1 Exploring the Possible Pathways of DNA Polymerase λ’s Nucleotidyl Transfer Reaction Meredith Foley Schlick Lab Retreat -- February 9, 2008 Chemistry

2 Available Data on the Reaction Pathway Using k pol values, the activation energy of the reaction can be estimated from transition state theory (pol λ: kcal/mol; pol β: 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

3 Methodology Model built from ternary complex with misaligned DNA (2.00 Å; pdb: 2bcv) Solvated complex in a box with 150 mM ionic strength Equilibrated system in CHARMM Reduced model for QM/MM calculations and added link atoms 3 movement areas defined (free, semi-fixed, and fixed) MM region treated with CHARMM ff QM region treated with HF/3-21G basis set QM/MM equilibration performed using CHARMM/Gamess-UK Reaction pathways followed using a constrained minimization approach Fixed Semi- Fixed Free 7 Å 13 Å

4 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

5 O3′ Attack on Pα Reaction Coordinate: O3’-Pa-O3A Energy (kcal/mol) Start End As O3′ attacks Pα, the cat Mg—dTTP:O1A distance decreases while the O3′--cat Mg distance increases (Mg doesn’t need to stabilize oxyanion) Pα-O3A breaks H3T is closer to D490:OD1 than O5′ D490

6 Proton Transfer to Asp490 Energy (kcal/mol) Reaction Coordinate: O3′-H3T-Asp490:OD1 Start End O3′-Pα increases H3T breaks from O5′ O3′-Pα ↑ cat Mg-O1A ↓ O3′-cat Mg ↑ O3′-cat Mg ↓ cat Mg-O1A ↓ Cat Mg helps to stabilize oxyanion O3′ as H3T is transferred to Asp490:OD1 O3′-Pα decreases as H3T is transferred to Asp490:OD1

7 Proton Transfer to dTTP:O2A Energy (kcal/mol) Reaction Coordinate: O3’-H3T-dTTP:O2A Start End H3T moving away from O5′ H3T equidistant from O3′ and O2A O3′-Pα distance decreases during the proton transfer O3′-cat Mg distance increases until H3T is transferred to O2A. Then, it decreases

8 Proton Transfer to Asp429 Energy (kcal/mol) Reaction Coordinate: O3′-H3T-Asp429:OD1 Start End H3T is equidistant from O3′ and Asp:OD1 H3T rotates to Asp:OD1; O3′-Pα increases O3′-cat Mg decreases O3′-Pα distance increases as H3T rotates toward Asp429, but then decreases as proton is transferred

9 Proton Transfer to Water Energy (kcal/mol) Reaction Coordinate: O3′-H3T-Water1:OH2 Start End O3′-Pα distance increases until H3T rotates away from O5′ toward Water1 H3T breaks away from O5′ Cat Mg – O3′ distance decreases

10 Future Work – Build New Models Continue following reaction pathways following proton transfer and O3′ attack Improve starting geometry (e.g., using models at left) Refine favored pathways with a larger basis set and smaller step size Consider simultaneous proton transfer and O3’ attack Proton transfer alone causes rearrangement of the catalytic ion D490 Wat H3T toward Wat1H3T toward Asp490

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12 Possible Step 2 – to Another Water Molecule Energy (kcal/mol) Rxn Coordinate: Wat1:OH2-Wat1:H1-Wat2:OH2 Start End

13 Possible Step 2 – O3′ Attack on Pα Energy (kcal/mol) Reaction Coordinate: O3′-Pα-O3A Start End

14 Pol β 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 with 21.5 kcal/mol higher energy than the reactant Lin, Pedersen, Batra, Beard, Wilson, Pedersen, 2006, PNAS 103:

15 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

16 Alberts & Schlick 2006

17 Transition State Theory Insertion rate constant of reaction = k pol k pol = vexp[−ΔG ‡ /RT] At 25°C, v = 6.212x10 12 s −1 (v = kT/h)