2. Introduction. Zn 2+ homeostasis is regulated at the transcriptional level by the DNA-binding protein SmtB. Manipulation of Zn 2+ 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 Zn 2+ & interactions between SmtB & DNA.

3. Currently Proposed Mechanism. SmtB DNA SmtA SmtB DNA SmtA Zn 2+ DNA SmtA SmtB Zn 2+  SmtB bound to DNA in low Zn 2+ ion levels.  SmtA removes free Zn 2+ ions.  Zn 2+ ions bind to SmtB.  Zn 2+ ion levels increase.  Zn 2+ ions bind to SmtB inducing dissociation.  SmtA is synthesized. Proposed mechanism derived from experimental observations. **Unpublished data from Frances Kondrat, and co-workers, Biological Sciences, University of Warwick.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 Zn 2+ ions in line with experimental observations. Key residues identified as Cys-61 & His-97. Each model is a dimer comprising one half of the overall SmtB tetrameric structure. Apoprotein SmtB 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 Zn 2+ 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 Zn 2+ systems modeled for 10 ns. Movement of Zn 2+ ions monitored. SmtB 1 Zn 2+ simulation. SmtB Apoprotein simulation.

10. DNA & Protein Production Runs. Apoprotein system modeled for 10 ns. 1 & 2 Zn 2+ systems modeled for 8 ns & 5 ns respectively. Movement of Zn 2+ ions monitored. Assess effect of DNA upon protein structure. Assess whether simulations agree with experimental evidence. Snapshots of 1 Zn 2+ 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 C α. Suppression of residues surrounding the His-97 by DNA & Zn 2+.

13. H-bonding. Some hydrogen bond formation in the Apoprotein & 1 Zn 2+ systems. Many other types of analysis were employed to assess the properties & interactions of DNA, Zn 2+ & protein.

14. Further Work. Employ full 26bp DNA model in aforementioned systems. Use different orientation & positioning of DNA with respect to protein. Identify the role of Zn 2+ in more detail. Employ the 26 bp DNA model in each aforementioned system. Alternative DNA orientation and positioning. Application to other transcriptional regulation systems. DNA 26 bp model.

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 Zn 2+ ions. More hydrogen bond formation between DNA & Apoprotein than Zn 2+ bound, supporting the proposed mechanism. Snapshot of 14 bp DNA & 1 Zn 2+ Protein model after 7 ns simulation.

16. Acknowledgements. Dr. Rebecca Notman. Dr. Kostas Thalassinos. 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 cyanobacterial metallothionein 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