Exploring the Possible Pathways of DNA Polymerase λ ’s Nucleotidyl Transfer Reaction

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

2. 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

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 Semi-Fixed Fixed 13 Å 7 Å Free

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 Pα-O3A breaks Start H3T is closer to D490:OD1 than O5′ Energy (kcal/mol) End D490 Reaction Coordinate: O3’-Pa-O3A • 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)

6. Proton Transfer to Asp490 Start H3T breaks from O5′ O3′-Pα ↑ cat Mg-O1A ↓ O3′-cat Mg ↑ O3′-cat Mg ↓ cat Mg-O1A ↓ Energy (kcal/mol) O3′-Pα increases End 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

7. Proton Transfer to dTTP:O2A Start H3T equidistant from O3′ and O2A H3T moving away from O5′ Energy (kcal/mol) End Reaction Coordinate: O3’-H3T-dTTP: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 Start H3T is equidistant from O3′ and Asp:OD1 Energy (kcal/mol) H3T rotates to Asp:OD1; O3′-Pα increases O3′-cat Mg decreases End Reaction Coordinate: O3′-H3T-Asp429:OD1 • O3′-Pα distance increases as H3T rotates toward Asp429, but then decreases as proton is transferred

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

10. Future Work – Build New Models H3T toward Wat1 • 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 H3T toward Asp490 2.8 3.0 1.6 1.65 2.1 Wat1 2.1 2.2 2.1 D490

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

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

14. 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 with 21.5 kcal/mol higher energy than the reactant Lin, Pedersen, Batra, Beard, Wilson, Pedersen, 2006, PNAS 103:13294-13299

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 = kpol • kpol = vexp[−ΔG‡/RT] • At 25°C, v = 6.212x1012s−1 (v = kT/h)