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Computational Study of Escherichia coli Signal Recognition Particle GTPases

Computational Study of Escherichia coli Signal Recognition Particle GTPases. BZH. Kelly Elkins. GPBM 2002: June 25, 2002. Why Modelling?. GTACTTACCCTAGTAC CATGAATGGGATCATG. ?. Gene Structure Function. Outline.

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Computational Study of Escherichia coli Signal Recognition Particle GTPases

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  1. Computational Study of Escherichia coliSignal Recognition Particle GTPases BZH Kelly Elkins GPBM 2002: June 25, 2002

  2. Why Modelling? GTACTTACCCTAGTAC CATGAATGGGATCATG ? Gene Structure Function

  3. Outline • What is the signal recognition particle? …proteins necessary for the proper export/transport of secretory and membrane proteins. A computational study of a protein-protein complex: Build a model of Ffh Evaluate proposed SRP:SR interaction model Is model valid in an apo-form? with Mg2+ bound? with GTP-Mg2+? Other possible models? Conclusions

  4. Signal Recognition Particle • Universally conserved system for protein trafficking • In humans, 6 proteins and 1 RNA • In E. coli: • 2 proteins: SRP and receptor; 1 RNA: 4.5S RNA • Both are GTPases • Ffh (SRP) 48 kDa GTPase protein • FtsY (receptor) GTPase protein 1FTS.pdb- G. Montoya, et al. (1997) Nature, 385, 365-368. 1DUL.pdb- R.T. Batey, et al. (2000) Science, 287, 1232-1239.

  5. E. coli Ffh SRP model Comparative Modelling pdb structures: www.rcsb.org/pdb/

  6. Ffh Model • Swiss Model • 1) First Approach Mode- templates: • -1JPJ.pdb (T. aquaticus Ffh • NG fragment bound GMPPNP) • -1FTS.pdb (E. coli apo- • FtsY NG fragment) • -1NG1.pdb (T. aquaticus Ffh • NG fragment bound GDP-Mg2+) • -1J8M.pdb (A. ambivalens • apo-Ffh NG fragment) • Optimize Project Mode- adjusted • sequence alignment with Swiss PDB Viewer to retain secondary structure elements • -Same templates • Checked Model with Procheck • and Whatif 1FTS.pdb- G. Montoya, et al. (1997) Nature, 385, 365-368. 1NG1.pdb- D.M. Freymann, et al. (1999) Nature Struct. Biol., 6, 793-801. 1JPJ.pdb- S. Padmanabhan, & D.M. Freymann, (2001) Structure (Camb.), 9, 859-867. 1J8M.pdb- G. Montoya, et al. (2000) Structure, 8, 515-525.

  7. Ffh Model Superimposition of the Ffh model on the 4 templates RMSD: 1NG1- 2.93 Angstroms 1J8M- 0.96 Angstroms 1JPJ- 3.04 Angstroms 1FTS- 2.94 Angstroms

  8. Evaluation of a Proposed Protein-Protein Interaction Model Proposed Model: Ffh-FtsY complex superimposed on 1N2C.pdb (nitrogenase iron protein) by structural similarity (Montoya, G., te Kaat, K., Moll, R., Schaefer, G., & Sinning, I. (2000). Structure, 8, 515-525.) Calculated the superimposed proposed model- sup2pdbs program (R.Gabdoulline)- Ca superimposition

  9. GTP-Mg2+ Docking • Superimposition • 42 GTP molecules- Protein Data Bank (23 Jan. 2002) • Superimposed GTPs with superimp program (G.M. Ullmann)- like atoms of 2 molecules are superimposed • Superimposed all GTPs into Ffh model and FtsY using 1NG1.pdb GDP as template • Mg2+ placement according to 1NG1.pdb GDP-Mg2+ structure • Energy minimized apo, Mg2+, and GTP-Mg2+ docked forms using AMBER7 • Visualize superimposed GTPs using Molsurfer • Electrostatics calculations of the complexes using UHBD with CHARMm forcefield parameters (unminimized forms) J.D. Madura, et al. (1994) Biological applications of electrostatic calculations and brownian dynamics simulations. In: “Reviews in Computational Chemistry, Volume V“, Lipkowitz, K.B., & Boyd, D. (Eds.), VCH Publishers, Inc., New York. R.R. Gabdoulline & R.C. Wade, (1997) Biophys. J., 72, 1917-1929.R.R. Gabdoulline & R.C. Wade, (2001) J. Mol. Biol., 306, 1139-1155.R.R. Gabdoulline& R.C. Wade, (1999) TIBS, 24, 285-287.

  10. GTP-Mg2+ Docking GTP-Mg2+ docked to Ffh

  11. Evaluation of a Proposed Protein-Protein Interaction Model SDA (Simulated Diffusional Association)- ab initio models unrestricted search search restricted to GTP-binding region UHBD calculations- compare electrostatic surfaces, charged regions DALI- propose other homology models (http://www2.ebi.ac.uk/dali/) Energy minimization with AMBER7- relieve bad contacts

  12. Evaluation of a Proposed Protein-Protein Interaction Model Hydrophobic patches using Molsurfer

  13. Future Work • Transform pdb coordinates and rotate according to DALI output to examine proposed alternate homology models with UHBD and Molsurfer • Evaluate ab initio models produced by SDA and by a hydrophobic patch pairing • Model the M domain of Ffh and the 4.5S RNA into the associated complex • Alternative GTP-Mg2+ placement using GRID20

  14. Conclusions • We have made a homology model of E. coli Ffh • We have docked GTP-Mg2+ to both Ffh and FtsY • The energy-minimized Ffh:FtsY complex produced by homology with the nitrogenase iron protein homodimer is not viable, but may need only small adjustments to relieve bad side- chain contacts and to obtain better hydrophobic contacts • The electrostatics calculations indicate that the charge landscapes of Ffh and FtsY are very complex and that hydrophobic residues must also mediate complex formation

  15. Acknowledgements Rebecca Wade, European Media Laboratory Irmi Sinning, University of Heidelberg Timm Essigke Razif Gabdoulline Ting Wang J. William Fulbright Foreign Scholarship Board and the German Fulbright Kommission Klaus Tschira Stiftung (KTS)

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