Robert Deller , Rebecca Notman & Kostas Thalassinos .

1. Molecular Dynamics investigation of DNA-Protein interactions involved in transcriptional regulation. Robert Deller, Rebecca Notman & Kostas Thalassinos.

2. Introduction. • Zn2+ homeostasis is regulated at the transcriptional level by the DNA-binding protein SmtB. • Manipulation of Zn2+ homeostasis could act as a potent anti-microbial mechanism. • Molecular dynamics provides a method of exploring the interactions between DNA & protein. • Investigate the role of Zn2+ & interactions between SmtB & DNA.

3. DNA DNA DNA SmtA SmtA SmtB SmtA SmtB Zn2+ Zn2+ Currently Proposed Mechanism. Proposed mechanism derived from experimental observations. SmtB bound to DNA in low Zn2+ ion levels. Zn2+ ion levels increase. Zn2+ ions bind to SmtBinducing dissociation. SmtA is synthesized. SmtB SmtA removes free Zn2+ ions. Zn2+ ions bind to SmtB. **Unpublished data from Frances Kondrat, and co-workers, Biological Sciences, University of Warwick.

4. Protein Models. • Three protein models based upon two existing PDB structures (1R1T & 1R23). • Each model contains either 0, 1 or 2 Zn2+ ions in line with experimental observations. • Key residues identified as Cys-61 & His-97. • Each model is a dimercomprising one half of the overall SmtB tetramericstructure. ApoproteinSmtB model.

5. DNA Models. • Previously identified 14 bp & 26 bp sequences suspected to be the binding sites of SmtB. • 14 bp & 26 bp sequences created and equilibrated. • 14 bp sequence (6-2-6 inverted repeat) chosen to partake in molecular dynamics simulations. DNA 14 bp model.

6. DNA & Protein Models. • Combined 14 bp DNA model with each protein model. • DNA & protein hybrid models created using interactions predicted from experiments. • Close to maximum model size that can be simulated at appreciable rates. DNA & 1 Zn2+SmtB model.

7. Equilibration. • Equilibration comprises of three distinctive steps. • Energy minimisation to ensure the system is fully relaxed. • NVT equilibration to stabilize the temperature of the system. • NPT equilibration to stabilize the pressure (density) of the system.

8. DNA MD Production Runs. • Comparative assessment of DNA model stability in the absence of protein. • 14 bp and 26 bp DNA modeled for 12 ns & 4 ns respectively. • Act as a control for hybrid systems. • Large level of flexibility in 14 bp model reduced in 26 bp model. 14 bp DNA simulation.

9. Protein MD Production Runs. • Inherent stability of each protein in solution is assessed as a comparison for the hybrid systems. • Apoprotein system modeled for 6 ns. • 1 & 2 Zn2+ systems modeled for 10 ns. • Movement of Zn2+ ions monitored. SmtBApoprotein simulation. SmtB 1 Zn2+ simulation.

10. DNA & Protein Production Runs. • Apoprotein system modeled for 10 ns. • 1 & 2 Zn2+ systems modeled for 8 ns & 5 ns respectively. • Movement of Zn2+ ions monitored. • Assess effect of DNA upon protein structure. • Assess whether simulations agree with experimental evidence. • Snapshots of 1 Zn2+ Protein & DNA MD simulation ranging from 0 – 6 ns at 2 ns intervals. 0 ns 2 ns 4 ns 6 ns

11. RMSD. • RMSD (root mean squared deviation) of the protein backbone with reference to the starting state (after equilibration). • Small differences between DNA & Protein & Protein models. • No convergence in DNA & protein model in this time frame.

12. Radius of Gyration & RMSF. • Radius of gyration is an indicator of protein compactness. • RMSF (root mean squared fluctuation) of each Cα. • Suppression of residues surrounding the His-97 by DNA & Zn2+.

13. H-bonding. • Some hydrogen bond formation in the Apoprotein & 1 Zn2+ systems. • Many other types of analysis were employed to assess the properties & interactions of DNA, Zn2+ & protein.

14. Further Work. • Identify the role of Zn2+ in more detail. • Employ the 26 bp DNA model in each aforementioned system. • AlternativeDNA orientation and positioning. • Application to other transcriptional regulation systems. • Employ full 26bp DNA model in aforementioned systems. • Use different orientation & positioning of DNA with respect to protein. DNA 26 bpmodel.

15. Conclusions. • Limited gross changes observed in the time period assessed. • Fluctuations of several residues around His-97 reduced by the presence of DNA and/or Zn2+ ions. • More hydrogen bond formation between DNA & Apoprotein than Zn2+ bound, supporting the proposed mechanism. Snapshot of 14 bp DNA & 1 Zn2+ Protein model after 7 ns simulation.

16. Acknowledgements. • Dr. RebeccaNotman. • Dr. KostasThalassinos. • Prof. Mike Allen. • Centre for Scientific Computing (CSC). • Molecular Organisation & Assembly of Cells DTC (MOAC). • Engineering & Physical Sciences Research Council (EPSRC).

17. References. • Cook, W. J.; Kar, S. R.; Taylor, K. B.; Hall, L. M. Crystal structure of the cyanobacterialmetallothionein repressor SmtB: A model for metalloregulatory proteins, J. Mol. Biol. 1998, 275, 337-346. • MacKerell, A. D.; Nilsson, L. Molecular dynamics simulations of nucleic acid-protein complexes, Curr. Opin. Struct. Biol. 2008, 18, 194-199. • Unpublished data from Frances Kondrat, and co-workers, Biological Sciences, University of Warwick. • VanZile, M. L.; Chen, X. H.; Giedroc, D. P. Allosteric negative regulation of smt O/P binding of the zinc sensor, SmtB, by metal ions: A coupled equilibrium analysis, Biochemistry 2002, 41, 9776-9786