BZH
Computational Study of Escherichia coli
Signal Recognition Particle GTPases
Kelly Elkins
GPBM 2002: June 25, 2002
Why Modelling?
GTACTTACCCTAGTAC
CATGAATGGGATCATG
Gene
?
Structure
Function
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
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
Comparative Modelling
Protein
Organism
Percent Identity
Ffh
Escherichia coli
100
Ffh
SRP54
FtsY
Thermus aquaticus
Acidianus ambivalens
(Desulfurolobus ambivalens)
(archaebacteria)
Escherichia coli
46
.pdb Structure
M domain in 1DUL with 4.5S RNA,
homology model KFFH
1NG1, GDP-Mg2+, NG domain
2NG1,
3NG1,
1FFH,
2FFH,
1JPJ,
GDP, NG domain
apo, NG domain
apo, NG domain,
apo, whole NG+M domain, A48T
GMPPNP, NG-domain
1J8M, apo, NG domain
35
33
1FTS, apo, NG domain
pdb structures: www.rcsb.org/pdb/
Ffh Model
Alignment used for E. coli Ffh model of NG-domain
Swiss Model
1) First Approach Mode- templates:
-1JPJ.pdb (T. aquaticus Ffh
NG fragment bound GMPPNP)
-1FTS.pdb (E. coli apoFtsY NG fragment)
-1NG1.pdb (T. aquaticus Ffh
NG fragment bound GDP-Mg2+)
-1J8M.pdb (A. ambivalens
apo-Ffh NG fragment)
2)
3)
Optimize Project Mode- adjusted
sequence alignment with Swiss PDB
Viewer to retain secondary structure
elements
-Same templates
Checked Model with Procheck
and Whatif
E.
A.
T.
T.
E.
coli Ffh KFFH
ambivalens SRP54 1J8M
aquaticus Ffh 1JPJ
aquaticus Ffh 1NG1
coli FtsY 1FTS
5
3
1
1
201
LT
LL
MFQQ----LS
MFQQ----LS
RSLL----KT
E.
A.
T.
T.
E.
coli Ffh KFFH
ambivalens SRP54 1J8M
aquaticus Ffh 1JPJ
aquaticus Ffh 1NG1
coli FtsY 1FTS
47
45
47
47
244
VVR-FINRVK
LVFSLTNKIK
VARDFVERVR
VARDFVERVR
TTRKIITNLT
E.
A.
T.
T.
E.
coli Ffh KFFH
ambivalens SRP54 1J8M
aquaticus Ffh 1JPJ
aquaticus Ffh 1NG1
coli FtsY 1FTS
95
94
95
95
288
EKAVGHEVNK SLTPGQEFVK
ERLKNEKPPT YIERREWFIK
EEALGKQVLE SLTPAEVILA
EEALGKQVLE SLTPAEVILA
EGASRKQLR- ---DAEALYG
|--P-loop-|
|--------G1--------|
AAQPP-AVVL MAGLQGAGKT TSVGKLGKFL
PDKIP-YVIM LVGVQGTGKT TTAGKLAYFY
LKDR--NLWF LVGLQGSGKT TTAAKLALYY
LK-DR-NLWF LVGLQGSGKT TTAAKLALYY
VEGKAPFVIL MVGVNGVGKT TTIGKLARQF
E.
A.
T.
T.
E.
coli Ffh KFFH
ambivalens SRP54 1J8M
aquaticus Ffh 1JPJ
aquaticus Ffh 1NG1
coli FtsY 1FTS
144
142
142
142
337
---|
IKQLETLAEQ
LEQLQQLGQQ
REQLRLLGEK
REQLRLLGEK
VEQLQVWGQR
VGVDFFPSDV
IGVPVYGEPG
VGVPVLEVMD
VGVPVLEVMD
NNIPVIAQH-
E.
A.
T.
T.
