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Meredith

VIEWS: 2 PAGES: 17

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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

								
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