Computational Study of Escherichia coli

Signal Recognition Particle GTPases

Kelly Elkins

BZH

GPBM 2002: June 25, 2002

Why Modelling?

Gene Structure Function

?GTACTTACCCTAGTACCATGAATGGGATCATG

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 FfhEvaluate proposed SRP:SR interaction modelIs model valid in an apo-form? with Mg2+ bound?

with GTP-Mg2+?Other possible models?Conclusions

Signal Recognition Particle

Universally conserved system for protein traffickingIn humans, 6 proteins and 1 RNA

In E. coli: 2 proteins: SRP and receptor; 1 RNA: 4.5S RNABoth are GTPasesFfh (SRP) 48 kDa GTPase proteinFtsY (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 modelComparative Modelling

pdb structures: www.rcsb.org/pdb/

Protein Organism Percent Identity .pdb Structure

Ffh Escherichia coli 100M domain in 1DUL with 4.5S RNA, homology model KFFH

Ffh Thermus aquaticus 46

1NG1, GDP-Mg2 +

, NG domain 2NG1, GDP, NG domain 3NG1, apo, NG domain 1FFH, apo, NG domain, 2FFH, apo, whole NG+M domain, A48T 1JPJ, GMPPNP, NG-domain

SRP54Acidianus ambivalens

(Desulfurolobus ambivalens) (archaebacteria)

351J8M, apo, NG domain

FtsY Escherichia coli 33 1FTS, apo, NG domain

Ffh Model

Swiss Model1) 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)

2) Optimize Project Mode- adjusted sequence alignment with Swiss PDB Viewer to retain secondary structure elements

-Same templates

3) Checked Model with Procheck and Whatif

Alignment used for E. coli Ffh model of NG-domain ‘ALLEADV’ E. coli Ffh KFFH 5 LT DRLSRTLRNI SGRGRLTEDN VKDTLREVRM ALLEADVALP A. ambivalens SRP54 1J8M 3 LL DNLRDTVRKF LTGSSSYDKA VEDFIKELQK SLISADVNVK T. aquaticus Ffh 1JPJ 1 MFQQ----LS ARLQEAIGRL RGRGRITEED LKATLREIRR ALMDADVNLE T. aquaticus Ffh 1NG1 1 MFQQ----LS ARLQEAIGRL RGRGRITEED LKATLREIRR ALMDADVNLE E. coli FtsY 1FTS 201 RSLL----KT KENLGSGFIS LFRG--KKID DDLFEELEEQ -LLIADVGVE E. coli Ffh KFFH 47 VVR-FINRVK EKAVGHEVNK SLTPGQEFVK IVRNELVAAM GEEN-QTLNL A. ambivalens SRP54 1J8M 45 LVFSLTNKIK ERLKNEKPPT YIERREWFIK IVYDELSNLF GGDK-EPKVI T. aquaticus Ffh 1JPJ 47 VARDFVERVR EEALGKQVLE SLTPAEVILA TVYEALKEAL GGE--ARLPV T. aquaticus Ffh 1NG1 47 VARDFVERVR EEALGKQVLE SLTPAEVILA TVYEALKEAL GGEA--RLPV E. coli FtsY 1FTS 244 TTRKIITNLT EGASRKQLR- ---DAEALYG LLKEEMGE-- ILAKVDEPLN |--P-loop-| |--------G1--------| |---G2--- E. coli Ffh KFFH 95 AAQPP-AVVL MAGLQGAGKT TSVGKLGKFL REKHKKKVLV VSADVYRPAA A. ambivalens SRP54 1J8M 94 PDKIP-YVIM LVGVQGTGKT TTAGKLAYFY KKK-GFKVGL VGADVYRPAA T. aquaticus Ffh 1JPJ 95 LKDR--NLWF LVGLQGSGKT TTAAKLALYY KGK-GRRPLL VAADTQRPAA T. aquaticus Ffh 1NG1 95 LK-DR-NLWF LVGLQGSGKT TTAAKLALYY KGK-GRRPLL VAADTQRPAA E. coli FtsY 1FTS 288 VEGKAPFVIL MVGVNGVGKT TTIGKLARQF EQQ-GKSVML AAGDTFRAAA

