introduction. zn 2+ homeostasis is regulated at the transcriptional level by the dna-binding protein...
TRANSCRIPT
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Molecular Dynamics investigation of DNA-Protein
interactions involved in transcriptional regulation.
Robert Deller, Rebecca Notman & Kostas Thalassinos.
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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.
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Currently Proposed Mechanism.
SmtB
DNASmtA
SmtB
DNASmtA Zn2+
DNASmtA
SmtB
Zn2+
SmtB bound to DNA in low Zn2+ ion levels.
SmtA removes free Zn2+ ions.Zn2+ ions bind to SmtB.
Zn2+ ion levels increase.Zn2+ 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**Unpublished data from Frances Kondrat, and co-workers, Biological Sciences, University of Warwick.
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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 dimer comprising one half of the overall SmtB tetrameric structure. Apoprotein SmtB model.
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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.
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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.
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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.
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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.
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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. SmtB 1 Zn2+ simulation.
SmtB Apoprotein simulation.
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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 ns6 ns
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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.
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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+.
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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.
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Further Work.
Employ full 26bp DNA model in aforementioned systems.
Use different orientation & positioning of DNA with respect to protein.
• Identify the role of Zn2+ 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.
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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.
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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).
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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