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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
Computational Study of Escherichia coliSignal Recognition Particle GTPases

BZH

Kelly Elkins

GPBM 2002: June 25, 2002


Why modelling
Why Modelling?

GTACTTACCCTAGTAC

CATGAATGGGATCATG

?

Gene Structure Function


Outline
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


Signal recognition particle
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.


E coli ffh srp model
E. coli Ffh SRP model

Comparative Modelling

pdb structures: www.rcsb.org/pdb/


Ffh model
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.


Ffh model1
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


Evaluation of a proposed protein protein interaction model
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


Gtp mg 2 docking
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.


Gtp mg 2 docking1
GTP-Mg2+ Docking

GTP-Mg2+ docked to Ffh


Evaluation of a proposed protein protein interaction model1
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



Future work
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


Conclusions
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


Acknowledgements
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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