---| |--G3--

E. coli Ffh KFFH 144 IKQLETLAEQ VGVDFFPSDV GQK-PVDIVN AALK-EAKLK F-YDVLLVDT A. ambivalens SRP54 1J8M 142 LEQLQQLGQQ IGVPVYGEPG EKD-VVGIAK RGVE-KFLSE K-MEIIIVDT T. aquaticus Ffh 1JPJ 142 REQLRLLGEK VGVPVLEVMD GES--PESIR RRVEEKA-RL EARDLILVDT T. aquaticus Ffh 1NG1 142 REQLRLLGEK VGVPVLEVMD GES--PESIR RRVEE-KARL EARDLILVDT E. coli FtsY 1FTS 337 VEQLQVWGQR NNIPVIAQH- TGADSASVIF DAIQ-AAKAR N-IDVLIADT -----| E. coli Ffh KFFH 191 AG--RLHVDE AMMDEI-KQV HASIN----- -PVETLFVVD AMTGQ---DA A. ambivalens SRP54 1J8M 189 AGRHGYGEEA ALLEEM-KNI YEAIK----- -PDEVTLVID ASIGQ---KA T. aquaticus Ffh 1JPJ 189 AGRLQID--- EPLMGELARL KEVLG----- -PDEVLLVLD AMTGQ---EA T. aquaticus Ffh 1NG1 189 AGRLQID--- EPLMGELARL KEVLG----- -PDEVLLVLD AMTGQ---EA E. coli FtsY 1FTS 384 AGRLQNK--- SHLMEELKKI VRVMKKLDVE APHEVMLTID ASTGQNAVSQ |--------G4--------| |closing E. coli Ffh KFFH 229 ANTAKAFNEA LPLTGVVLTK VDGDARGGAA LSIRHIT-GK PIKFLGVGEK A. ambivalens SRP54 1J8M 229 YDLASKFNQA SKIGTIIITK MDGTAKGGGA LSAVAAT-GA TIKFIGTGEK T. aquaticus Ffh 1JPJ 227 LSVARAFDEK VGVTGLVLTK LDGD-ARGGA ALSARHVTGK PIYFAGVSEK T. aquaticus Ffh 1NG1 227 LSVARAFDEK VGVTGLVLTK LDGD-ARGGA ALSARHVTGK PIYFAGVSEK E. coli FtsY 1FTS 431 AKLFHE---A VGLTGITLTK LDGTAKGGV- IFSVADQFGI PIRYIGVGER loop| E. coli Ffh KFFH 278 TEALEPFHPD RIASR--ILG MGD 298 A. ambivalens SRP54 1J8M 278 IDELEVFNPR RFVAR--L-H HHH 297 T. aquaticus Ffh 1JPJ 276 PEGLEPFYPE RLAGR--ILG M-- 294 T. aquaticus Ffh 1NG1 276 PEGLEPFYPE RLAGRILG-M --- 294 E. coli FtsY 1FTS 477 IEDLRPFKAD DFIEAL--FA R-- 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 Angstroms1J8M- 0.96 Angstroms1JPJ- 3.04 Angstroms1FTS- 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)-

C superimposition

GTP-Mg2+ Docking

Superimposition42 GTP molecules- Protein Data Bank (23 Jan. 2002)Superimposed GTPs with superimp program (G.M. Ullmann)-

like atoms of 2 molecules are superimposedSuperimposed all GTPs into Ffh model and FtsY using

1NG1.pdb GDP as templateMg2+ placement according to 1NG1.pdb GDP-Mg2+ structureEnergy minimized apo, Mg2+, and GTP-Mg2+ docked forms

using AMBER7Visualize 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

UHBD calculations- compare electrostatic surfaces, charged regions

SDA (Simulated Diffusional Association)- ab initio modelsunrestricted searchsearch restricted to GTP-binding region