E.
coli Ffh KFFH
ambivalens SRP54 1J8M
aquaticus Ffh 1JPJ
aquaticus Ffh 1NG1
coli FtsY 1FTS
191
189
189
189
384
-----|
AG--RLHVDE
AGRHGYGEEA
AGRLQID--AGRLQID--AGRLQNK---
AMMDEI-KQV
ALLEEM-KNI
EPLMGELARL
EPLMGELARL
SHLMEELKKI
E.
A.
T.
T.
E.
coli Ffh KFFH
ambivalens SRP54 1J8M
aquaticus Ffh 1JPJ
aquaticus Ffh 1NG1
coli FtsY 1FTS
229
229
227
227
431
E.
A.
T.
T.
E.
coli Ffh KFFH
ambivalens SRP54 1J8M
aquaticus Ffh 1JPJ
aquaticus Ffh 1NG1
coli FtsY 1FTS
278
278
276
276
477
DRLSRTLRNI
DNLRDTVRKF
ARLQEAIGRL
ARLQEAIGRL
KENLGSGFIS
SGRGRLTEDN
LTGSSSYDKA
RGRGRITEED
RGRGRITEED
LFRG--KKID
‘ALLEADV’
VKDTLREVRM ALLEADVALP
VEDFIKELQK SLISADVNVK
LKATLREIRR ALMDADVNLE
LKATLREIRR ALMDADVNLE
DDLFEELEEQ -LLIADVGVE
IVRNELVAAM
IVYDELSNLF
TVYEALKEAL
TVYEALKEAL
LLKEEMGE--
GEEN-QTLNL
GGDK-EPKVI
GGE--ARLPV
GGEA--RLPV
ILAKVDEPLN
REKHKKKVLV
KKK-GFKVGL
KGK-GRRPLL
KGK-GRRPLL
EQQ-GKSVML
|---G2--VSADVYRPAA
VGADVYRPAA
VAADTQRPAA
VAADTQRPAA
AAGDTFRAAA
GQK-PVDIVN
EKD-VVGIAK
GES--PESIR
GES--PESIR
TGADSASVIF
AALK-EAKLK
RGVE-KFLSE
RRVEEKA-RL
RRVEE-KARL
DAIQ-AAKAR
|--G3-F-YDVLLVDT
K-MEIIIVDT
EARDLILVDT
EARDLILVDT
N-IDVLIADT
HASIN----YEAIK----KEVLG----KEVLG----VRVMKKLDVE
-PVETLFVVD
-PDEVTLVID
-PDEVLLVLD
-PDEVLLVLD
APHEVMLTID
AMTGQ---DA
ASIGQ---KA
AMTGQ---EA
AMTGQ---EA
ASTGQNAVSQ
ANTAKAFNEA
YDLASKFNQA
LSVARAFDEK
LSVARAFDEK
AKLFHE---A
|--------G4--------|
LPLTGVVLTK VDGDARGGAA LSIRHIT-GK
SKIGTIIITK MDGTAKGGGA LSAVAAT-GA
VGVTGLVLTK LDGD-ARGGA ALSARHVTGK
VGVTGLVLTK LDGD-ARGGA ALSARHVTGK
VGLTGITLTK LDGTAKGGV- IFSVADQFGI
|closing
PIKFLGVGEK
TIKFIGTGEK
PIYFAGVSEK
PIYFAGVSEK
PIRYIGVGER
loop|
TEALEPFHPD
IDELEVFNPR
PEGLEPFYPE
PEGLEPFYPE
IEDLRPFKAD
RIASR--ILG
RFVAR--L-H
RLAGR--ILG
RLAGRILG-M
DFIEAL--FA
MGD
HHH
M---R--
298
297
294
294
495
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 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
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-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-Mg2+ Docking
GTP-Mg2+ docked to Ffh
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
Evaluation of a Proposed Protein-Protein
Interaction Model
Hydrophobic patches using Molsurfer
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
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 sidechain 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
Rebecca Wade, European Media Laboratory
Irmi Sinning, University of Heidelberg
Timm Essigke
Razif Gabdoulline
Ting Wang
GTP-binding and Association of the Escherichia
coli Signal2 Recognition Particle,
Ffh, and its Receptor, FtsY
1*
1
Kelly M. Elkins , Irmgard Sinning , and Rebecca C. Wade
BZH
Abstract
Results
The signal recognition particle (SRP) is a universally conserved system for protein
trafficking. Many SRPs are GTP-binding proteins. Their crucial role in ensuring that
proteins are not misplaced into the wrong cellular location makes them potential
targets for drug design. The aim of our work is to derive structural models of the
interactions of the SRP and its receptor. This is being done for the SRP system from
Escherichia coli, which is much simpler than that found in humans and thus
provides a good model system. The E. coli SRP is a ribonucleoprotein complex
composed of a 48 kDa GTPase protein and a 4.5 S RNA. While the crystal structure
of the E. coli SRP receptor FtsY, also a GTPase, has been solved, the structure of
the E. coli SRP protein itself has not. Consequently, we have used comparative
modelling techniques to build a model of the E. coli SRP protein, Ffh,
2+ on the basis
of SRP structures from other organisms and to dock GTP and Mg into their
hypothesized sites on both Ffh and FtsY. These modelled structures are being
used to build a model of the active SRP:SRP receptor complex. This model
should prove useful in understanding how the components of the SRP interact
to direct protein traffic within and out of the cell.
