Robert Deller , Rebecca Notman & Kostas Thalassinos .
description
Transcript of Robert Deller , Rebecca Notman & Kostas Thalassinos .
![Page 1: Robert Deller , Rebecca Notman & Kostas Thalassinos .](https://reader034.fdocuments.in/reader034/viewer/2022042822/56815fc0550346895dcebc50/html5/thumbnails/1.jpg)
Molecular Dynamics investigation of DNA-Protein
interactions involved in transcriptional regulation.
Robert Deller, Rebecca Notman & Kostas Thalassinos.
![Page 2: Robert Deller , Rebecca Notman & Kostas Thalassinos .](https://reader034.fdocuments.in/reader034/viewer/2022042822/56815fc0550346895dcebc50/html5/thumbnails/2.jpg)
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.
![Page 3: Robert Deller , Rebecca Notman & Kostas Thalassinos .](https://reader034.fdocuments.in/reader034/viewer/2022042822/56815fc0550346895dcebc50/html5/thumbnails/3.jpg)
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 **Unpublished data from Frances Kondrat, and co-workers, Biological Sciences, University of Warwick.
![Page 4: Robert Deller , Rebecca Notman & Kostas Thalassinos .](https://reader034.fdocuments.in/reader034/viewer/2022042822/56815fc0550346895dcebc50/html5/thumbnails/4.jpg)
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.
![Page 5: Robert Deller , Rebecca Notman & Kostas Thalassinos .](https://reader034.fdocuments.in/reader034/viewer/2022042822/56815fc0550346895dcebc50/html5/thumbnails/5.jpg)
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.
![Page 6: Robert Deller , Rebecca Notman & Kostas Thalassinos .](https://reader034.fdocuments.in/reader034/viewer/2022042822/56815fc0550346895dcebc50/html5/thumbnails/6.jpg)
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.
![Page 7: Robert Deller , Rebecca Notman & Kostas Thalassinos .](https://reader034.fdocuments.in/reader034/viewer/2022042822/56815fc0550346895dcebc50/html5/thumbnails/7.jpg)
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.
![Page 8: Robert Deller , Rebecca Notman & Kostas Thalassinos .](https://reader034.fdocuments.in/reader034/viewer/2022042822/56815fc0550346895dcebc50/html5/thumbnails/8.jpg)
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.
![Page 9: Robert Deller , Rebecca Notman & Kostas Thalassinos .](https://reader034.fdocuments.in/reader034/viewer/2022042822/56815fc0550346895dcebc50/html5/thumbnails/9.jpg)
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.
![Page 10: Robert Deller , Rebecca Notman & Kostas Thalassinos .](https://reader034.fdocuments.in/reader034/viewer/2022042822/56815fc0550346895dcebc50/html5/thumbnails/10.jpg)
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 ns2 ns
4 ns6 ns
![Page 11: Robert Deller , Rebecca Notman & Kostas Thalassinos .](https://reader034.fdocuments.in/reader034/viewer/2022042822/56815fc0550346895dcebc50/html5/thumbnails/11.jpg)
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.
![Page 12: Robert Deller , Rebecca Notman & Kostas Thalassinos .](https://reader034.fdocuments.in/reader034/viewer/2022042822/56815fc0550346895dcebc50/html5/thumbnails/12.jpg)
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+.
![Page 13: Robert Deller , Rebecca Notman & Kostas Thalassinos .](https://reader034.fdocuments.in/reader034/viewer/2022042822/56815fc0550346895dcebc50/html5/thumbnails/13.jpg)
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.
![Page 14: Robert Deller , Rebecca Notman & Kostas Thalassinos .](https://reader034.fdocuments.in/reader034/viewer/2022042822/56815fc0550346895dcebc50/html5/thumbnails/14.jpg)
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.
![Page 15: Robert Deller , Rebecca Notman & Kostas Thalassinos .](https://reader034.fdocuments.in/reader034/viewer/2022042822/56815fc0550346895dcebc50/html5/thumbnails/15.jpg)
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.
![Page 16: Robert Deller , Rebecca Notman & Kostas Thalassinos .](https://reader034.fdocuments.in/reader034/viewer/2022042822/56815fc0550346895dcebc50/html5/thumbnails/16.jpg)
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).
![Page 17: Robert Deller , Rebecca Notman & Kostas Thalassinos .](https://reader034.fdocuments.in/reader034/viewer/2022042822/56815fc0550346895dcebc50/html5/thumbnails/17.jpg)
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