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 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

A l i g n m e n t u s e d f o r E . c o l i F f h m o d e l o f N G - d o m a i n ‘ALLEADV’ E. coli Ffh KFFH 5 LT DRLSRTLRNI SGRGRLTEDN VKDTLREVRM ALLEADVALP A. ambivalens SRP54 1J8M 3 LL DNLRDTVRKF LTGSSSYDKA VEDFIKELQK SLISADVNVK T. aquaticus Ffh 1JPJ 1 MFQQ----LS ARLQEAIGRL RGRGRITEED LKATLREIRR ALMDADVNLE T. aquaticus Ffh 1NG1 1 MFQQ----LS ARLQEAIGRL RGRGRITEED LKATLREIRR ALMDADVNLE E. coli FtsY 1FTS 201 RSLL----KT KENLGSGFIS LFRG--KKID DDLFEELEEQ -LLIADVGVE E. coli Ffh KFFH 47 VVR-FINRVK EKAVGHEVNK SLTPGQEFVK IVRNELVAAM GEEN-QTLNL A. ambivalens SRP54 1J8M 45 LVFSLTNKIK ERLKNEKPPT YIERREWFIK IVYDELSNLF GGDK-EPKVI T. aquaticus Ffh 1JPJ 47 VARDFVERVR EEALGKQVLE SLTPAEVILA TVYEALKEAL GGE--ARLPV T. aquaticus Ffh 1NG1 47 VARDFVERVR EEALGKQVLE SLTPAEVILA TVYEALKEAL GGEA--RLPV E. coli FtsY 1FTS 244 TTRKIITNLT EGASRKQLR- ---DAEALYG LLKEEMGE-- ILAKVDEPLN |--P-loop-| |--------G1--------| |---G2--- E. coli Ffh KFFH 95 AAQPP-AVVL MAGLQGAGKT TSVGKLGKFL REKHKKKVLV VSADVYRPAA A. ambivalens SRP54 1J8M 94 PDKIP-YVIM LVGVQGTGKT TTAGKLAYFY KKK-GFKVGL VGADVYRPAA T. aquaticus Ffh 1JPJ 95 LKDR--NLWF LVGLQGSGKT TTAAKLALYY KGK-GRRPLL VAADTQRPAA T. aquaticus Ffh 1NG1 95 LK-DR-NLWF LVGLQGSGKT TTAAKLALYY KGK-GRRPLL VAADTQRPAA E. coli FtsY 1FTS 288 VEGKAPFVIL MVGVNGVGKT TTIGKLARQF EQQ-GKSVML AAGDTFRAAA ---| |--G3-- E. coli Ffh KFFH 144 IKQLETLAEQ VGVDFFPSDV GQK-PVDIVN AALK-EAKLK F-YDVLLVDT A. ambivalens SRP54 1J8M 142 LEQLQQLGQQ IGVPVYGEPG EKD-VVGIAK RGVE-KFLSE K-MEIIIVDT T. aquaticus Ffh 1JPJ 142 REQLRLLGEK VGVPVLEVMD GES--PESIR RRVEEKA-RL EARDLILVDT T. aquaticus Ffh 1NG1 142 REQLRLLGEK VGVPVLEVMD GES--PESIR RRVEE-KARL EARDLILVDT E. coli FtsY 1FTS 337 VEQLQVWGQR NNIPVIAQH- TGADSASVIF DAIQ-AAKAR N-IDVLIADT -----| E. coli Ffh KFFH 191 AG--RLHVDE AMMDEI-KQV HASIN----- -PVETLFVVD AMTGQ---DA A. ambivalens SRP54 1J8M 189 AGRHGYGEEA ALLEEM-KNI YEAIK----- -PDEVTLVID ASIGQ---KA T. aquaticus Ffh 1JPJ 189 AGRLQID--- EPLMGELARL KEVLG----- -PDEVLLVLD AMTGQ---EA T. aquaticus Ffh 1NG1 189 AGRLQID--- EPLMGELARL KEVLG----- -PDEVLLVLD AMTGQ---EA E. coli FtsY 1FTS 384 AGRLQNK--- SHLMEELKKI VRVMKKLDVE APHEVMLTID ASTGQNAVSQ |--------G4--------| |closing E. coli Ffh KFFH 229 ANTAKAFNEA LPLTGVVLTK VDGDARGGAA LSIRHIT-GK PIKFLGVGEK A. ambivalens SRP54 1J8M 229 YDLASKFNQA SKIGTIIITK MDGTAKGGGA LSAVAAT-GA TIKFIGTGEK T. aquaticus Ffh 1JPJ 227 LSVARAFDEK VGVTGLVLTK LDGD-ARGGA ALSARHVTGK PIYFAGVSEK T. aquaticus Ffh 1NG1 227 LSVARAFDEK VGVTGLVLTK LDGD-ARGGA ALSARHVTGK PIYFAGVSEK E. coli FtsY 1FTS 431 AKLFHE---A VGLTGITLTK LDGTAKGGV- IFSVADQFGI PIRYIGVGER loop| E. coli Ffh KFFH 278 TEALEPFHPD RIASR--ILG MGD 298 A. ambivalens SRP54 1J8M 278 IDELEVFNPR RFVAR--L-H HHH 297 T. aquaticus Ffh 1JPJ 276 PEGLEPFYPE RLAGR--ILG M-- 294 T. aquaticus Ffh 1NG1 276 PEGLEPFYPE RLAGRILG-M --- 294 E. coli FtsY 1FTS 477 IEDLRPFKAD DFIEAL--FA R-- 495

