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

FixedSemi-Fixed

Free

7 Å

13 Å

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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

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O3′ Attack on Pα

-2331400

-2331380

-2331360

-2331340

-2331320

-2331300

-2331280

-2331260

-2331240

-2331220

-2331200

-2331180

-2331160

-2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5

O3'-Pa-O3A

En

erg

y (k

cal/m

ol)

Reaction Coordinate: O3’-Pa-O3A

Ene

rgy

(kca

l/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

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Proton Transfer to Asp490

-2331340

-2331330

-2331320

-2331310

-2331300

-2331290

-2331280

-2331270

-2331260

-2331250

-2331240

-2331230

-2331220

-2331210

-2331200

-2331190

-2331180

-3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5

Ene

rgy

(kca

l/mol

)

Reaction Coordinate: O3′-H3T-Asp490:OD1

Start

EndO3′-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

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Proton Transfer to dTTP:O2A

-2331280

-2331270

-2331260

-2331250

-2331240

-2331230

-2331220

-2331210

-2331200

-2331190

-2331180

-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5

Ene

rgy

(kca

l/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

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Proton Transfer to Asp429

-2331440

-2331420

-2331400

-2331380

-2331360

-2331340

-2331320

-2331300

-2331280

-2331260

-2331240

-2331220

-2331200

-2331180

-2331160

-2 -1.5 -1 -0.5 0 0.5 1 1.5

Ene

rgy

(kca

l/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

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Proton Transfer to Water

-2331310

-2331300

-2331290

-2331280

-2331270

-2331260

-2331250

-2331240

-2331230

-2331220

-2331210

-2331200

-2331190

-2331180

-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5

Ene

rgy

(kca

l/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

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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

Wat1

2.8 3.01.6

1.652.2 2.1

2.12.1

H3T toward Wat1 H3T toward Asp490

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12

Possible Step 2 – to Another Water Molecule

-2331306

-2331304

-2331302

-2331300

-2331298

-2331296

-2331294

-2331292

0 0.2 0.4 0.6 0.8 1 1.2

Ene

rgy

(kca

l/mol

)Rxn Coordinate: Wat1:OH2-Wat1:H1-Wat2:OH2

StartEnd

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Possible Step 2 – O3′ Attack on Pα

-2331340

-2331330

-2331320

-2331310

-2331300

-2331290

-2331280

-2331270

-2331260

-2331250

-2 -1.5 -1 -0.5 0 0.5 1 1.5 2

Ene

rgy

(kca

l/mol

)

Reaction Coordinate: O3′-Pα-O3A

Start

End

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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

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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

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Alberts & Schlick 2006

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Transition State Theory

• Insertion rate constant of reaction = kpol

• kpol = vexp[−ΔG‡/RT]

• At 25°C, v = 6.212x1012s−1 (v = kT/h)