Protein
Organism
Ffh Escherichia coli
Ffh
Percent Identity
100
Thermus aquaticus
SRP54 Acidianus ambivalens
(Desulfurolobus ambivalens)
(archaebacteria)
FtsY Escherichia coli
46
35
33
.pdb Structure
M domain in 1DUL with 4.5S RNA,
homology model KFFH
Ref.
(this
work)
1NG1, GDP-Mg2+, NG domain
(2)
2NG1, GDP, NG domain
(2)
3NG1, apo, NG domain
(2)
1FFH, apo, NG domain
2FFH, apo, whole NG+M domain, A48T (5)
1JPJ, GMPPNP, NG-domain
(3)
1J8M, apo, NG-domain
(4)
1FTS, apo, NG domain
(1)
Table 1: Swiss-Model (http://www.expasy.ch/swissmod/SWISS-MODEL.html) Predict Protein maximum homology alignment
summary for E. coli Ffh and bacterial/archaebacterial signal recognition particle proteins and receptors for which crystal structures are
known and were used to make the homology model KFFH (red). Colored in blue are the structures used to model/insert the M-domain
and 4.5S RNA to KFFH.
2+
Alignment used for E. coli Ffh model of NG-domain
E. coli Ffh KFFH
A. ambivalens SRP54 1J8M
T. aquaticus Ffh 1JPJ
T. aquaticus Ffh 1NG1
E. coli FtsY 1FTS
E. coli Ffh KFFH
A. ambivalens SRP54 1J8M
T. aquaticus Ffh 1JPJ
T. aquaticus Ffh 1NG1
E. coli FtsY 1FTS
E. coli Ffh KFFH
A. ambivalens SRP54 1J8M
T. aquaticus Ffh 1JPJ
T. aquaticus Ffh 1NG1
E. coli FtsY 1FTS
Figure 2: GTP-Mg is docked into the E. coli Ffh
model. Residues which are within ligating distance
E. coli Ffh KFFH
are shown, including: Gln108, Gly109, Ala110,
A. ambivalens SRP54 1J8M
Gly111, Lys112, Thr113, Thr114, Lys118, Arg140,
T. aquaticus Ffh 1JPJ
Asp189, Lys248, Asp250, & Gly275.
T. aquaticus Ffh 1NG1
Make a homology model of the NG-domain of the E. coli SRP, Ffh
Calculate pKas of Ffh and FtsY: are there residues
where the pKas are shifted?
2+
What roles do these residues play in GTP-Mg binding or protein-protein association?
E. coli FtsY 1FTS
E. coli Ffh KFFH
A. ambivalens SRP54 1J8M
T. aquaticus Ffh 1JPJ
T. aquaticus Ffh 1NG1
E. coli FtsY 1FTS
2+
Dock GTP-Mg to E. coli Ffh, and its receptor, FtsY
Compute the electrostatic potential using the Poisson-Bolzmann equation
Dock the two proteins
Evaluate the docked complex
Evaluate conformational changes
Figure 1: Homology model of the NG-domain
of the Escherichia coli signal recognition
particle Ffh(red trace) superimposed on
the templates (blue traces).