P r o t e i n O r g a n i s m P e r c e n t I d e n t i t y . p d b S t r u c t u r e R e f . Ffh Escherichia coli 100 M domain in 1DUL with 4.5S RNA, (this homology model KFFH work) Ffh Thermus aquaticus 46 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) SRP54 Acidianus ambivalens 35 1J8M, apo, NG-domain (4) (Desulfurolobus ambivalens) (archaebacteria) FtsY Escherichia coli 33 1FTS, apo, NG domain (1) T a b l e 1 : S w is s - M o d e l ( h t t p : / / w w w . e x p a s y . c h / s w i s s m o d / S W I S S - M O D E L . h t m l ) P r e d i c t P r o t e i n m a x i m u m h o m o l o g y a l i g n m e n t s u m m a r y f o r E . c o l i F f h a n d b a c t e r i a l / a r c h a e b a c t e r i a l s i g n a l r e c o g n i t i o n p a r t i c l e p r o t e i n s a n d r e c e p t o r s f o r w h i c h c r y s t a l s t r u c t u r e s a r e k n o w n a n d w e r e u s e d t o m a k e t h e h o m o l o g y m o d e l K F F H ( r e d ) . C o l o r e d i n b l u e a r e t h e s t r u c t u r e s u s e d t o m o d e l / i n s e r t t h e M - d o m a i n a n d 4 . 5 S R N A t o K F F H .

K e l l y M . E l k i n s , I r m g a r d S i n n i n g , a n d R e b e c c a C . W a d e1 * 2 1G T P - b i n d i n g a n d A s s o c i a t i o n o f t h e i g n a l R e c o g n i t i o n P a r t i c l e , F f h , a n d i t s R e c e p t o r , F t s YE s c h e r i c h i a c o l i SA b s t r a c t

