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Exploring the Possible Pathways of DNA
Polymerase λ’s Nucleotidyl Transfer Reaction
Meredith Foley
Schlick Lab Retreat -- February 9, 2008
Chemistry
1
Available Data on the Reaction Pathway
• Using kpol values, the activation energy of the reaction
can be estimated from transition state theory (pol λ: 16-
17 kcal/mol; pol β: 16-18 kcal/mol)
• Many computational studies have focused on pol β’s
reaction using both QM and QM/MM methods
• Among the QM/MM-determined reaction mechanisms for
pol β, initial O3′ proton transfer to a water or a catalytic
aspartate are the most favorable
• For pol λ, I have considered 4 different initial proton
transfer pathways as well as the case when O3′ attacks
Pα without forcing O3′ proton transfer
2
Methodology
• Model built from ternary complex with
misaligned DNA (2.00 Å; pdb: 2bcv)
• Solvated complex in a box with
150 mM ionic strength Semi- Fixed
• Equilibrated system in CHARMM Fixed 13 Å
• Reduced model for QM/MM
calculations and added link atoms
• 3 movement areas defined (free,
semi-fixed, and fixed) 7Å
• MM region treated with CHARMM ff
• QM region treated with HF/3-21G
basis set Free
• QM/MM equilibration performed using
CHARMM/Gamess-UK
• Reaction pathways followed using a
constrained minimization approach 3
Active Site Model
• 75 atoms including 6 link
atoms in the QM region
• −3 charge in QM region
• O3′H (H3T atom) points
toward O5′ (not a viable
pathway)
• Used this structure as a
starting point for all
reaction mechanisms
explored
4
O3′ Attack on Pα
-2331160
-2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5
-2331180
Pα-O3A
Start
-2331200 breaks
H3T is -2331220
closer to
Energy (kcal/mol)
-2331240
D490:OD1
Energy (kcal/mol)
-2331260
than O5′
-2331280
-2331300
-2331320
-2331340
-2331360
End
-2331380
-2331400
O3'-Pa-O3A
D490 Reaction Coordinate: O3’-Pa-O3A
• As O3′ attacks Pα, the cat Mg—dTTP:O1A distance decreases while the 5
O3′--cat Mg distance increases (Mg doesn’t need to stabilize oxyanion)
Proton Transfer to Asp490
-2331180
-3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5
Start -2331190
-2331200
H3T -2331210
breaks O3′-Pα ↑
Energy (kcal/mol)
-2331220
-2331230
from O5′ cat Mg-O1A ↓ -2331240
O3′-cat Mg ↑
-2331250
-2331260 O3′-cat Mg ↓
cat Mg-O1A ↓
-2331270
-2331280
-2331290
O3′-Pα -2331300
-2331310
increases
-2331320 End
-2331330
-2331340
Reaction Coordinate: O3′-H3T-Asp490:OD1
• Cat Mg helps to stabilize oxyanion O3′ as H3T
is transferred to Asp490:OD1
• O3′-Pα decreases as H3T is transferred to
Asp490:OD1 6
Proton Transfer to dTTP:O2A
-2331180
-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5
-2331190
Start
-2331200
Energy (kcal/mol)
-2331210 H3T
H3T moving
equidistant
away from -2331220
from O3′
O5′ -2331230
and O2A
-2331240
-2331250
-2331260
End
-2331270
-2331280
Reaction Coordinate: O3’-H3T-dTTP:O2A
• O3′-Pα distance decreases during the
proton transfer
• O3′-cat Mg distance increases until H3T 7
is transferred to O2A. Then, it decreases
Proton Transfer to Asp429
-2331160
-2 -1.5 -1 -0.5 0 0.5 1 1
-2331180
Start -2331200 H3T is
-2331220
equidistant
Energy (kcal/mol)
-2331240
-2331260 from O3′ and
-2331280
Asp:OD1
-2331300
-2331320
H3T rotates -2331340
to Asp:OD1; -2331360
-2331380
O3′-Pα O3′-cat Mg
-2331400
End
increases decreases
-2331420
-2331440
Reaction Coordinate: O3′-H3T-Asp429:OD1
• O3′-Pα distance increases as H3T rotates
toward Asp429, but then decreases as proton
is transferred
8
Proton Transfer to Water
-2331180
-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5
-2331190
Start
H3T -2331200
Cat Mg – O3′
Energy (kcal/mol)
-2331210
breaks -2331220
distance
away -2331230
decreases
from -2331240
-2331250
O5′ -2331260
-2331270
-2331280
-2331290
-2331300
End
-2331310
Reaction Coordinate: O3′-H3T-Water1:OH2
• O3′-Pα distance increases until H3T
rotates away from O5′ toward Water1
9
Future Work – Build New Models
• Continue following
reaction pathways
H3T toward Wat1 H3T toward Asp490 following proton transfer
and O3′ attack
• Improve starting
geometry (e.g., using
models at left)
2.8 3.0 • Refine favored pathways
1.6 with a larger basis set
Wat1 1.65 2.1
2.2 2.1 2.1 and smaller step size
• Consider simultaneous
proton transfer and O3’
D490 attack
• Proton transfer alone
causes rearrangement
of the catalytic ion 10
11
Possible Step 2 – to Another Water Molecule
-2331292
0 0.2 0.4 0.6 0.8 1 1.2
-2331294
Start
Energy (kcal/mol)
-2331296
End
-2331298
-2331300
-2331302
-2331304
-2331306
Rxn Coordinate: Wat1:OH2-Wat1:H1-Wat2:OH2
12
Possible Step 2 – O3′ Attack on Pα
-2331250
-2 -1.5 -1 -0.5 0 0.5 1 1.5 2
-2331260
Energy (kcal/mol)
-2331270
-2331280
-2331290
Start
-2331300
-2331310
End
-2331320
-2331330
-2331340
Reaction Coordinate: O3′-Pα-O3A
13
Pol β -- 2006
• Rotated O3′H toward Asp256 to
obtain initial geometry
• γ-phosphate oxygen protonated
• 64 atoms in QM region with −2
charge
• ONIOM method (QM region:
B3LYP and 6-31G*; MM region:
Amber ff)
• Followed O3’-Pα-O3A reaction
coordinate with 0.10 Å step size
• Estimate that TS occurs at O3′-
Pα = 2.2 Å and Pα-O3A = 1.9 Å
Lin, Pedersen, Batra, Beard, Wilson, with 21.5 kcal/mol higher energy
Pedersen, 2006, PNAS 103:13294-13299 than the reactant
14
Radhakrishnan & Schlick 2006
• G:C and G:A systems (1bpy) equilibrated in CHARMM; aspartates and dNTPs are
unprotonated
• QM region has 86 atoms (includes S180 and R183); -1 charge
• Reduced waters to 3 solvation shells in QM/MM model; added SLA
• QM region:B3LYP and 6-311G; MM region: CHARMM ff
• QM/MM equilibration followed by five 1 ps trajectories along O3’-Pa-O3A
coordinate; O3’-cat Mg restrained to 2 A
• From trajectories, 50 snapshots were chosen and minimized without constraints; in
both systems 6 different states were obtained; G:C free energy of activation at least
17 kcal/mol, G:A free energy of activation at least 21 kcal/mol 15
Alberts & Schlick 2006
16
Transition State Theory
• Insertion rate constant of reaction = kpol
• kpol = vexp[−ΔG‡/RT]
• At 25°C, v = 6.212x1012s−1 (v = kT/h)
17