1.Montoya, G., Svensson, C., Luirink, J., & Sinning, I. (1997). Crystal structure of the
NG domain from the signal recognition particle receptor FtsY. Nature, 385, 365-368.
2.Freymann, D.M., Keenan, R.J., Stroud, R.M., & Walter, P. (1999).
Functional changes in the
2+
structure of the SRP GTPase on the binding of GDP and Mg GDP. Nature Struct. Biol., 6, 793-801.
3.Padmanabhan, S., & Freymann, D.M. (2001). The conformation of bound GMPPNP suggests a
mechanism for gating the active site of the SRP GTPase. Structure (Camb.), 9, 859-867.
4.Montoya, G., te Kaat, K., Moll, R., Schaefer, G., & Sinning, I. (2000). The crystal structure of the
conserved GTPase of SRP54 from the archeon Acidianus ambivalens and its comparison with
related structures suggests a model for the SRP:SRP receptor complex. Structure, 8, 515-525.
5.Keenan, R.J., Freymann, D.M., Walter, P., & Stroud, R.M. (1998). Crystal structure of the signal
sequence binding subunit of the signal recognition particle. Cell, 94, 181-191.
6.Batey, R.T., Rambo, R.P., Lucast, L., Rha, B., & Doudna, J.A. (2000). Crystal structure of the
ribonucleoprotein core of the signal recognition particle. Science, 287, 1232-1239.
7.Demchuk, E. & Wade, R.C. (1996). Improving the continuum dielectric approach to calculating
pKas of ionizable groups in proteins. J. Phys. Chem., 100, 17373-17387.
8.Madura, J.D., Davis, M.E., Gilson, M.K., Wade, R.C., Luty, B.A., & McCammon, J.A. (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.
9.Gabdoulline, R.R. & Wade, R.C. (1997). Simulation of the diffusional association of
barnase and barstar. Biophys. J., 72, 1917-1929.
10.Gabdoulline, R.R. & Wade, R.C. (2001). Protein-protein association: investigation of factors
influencing association rates by brownian dynamics simulations. J. Mol. Biol., 306, 1139-1155.
‘ALLEADV’
5
LT DRLSRTLRNI SGRGRLTEDN VKDTLREVRM ALLEADVALP
3
LL DNLRDTVRKF LTGSSSYDKA VEDFIKELQK SLISADVNVK
1 MFQQ----LS ARLQEAIGRL RGRGRITEED LKATLREIRR ALMDADVNLE
1 MFQQ----LS ARLQEAIGRL RGRGRITEED LKATLREIRR ALMDADVNLE
201 RSLL----KT KENLGSGFIS LFRG--KKID DDLFEELEEQ -LLIADVGVE
47 VVR-FINRVK EKAVGHEVNK SLTPGQEFVK IVRNELVAAM GEEN-QTLNL
45 LVFSLTNKIK ERLKNEKPPT YIERREWFIK IVYDELSNLF GGDK-EPKVI
47 VARDFVERVR EEALGKQVLE SLTPAEVILA TVYEALKEAL GGE--ARLPV
47 VARDFVERVR EEALGKQVLE SLTPAEVILA TVYEALKEAL GGEA--RLPV
244 TTRKIITNLT EGASRKQLR- ---DAEALYG LLKEEMGE-- ILAKVDEPLN
|--P-loop-|
|--------G1--------|
|---G2--95 AAQPP-AVVL MAGLQGAGKT TSVGKLGKFL REKHKKKVLV VSADVYRPAA
94 PDKIP-YVIM LVGVQGTGKT TTAGKLAYFY KKK-GFKVGL VGADVYRPAA
95 LKDR--NLWF LVGLQGSGKT TTAAKLALYY KGK-GRRPLL VAADTQRPAA