C o n c l u s i o n s & O u t l o o k

B Z H

1 . M o n t o y a , G . , S v e n s s o n , C . , L u i r i n k , J . , & S i n n i n g , I . ( 1 9 9 7 ) . C r y s t a l s t r u c t u r e o f t h e N G d o m a i n f r o m t h e s i g n a l r e c o g n i t i o n p a r t i c l e r e c e p t o r F t s Y . , , 3 6 5 - 3 6 8 . 2 . F r e y m a n n , D . M . , K e e n a n , R . J . , S t r o u d , R . M . , & W a l t e r , P . ( 1 9 9 9 ) . F u n c t i o n a l c h a n g e s i n t h e s t r u c t u r e o f t h e S R P G T P a s e o n t h e b i n d i n g o f G D P a n d M g G D P . . , , 7 9 3 - 8 0 1 . 3 . P a d m a n a b h a n , S . , & F r e y m a n n , D . M . ( 2 0 0 1 ) . T h e c o n f o r m a t i o n o f b o u n d G M P P N P s u g g e s t s a m e c h a n i s m f o r g a t i n g t h e a c t i v e s i t e o f t h e S R P G T P a s e . , , 8 5 9 - 8 6 7 . 4 . M o n t o y a , G . , t e K a a t , K . , M o l l , R . , S c h a e f e r , G . , & S i n n i n g , I . ( 2 0 0 0 ) . T h e c r y s t a l s t r u c t u r e o f t h e c o n s e r v e d G T P a s e o f S R P 5 4 f r o m t h e a r c h e o n a n d i t s c o m p a r i s o n w i t h r e l a t e d s t r u c t u r e s s u g g e s t s a m o d e l f o r t h e S R P : S R P r e c e p t o r c o m p l e x . , , 5 1 5 - 5 2 5 . 5 . K e e n a n , R . J . , F r e y m a n n , D . M . , W a l t e r , P . , & S t r o u d , R . M . ( 1 9 9 8 ) . C r y s t a l s t r u c t u r e o f t h e s i g n a l s e q u e n c e b i n d i n g s u b u n i t o f t h e s i g n a l r e c o g n i t i o n p a r t i c l e . , , 1 8 1 - 1 9 1 . 6 . B a t e y , R . T . , R a m b o , R . P . , L u c a s t , L . , R h a , B . , & D o u d n a , J . A . ( 2 0 0 0 ) . C r y s t a l s t r u c t u r e o f t h e r i b o n u c l e o p r o t e i n c o r e o f t h e s i g n a l r e c o g n i t i o n p a r t i c l e . , , 1 2 3 2 - 1 2 3 9 .7 . D e m c h u k , E . & W a d e , R . C . ( 1 9 9 6 ) . I m p r o v i n g t h e c o n t i n u u m d i e l e c t r i c a p p r o a c h t o c a l c u l a t i n g p K a s o f i o n i z a b l e g r o u p s i n p r o t e i n s . . , , 1 7 3 7 3 - 1 7 3 8 7 .8 . M a d u r a , J . D . , D a v i s , M . E . , G i l s o n , M . K . , W a d e , R . C . , L u t y , B . A . , & M c C a m m o n , J . A . ( 1 9 9 4 ) . B i o l o g i c a l a p p l i c a t i o n s o f e l e c t r o s t a t i c c a l c u l a t i o n s a n d b r o w n i a n d y n a m i c s s i m u l a t i o n s . I n : “ “ , L i p k o w i t z , K . B . , & B o y d , D . ( E d s . ) , V C H P u b l i s h e r s , I n c . , N e w Y o r k . 9 . G a b d o u l l i n e , R . R . & W a d e , R . C . ( 1 9 9 7 ) . S i m u l a t i o n o f t h e d i f f u s i o n a l a s s o c i a t i o n o f b a r n a s e a n d b a r s t a r . . , , 1 9 1 7 - 1 9 2 9 .1 0 . G a b d o u l l i n e , R . R . & W a d e , R . C . ( 2 0 0 1 ) . P r o t e i n - p r o t e i n a s s o c i a t i o n : i n v e s t i g a t i o n o f f a c t o r s i n f l u e n c i n g a s s o c i a t i o n r a t e s b y b r o w n i a n d y n a m i c s s i m u l a t i o n s . . , , 1 1 3 9 - 1 1 5 5 .

