[PPT] - Unraveling functional hole hopping pathways in the PowerPoint Presentation

SLIDE 1

Unraveling functional hole hopping pathways in the [4Fe4S]-containing DNA primase

Darius Teo, Ph.D. candidate Beratan group, Duke University

“B “Blue ue Wat aters rs has has enabl nabled d me me to de develop p forc rce field d parame parameters rs for r [F [Fe4S4]2+

2+/3+ 3+ cl

cluster and EH EHPath.py.” .”

SLIDE 2

Eme Emergi ging g rol

les of
f Fe-S

S cluster enzym ymes in DNA NA replication and repair

Fuss, J. O. et al., 2015. Biochim. Biophys. Acta -Molecular Cell Research, 1853(6), pp.1253-1271.

Handover of the primer from p58c of primase to p180core of Polα.

SLIDE 3

Perera, R.L., Torella, R., Klinge, S., Kilkenny, M.L., Maman, J.D. and Pellegrini, L., 2013. Elife, 2.

RN RNA-DN DNA A pri rimer er synth thes esis duri ring DN DNA A rep eplicati tion of th the e lagging str trand

SLIDE 4

Pr Proposed mechanism of primer handoff drive ven by DNA charge transf sfer

O’Brien, E et al., 2017. Science, 355(6327), p.eaag1789.

SLIDE 5

DN DNA-bin indin ing, ch char arge tr tran ansfer–deficie icient t p58C 58C (prim imas ase) mutan ants ts

O’Brien, E et al., 2017. Science, 355(6327), p.eaag1789.

How does the mutation affect RNA/DNA-protein binding and charge transfer rates?

PDB 5F0Q

SLIDE 6

Objectives

1) Develop AMBER force field parameters for the [4Fe4S] cluster in 2+/3+ state.

Broken-symmetry DFT for geometry optimization
Generate force constants and RESP charges
Validate parameters using MD simulations

2) Charge transfer pathway analysis using a hopping program

EHPath.py

3) Examine binding between primase and RNA/DNA duplex

MMPBSA.py

SLIDE 7

Br Brok

ken-sy

symmetry method

Kitagawa, Y. et al., 2018. In Symmetry (Group Theory) and Mathematical Treatment in Chemistry.

SLIDE 8

Mo Mode deling ng and nd comput putationa nal setup up

Fe4S43+

Charge = -2 S = 9/2 Charge = -1 S = 4

PDB 5F0Q

Charge = -1 S = 9/2 Charge = -1 S = 9/2

Fe4S42+

6 assignments 3 assignments

B3LYP/6-31G**, COSMO

Fe-coordinated Cys are included in the treatment but not shown here

SLIDE 9

Structures RMSD (Å) 13+ 0.283 23+ 0.278 33+ 0.258 43+ 0.311 53+ 0.279 63+ 0.307

St Structural comparison of Fe4S43+

3+ DF

DFT stru tructu tures es with th crystal stru tructu ture

[Fe4S4] cluster of primase was likely crystallized in the oxidized state of 3+, as the (aerobic) sitting-drop vapor diffusion protocol was utilized and generated needle-like prisms over 2-4 days.

SLIDE 10

Ov Overvie iew of

f for
rce

ce fie ield ld par aram ameters

All-Atom Force Fields: e.g., CHARMM, AMBER, OPLS, GROMOS

2 0)

( r r kr

Bonds

2 0)

( q q

q

k

å

n n

n k ) (cos j Angles Torsions Nonbonded: Lennard-Jones

ij j i

r q q ú ú û ù ê ê ë é ÷ ÷ ø ö ç ç è æ

÷

÷ ø ö ç ç è æ

6 12 ij ij ij ij ij

r r s s e Electrostatic Sources of parameters:

Gas-phase QM
Macroscopic

properties via liquid state simulation, e.g., density, heat capacity, compressibility (esp. OPLS)

Spectroscopic and

crystallographic data (small molecules) q r f H N C O Matt Jacobson, UCSF

SLIDE 11

Seminario, J.M., 1996. Int. J. Quantum Chem., 60(7), pp.1271-1277.

Vi Visual Force Fi Field De Derivation To Toolkit (VFFDT)

Zheng, S. et al., 2016. J. Chem. Inf. Model., 56(4), pp.811-818.

Bon

nd an

and an angle le for

rce

ce con

nstan

ants ts

SLIDE 12

Dispersion and short-range

repulsion are then combined in the Lennard-Jones formula: A/r12 – B/r6

LJ parameters are scaled

according to formal charges

f Fe in the cluster
i.e., Fe2.5+ parameters are

derived as the average of the Fe2+ and Fe3+ parameters

Li, P. et al., 2013. JCTC, 9(6), pp.2733-2748. Li, P. et al., 2014. J. Phys. Chem. B, 119(3), pp.883-895.

12 12-6 6 Lennar ard-Jo Jone nes pa parameters

B3LYP/6-31G* in order for compatibility with ff99SB

RE RESP Char arges:

SLIDE 13

Va Validation of force field parameters for the [4Fe4S]3+

3+ cl

cluster

Cl Cluster r + Protein + DNA NA Us Using ‘average’ ’ parameters, Cl Cluster

1 2 3 20 40 60 80 100

RMSD (Å) Time (ns)

0.1 0.2 20 40 60 80 100

RMSD (Å) Time (ns)

SLIDE 14

EHPath.py

SLIDE 15

D A e- h+ kDA = ?

15 of 8

Char Charge trans ansfer be between n do dono nor and and acce acceptor

SLIDE 16

𝑙"# = 2π ħ 𝑊

"# )

1 4π𝜇"#𝑈 e

/ ∆1°3456 7 84569:;

16

𝜇"# - reorganization energy, depends on changes of solvation and donor/acceptor geometries upon CT. ∆𝐻° - free energy change of the CT reaction. 𝑊

"# - electronic coupling, decays with donor/acceptor distance.

Ma Marcus s theory of charge transfer

SLIDE 17

Forward & Backward Forward only Donor Bridge Acceptor

𝜐 = >

?@A B

𝜐? = >

?@A B/C

1 𝑙?→?3C >

E@A B/?/C

F

G@?3C B/E 𝑙G→G/C

𝑙G→G3C + 1 + 1 𝑙B→B3C 𝜐IJJKLM ≅ >

?@A B

1 𝑙?→?3C

Teo, R. D. et. al, 2019. Chem, 5(1), pp.122-137.

Kin Kinetic tic mod

del

l an and mean an resid idence ce tim time

SLIDE 18

Pa Pathway analysis in wi wild-ty type p5 p58c-DN DNA/RN RNA usin ing EH EHPath.py

Top hopping pathways % of pathways [4Fe4S]-DA7 1.2 [4Fe4S]-Y309-DA7 54.3 [4Fe4S]-Y309-M307-DA7 33.3 [4Fe4S]-Y309-W327-DA7 11.1

Us Using MD snapshots fr from 20 - 100ns 100ns, At At 20 ns, [4 [4Fe4S] Y309 Y309 W3 W327 M3 M307 DA DA7

SLIDE 19

MMPBSA.py

Miller III, B.R. et. al. JCTC, 8(9), pp.3314-3321.

SLIDE 20

Free energy calcu alcula lation tions usin ing MMP MMPBSA.p .py

Egas – molecular mechanical energies (bonded, electrostatic, VDW)
ΔGsolvation – polar (implicit solvent models) and non-polar
Ssolute – vibrational contribution calculated by normal mode analysis
r quasi-harmonic approximation
Single trajectory protocol (STP)