95 LK-DR-NLWF LVGLQGSGKT TTAAKLALYY KGK-GRRPLL VAADTQRPAA
288 VEGKAPFVIL MVGVNGVGKT TTIGKLARQF EQQ-GKSVML AAGDTFRAAA
---|
|--G3-144 IKQLETLAEQ VGVDFFPSDV GQK-PVDIVN AALK-EAKLK F-YDVLLVDT
142 LEQLQQLGQQ IGVPVYGEPG EKD-VVGIAK RGVE-KFLSE K-MEIIIVDT
142 REQLRLLGEK VGVPVLEVMD GES--PESIR RRVEEKA-RL EARDLILVDT
142 REQLRLLGEK VGVPVLEVMD GES--PESIR RRVEE-KARL EARDLILVDT
337 VEQLQVWGQR NNIPVIAQH- TGADSASVIF DAIQ-AAKAR N-IDVLIADT
-----|
191 AG--RLHVDE AMMDEI-KQV HASIN----- -PVETLFVVD AMTGQ---DA
189 AGRHGYGEEA ALLEEM-KNI YEAIK----- -PDEVTLVID ASIGQ---KA
189 AGRLQID--- EPLMGELARL KEVLG----- -PDEVLLVLD AMTGQ---EA
189 AGRLQID--- EPLMGELARL KEVLG----- -PDEVLLVLD AMTGQ---EA
384 AGRLQNK--- SHLMEELKKI VRVMKKLDVE APHEVMLTID ASTGQNAVSQ
E. coli Ffh KFFH
A. ambivalens SRP54 1J8M
T. aquaticus Ffh 1JPJ
T. aquaticus Ffh 1NG1
E. coli FtsY 1FTS
|--------G4--------|
|closing
229 ANTAKAFNEA LPLTGVVLTK VDGDARGGAA LSIRHIT-GK PIKFLGVGEK
229 YDLASKFNQA SKIGTIIITK MDGTAKGGGA LSAVAAT-GA TIKFIGTGEK
227 LSVARAFDEK VGVTGLVLTK LDGD-ARGGA ALSARHVTGK PIYFAGVSEK
227 LSVARAFDEK VGVTGLVLTK LDGD-ARGGA ALSARHVTGK PIYFAGVSEK
431 AKLFHE---A VGLTGITLTK LDGTAKGGV- IFSVADQFGI PIRYIGVGER
E. coli Ffh KFFH
A. ambivalens SRP54 1J8M
T. aquaticus Ffh 1JPJ
T. aquaticus Ffh 1NG1
E. coli FtsY 1FTS
loop|
278 TEALEPFHPD RIASR--ILG MGD 298
278 IDELEVFNPR RFVAR--L-H HHH 297
276 PEGLEPFYPE RLAGR--ILG M-- 294
276 PEGLEPFYPE RLAGRILG-M --- 294
477 IEDLRPFKAD DFIEAL--FA R-- 495
Table 2: Sequence alignment and template x-ray crystal structures used
to produce E. coli Ffh homology model.
Figure 4: Results of UHBD electrostatics calculations using CHARMm forcefield parameters.
The proteins are shown in the same orientation as they were positioned in the complex. Left:
E. coli Ffh, Right: E. coli FtsY. Contour levels for both proteins: -0.1 to 0.1. Positively
charged regions are shown in blue and negatively charged regions are shown in red.
Conclusions & Outlook
*We have made a homology2+ model of E. coli Ffh
*We have docked GTP-Mg to both Ffh and
FtsY
*We are evaluating a proposed model of the
Ffh-FtsY complex
*The electrostatics calculations indicate that the
positively charged Ffh and negatively charged
FtsY are somewhat complementary in the
orientation from the proposed model
*SDA (simulated diffusional association)
calculationsare currently underway to evaluate
other possible association modes for the 2
proteins
*The M domain of Ffh and the 4.5S RNA are
currently being modelled into the complex
J. William Fulbright Foreign Scholarship Board
and the German Fulbright Kommission
Klaus Tschira Stiftung (KTS)