1. SMA5422: Special Topics in Biotechnology Lecture 9: Computer modeling of biomolecules: Structure, motion, and binding. Chen Yu Zong Department of Computational Science National University of Singapore Singapore 119260

2. Key Points Protein and DNA structure Protein and DNA internal motions and functional relevance. Computer modeling strategies. Examples: protein transient opening and pathways for functional motions. Current development and and future applications.

3. Is protein structure static? Crystal X-ray structure of a calcium binding protein

4. Crystal X-ray structure of a calcium binding protein: Ribbon presentation

5. Crystal X-ray structure of a protein in two different conformations: Indication of a hinge motion

6. Crystal X-ray structure of a DNA double helix as part of a DNA-protein complex: Indication of a base flipping motion

7. Observation of protein internal motions:

8. Functionally Important Motions Hinge Motions and domain movement in Proteins: –Conformation change between active and inactive form, or for site opening and closure. –Protein activation or inactivation by signalling chemicals. –Energy-coupled motors X-ray crystallography indicates: –Many hinge motions involve local bond rotations (4 to 8 amino acid residues). Biochemistry 33:6739 (1994)

9. Observation of protein internal motions: Protein domain motion Related database

10. Functional Roles: Transient opening or closure for ligand binding and dissociation. Creation of transient escape channel. Exposure of active site. ATP driven protein movement. Binding induced protein closure.

11. Functionally Important Motions in DNA DNA Base flipping is essential for: –DNA methylation –DNA repair –Mismatch recognition –Initiation of transcription and replication X-ray crystallography indicates: –Base flipping involves localised bond rotation. Cell 82, 9 (1995).

12. Modeling Strategies: Molecular dynamics. Time-dependent motion trajectory based on laws of classical physics. Advantage: "Accurate" dynamics. Disadvantage: Short-time event only. Application: "All purpose", most widely used approach. Curr. Opin. Struct. Biol. 6, 232 (1996).

13. Modeling Strategies: Stochastic Dynamics. Solvent represented by frictional and stochastic forces. Advantage: Large scale motions, simplified treatment. Disadvantage: Limited applicability. Application: Protein transport, Domain motion, side-chain swing. Ann. Rev. Biochem 53, 263 (1983)

14. Modeling Strategies: Harmonic Dynamics. "Real" forces approximated by Hooke forces. Advantage: Long time dynamics, Completely solvable. Disadvantage: Restricted to vibration-like motions. Application: Force field, spectroscopy and theoretical analysis. Curr. Opin. Struct. Biol. 4, 285 (1994)

15. Modeling Strategies: Simplified Model Dynamics. Simplified structure or forces (e.g. HP lattice, spin glass model). Advantage: Long time and large scale dynamics. Disadvantage: Loss of important features. Application: Protein folding, mechanical models, solitons. Proc. Natl. Acad. Sci. USA 90, 1942 (1993); 89, 4918 (1992)

16. Modeling Strategies: Energy Landscape Along Motion Pathways. Generation of motion pathways by bond rotations. Molecular mechanics energy analysis. Advantage: Complete motion trajectories. Disadvantage: Limited to “straightforward” motions. Application: DNA base opening, base flipping motions. Phys. Rev. EPhys. Rev. E62, 1133-1137 (2000).

17. Molecular Dynamics: Basic Assumptions: Atoms interact with each other by empirical forces. Their motions governed by Newton's law.

18. Molecular Dynamics: Basic Equations: Forces = bond stretch + angle bending + torsion angle distortion + hydrogen bonding + van de Waals + electrostatic forces + hydrophobic + other solvent interactions Newton's law: Forces = mass x acceleration (F=ma)

19. Molecular Dynamics: Initial conditions at time t: r(t), v(t-dt/2) Atomic trajectory at t+dt: a(t) = F(r(t))/m v(t+dt/2) = v(t-dt/2) + a(t) dt r(t+dt) = r(t) + v(t+dt/2) dt

20. Simulation of open "back door" in acetylcholinesterase: Science 263, 1276 (1994) Why interested in this protein? Key role in signal control in nervous system. Target of Chinese natural product (Qian Ceng Ta). Nature Struct. Biol. 4, 57-62 (1997)

21. Simulation of open "back door" in acetylcholinesterase: Science 263, 1276 (1994) Special features: Active site is in a deep and narrow gorge. Strong electrostatic force attracts substrate into the gorge.

22. Simulation of open "back door" in acetylcholinesterase: Science 263, 1276 (1994) Question: How can a substrate and water escape from the active site?

23. Pathways for Functionally Important Motions DNA base flipping mechanism: –Enzyme induced?. –Enzyme captures a transiently opened base? X-ray crystallogaphy: –Structural information on both end of pathway. –The intermediate path remains unclear Curr. Opin. Struct. Biol. 7, 103 (1997) Cell 82, 9 (1995)

24. Mechanism of base flipping Scenario I: Protein induced flipping? Major groove blocked by enzyme, minor groove pathway? May explain why flipped base orients towards minor groove.. Cell 76, 357-369 (1994) Cell 82, 9-12 (1995)

25. Mechanism of base flipping Scenario II: Enzyme recognises and traps a transiently flipped base? Structure 2, 79 (1994). Base pair life time comparable to methyltransferase reaction time. All modelling studies consistently show base opens via major groove, yet to show how minor groove opening is possible. Proc. Natl. Acad. Sci. USA. 85, 7231 (1988) J. Biomol. Struct. Dyn. 15, 765 (1998)

26. Objective: –Major groove or minor groove pathway? Enzyme-captured or enzyme-induced flipping? Flipping motion and pathway modelling: –Identification of key rotatable bonds whose motions constructive to flipping. –Modelling of collective rotations of all bonds. Energy cost of motion. –Energy barrier along pathway computed by standard force fields. Testing Scenarios by Molecular Modelling

27. Modelling Strategy Phys. Rev. EPhys. Rev. E62, 1133-1137 (2000).

28. Modelling Strategy

29. Energy Functions in Molecular Mechanics Potential Energy Description: –Torsion (bond rotation) –Hydrogen bonding –van der Waals interactions –Electrostatic interactions –Empirical solvation free energy V =  torsions 1/2 V n [ 1 + cos(n  -  ') ] +  H bonds [ V 0 (1-e -a(r-r0) ) 2 - V 0 ] +  non bonded [ A ij /r ij 12 - B ij /r ij 6 + q i q j /  r r ij ] +  atoms i  i A i

30. Modelling Results on DNA Base Flipping

31. Energy Barrier for Base Flipping Phys. Rev. EPhys. Rev. E62, 1133-1137 (2000).

32. Maximum Extent of Base Flipping Along Both Grooves System NDB ID Flipped D major d minor Base (A) (A) Hhal Mtase GATAGCGCTATC PDEB08C1811.894.87 HhaI MTase CCATGCGCTGAC PDEB123C1812.253.23 Hhal Mtase GTCAGCGCATGG PD0017A1811.374.42 HeaIII Mtase ACCAGCAGGCCACCAGTG PDEB19C1012.011.23 B-form CCGGCGGCCGG BDL039C512.132.13 B-form CGCGAATTCGCG BDL001A514.171.47 B-form ACCGGCGCACA BDL035 C7 12.18 2.02 Phys. Rev. EPhys. Rev. E62, 1133-1137 (2000).

33. Current and Future Development: Challenge: Time -scale gap problem

34. Current and Future Development: Time-saving techniques in development: