | 0.0718 |
41 | H2'2 | HC | 0.0718 |
42 | O3' | OH | -0.6549 |
43 | H3T | HO | 0.4396 |
The charges for atoms from C5' (atom number 15) to H3T (atom number 43) were taken from the D-thymine with a 5'-phosphate group and a 3'-OH group in Amber99 force field (1). The charges for atoms O1G (atom number 1) to O2A (atom number 14) were taken from RESP charges of a model methyl triphosphate, which is built based on the 2',3'-dideoxyribocytidine (ddCTP) structure in the high-resolution x-ray crystal structure of the ternary (gapped DNA-ddCTP analog-pol b) complex (2).
References
1. Wang, J. M., Wolf, R.M., Caldwell, J. W., Kollman, P. A. & Case, D. A. (2004) J. Comput. Chem. 25, 1157-1174.
2. Batra, V. K., Beard, W. A., Shock, D. D., Krahn, J. M., Pedersen, L. C. & Wilson, S. H. (2006) Structure (London) 14, 757-766.
Supporting Text
Computational Details
The dynamic simulations and minimizations were performed as follows. First, the water molecules (with all other molecules fixed) were subjected to a cooling procedure under constant temperature and pressure (50 K, 1 atm) until the density of the system reached »0.99 g/cm3. This was followed by a 50-ps equilibration step at constant volume and temperature (298 K). Second, the whole system was energy-minimized to remove high energy contacts created by hydrogenation while the heavy atoms from the crystal structure were subjected to harmonic constraints with a force constant of 20 kcal/mol per Å2. The system was then slowly heated to 298 K in 150 ps, after which it remained at 298 K for another 200 ps before cooling to 100 K in the next 50 ps at constant volume. During this process, all heavy atoms from the crystal structure were subjected to harmonic constraints with a force constant of 2.0 kcal/mol per Å2. Finally, the total system underwent a full unconstrained minimization. This procedure reproduced the crystal structure very well.
In the ONIOM(QM:MM) calculation using Gaussian 03 (1), the distance constraints were applied by freezing the corresponding redundant coordinates. A step size of 0.10 Å was used in the potential energy surface scan. The optimization convergence criteria was set to a maximum step size of 0.01 au (0.00529 Å) and an rms force of 0.0017 au (2.0158 kcal/mol per Å) during the optimization of the relaxed geometries on potential energy surface; these criteria were 0.0018 au (0.00095 Å) and 0.0003 au (0.3557 kcal/mol per Å), respectively, for the full geometry optimization of the reactant, local minimum, and product states.
1. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Montgomery JA, Jr., Vreven T, Kudin KN, Burant JC, et al. (2004) Gaussian 03 (Gaussian, Wallingford CT), Revision C.02.