Unraveling functional hole hopping pathways in the - PowerPoint PPT Presentation

Unraveling functional hole hopping pathways in the [4Fe4S]-containing DNA primase “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 3+ cl [F [Fe 4 S 4 ] 2+ cluster and EH EHPath.py.” .” 2+/3+ Darius Teo, Ph.D. candidate Beratan group, Duke University

Eme Emergi ging g rol oles of of Fe-S S cluster enzym ymes in DNA NA replication and repair Handover of the primer from p58c of primase to p180core of Polα. Fuss, J. O. et al., 2015. Biochim. Biophys. Acta -Molecular Cell Research , 1853 (6), pp.1253-1271.

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

Pr Proposed mechanism of primer handoff drive ven by DNA charge transf sfer O’Brien, E et al., 2017. Science , 355 (6327), p.eaag1789.

DN DNA-bin indin ing, ch char arge tr tran ansfer–deficie icient t p58C 58C (prim imas ase) mutan ants ts How does the mutation affect RNA/DNA-protein binding and charge transfer rates? PDB 5F0Q O’Brien, E et al., 2017. Science , 355 (6327), p.eaag1789.

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

Br Brok oken-sy symmetry method Kitagawa, Y. et al., 2018. In Symmetry (Group Theory) and Mathematical Treatment in Chemistry .

Mo Mode deling ng and nd comput putationa nal setup up B3LYP/6-31G**, COSMO PDB 5F0Q Charge = -2 Charge = -1 S = 9/2 S = 9/2 Charge = -1 Charge = -1 S = 4 S = 9/2 Fe-coordinated Cys are Fe 4 S 43+ Fe 4 S 42+ included in the treatment but not shown here 6 assignments 3 assignments

Structural comparison of Fe 4 S 43+ St 3+ DF DFT stru tructu tures es with th crystal stru tructu ture Structures RMSD (Å) 1 3+ 0.283 [Fe 4 S 4 ] cluster of primase was likely crystallized in the oxidized state of 2 3+ 0.278 3+, as the (aerobic) sitting-drop vapor 3 3+ 0.258 diffusion protocol was utilized and 4 3+ 0.311 generated needle-like prisms over 2-4 days. 5 3+ 0.279 6 3+ 0.307

Ov Overvie iew of of for orce ce fie ield ld par aram ameters r 2 - Bonds k r ( r r 0 ) Sources of parameters: 2 q - q q Angles k ( 0 ) •Gas-phase QM q •Macroscopic (cos j Torsions å properties via liquid k n ) n n state simulation, e.g., f density, heat capacity, Nonbonded: é ù compressibility (esp. 12 6 æ ö æ ö s s N ij ij ê ç ÷ ç ÷ ú e - OPLS) Lennard-Jones ij ç ÷ ç ÷ ê ú r r H è ø è ø •Spectroscopic and ij ij ë û crystallographic data q q (small molecules) O i j Electrostatic r C ij All-Atom Force Fields: e.g., CHARMM, AMBER, OPLS, GROMOS Matt Jacobson, UCSF

Bon ond an and an angle le for orce ce con onstan ants ts Vi Visual Force Field De Fi Derivation Toolkit (VFFDT) To Seminario, J.M., 1996. Int. J. Quantum Chem. , 60 (7), pp.1271-1277. Zheng, S. et al., 2016. J. Chem. Inf. Model. , 56 (4), pp.811-818.

12-6 12 6 Lennar ard-Jo Jone nes pa parameters • Dispersion and short-range repulsion are then combined in the Lennard-Jones formula: A/r 12 – B/r 6 • LJ parameters are scaled according to formal charges of Fe in the cluster • i.e., Fe 2.5+ parameters are derived as the average of the Fe 2+ and Fe 3+ parameters RE RESP Char arges: B3LYP/6-31G* in order for compatibility with ff99SB 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.

3+ cl Va Validation of force field parameters for the [4Fe4S] 3+ cluster Using ‘average’ Us ’ parameters, 0.2 3 2 RMSD (Å) RMSD (Å) 0.1 1 0 0 0 20 40 60 80 100 0 20 40 60 80 100 Time (ns) Time (ns) Cl Cluster r + Protein + DNA NA Cl Cluster

EHPath.py

Char Charge trans ansfer be between n do dono nor and and acce acceptor e - D A h + k DA = ? 15 of 8

Marcus Ma s theory of charge transfer / ∆1°34 56 7 𝑙 "# = 2π 1 ) e 84 56 9 : ; 𝑊 "# ħ 4π𝜇 "# 𝑈 𝑊 "# - electronic coupling , decays with donor/acceptor distance. ∆𝐻° - free energy change of the CT reaction. 𝜇 "# - reorganization energy, depends on changes of solvation and donor/acceptor geometries upon CT. 16

Kin Kinetic tic mod odel l an and mean an resid idence ce tim time B/E 𝑙 G→G/C B B/C B/?/C 1 1 𝜐 = > 𝜐 ? = > > F + 1 + 𝑙 ?→?3C 𝑙 G→G3C 𝑙 B→B3C ?@A ?@A E@A G@?3C B Forward & Backward Forward only 1 𝜐 IJJKLM ≅ > Donor Bridge 𝑙 ?→?3C Acceptor ?@A Teo, R. D. et. al, 2019. Chem , 5 (1), pp.122-137.

Pa Pathway analysis in wi wild-ty type p5 p58c-DN DNA/RN RNA usin ing EH EHPath.py At 20 ns, At Using MD snapshots fr Us from 20 - 100ns 100ns, W3 W327 Top hopping pathways % of pathways Y309 Y309 [4Fe4S]-DA7 1.2 [4Fe4S]-Y309-DA7 54.3 [4Fe4S]-Y309-M307-DA7 33.3 [4 [4Fe4S] M307 M3 [4Fe4S]-Y309-W327-DA7 11.1 DA DA7

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

Free energy calcu alcula lation tions usin ing MMP MMPBSA.p .py • E gas – molecular mechanical energies (bonded, electrostatic, VDW) • ΔG solvation – polar (implicit solvent models) and non-polar • S solute – vibrational contribution calculated by normal mode analysis or quasi-harmonic approximation • Single trajectory protocol (STP) Miller III, B.R. et. al. JCTC , 8 (9), pp.3314-3321.

[4 [4Fe4S] S] 3+ 3+ -DNA DNA/RNA NA binding free ee en ener ergy (MM/PB PBSA SA) Differences (Complex - Receptor - Ligand): Energy Component Average Std. Dev. Std. Err. of Mean ------------------------------------------------------------------------------- VDWAALS -120.9776 8.1879 1.1465 EEL -3093.7918 82.3014 11.5245 EPB 3078.1227 80.0224 11.2054 ENPOLAR -12.2814 0.5799 0.0812 EDISPER 0.0000 0.0000 0.0000 DELTA G gas -3214.7693 82.0505 11.4894 DELTA G solv 3065.8414 79.9099 11.1896 DELTA TOTAL -148.9280 9.9197 1.3890 ------------------------------------------------------------------------------- Using Quasi-harmonic Entropy Approximation: DELTA G binding = -7.8911 -------------------------------------------------------------------------------

Ackn cknowled edgemen ements • Professor David Beratan • Professor Agostino Migliore • Dr. Victor Anisimov • Beratan group • Dr. Tomasz Janowski • Tom Milledge • Blue Waters and NCSA staff

Thank you for your attention!