N a t u r eN a t u r e S t r u c t . B i o lS t r u c t u r e ( C a m b . )A c i d i a n u s a m b i v a l e n s S t r u c t u r eC e l lS c i e n c eJ . P h y s . C h e mR e v i e w s i n C o m p u t a t i o n a l C h e m i s t r y , V o l u m e VB i o p h y s . J J . M o l . B i o l

3 8 5 69 89 42 8 71 0 07 2 3 0 6

2 +

T h e s i g n a l r e c o g n i t i o n p a r t i c l e ( S R P ) i s a u n i v e r s a l l y c o n s e r v e d s y s t e m f o r p r o t e i n t r a f f i c k i n g . M a n y S R P s a r e G T P - b i n d i n g p r o t e i n s . T h e i r c r u c i a l r o l e i n e n s u r i n g t h a t p r o t e i n s a r e n o t m i s p l a c e d i n t o t h e w r o n g c e l l u l a r l o c a t i o n m a k e s t h e m p o t e n t i a l t a r g e t s f o r d r u g d e s i g n . T h e a i m o f o u r w o r k i s t o d e r i v e s t r u c t u r a l m o d e l s o f t h e i n t e r a c t i o n s o f t h e S R P a n d i t s r e c e p t o r . T h i s i s b e i n g d o n e f o r t h e S R P s y s t e m f r o m , w h i c h i s m u c h s i m p l e r t h a n t h a t f o u n d i n h u m a n s a n d t h u s p r o v i d e s a g o o d m o d e l s y s t e m . T h e S R P i s a r i b o n u c l e o p r o t e i n c o m p l e x c o m p o s e d o f a 4 8 k D a G T P a s e p r o t e i n a n d a 4 . 5 S R N A . W h i l e t h e c r y s t a l s t r u c t u r e o f t h e S R P r e c e p t o r F t s Y , a l s o a G T P a s e , h a s b e e n s o l v e d , t h e s t r u c t u r e o f t h e S R P p r o t e i n i t s e l f h a s n o t . C o n s e q u e n t l y , w e h a v e u s e d c o m p a r a t i v e m o d e l l i n g t e c h n i q u e s t o b u i l d a m o d e l o f t h e S R P p r o t e i n , F f h , o n t h e b a s i s o f S R P s t r u c t u r e s f r o m o t h e r o r g a n i s m s a n d t o d o c k G T P a n d M g i n t o t h e i r h y p o t h e s i z e d s i t e s o n b o t h F f h a n d F t s Y . T h e s e m o d e l l e d s t r u c t u r e s a r e b e i n g u s e d t o b u i l d a m o d e l o f t h e a c t i v e S R P : S R P r e c e p t o r c o m p l e x . T h i s m o d e l s h o u l d p r o v e u s e f u l i n u n d e r s t a n d i n g h o w t h e c o m p o n e n t s o f t h e S R P i n t e r a c t t o d i r e c t p r o t e i n t r a f f i c w i t h i n a n d o u t o f t h e c e l l .E s c h e r i c h i a c o l i E . c o l i E . c o l iE . c o l i E . c o l i 2 + R e s u l t s

F i g u r e 1 : H o m o l o g y m o d e l o f t h e N G - d o m a i n o f t h e s i g n a l r e c o g n i t i o n p a r t i c l e F f h ( r e d t r a c e ) s u p e r i m p o s e d o n t h e t e m p l a t e s ( b l u e t r a c e s ) .E s c h e r i c h i a c o l i T a b l e 2 : S e q u e n c e a l i g n m e n t a n d t e m p l a t e x - r a y c r y s t a l s t r u c t u r e s u s e d t o p r o d u c e F f h h o m o l o g y m o d e l . E . c o l i

F i g u r e 2 : G T P - M g i s d o c k e d i n t o t h e F f h m o d e l . R e s i d u e s w h i c h a r e w i t h i n l i g a t i n g d i s t a n c ea r e s h o w n , i n c l u d i n g : G l n 1 0 8 , G l y 1 0 9 , A l a 1 1 0 , G l y 1 1 1 , L y s 1 1 2 , T h r 1 1 3 , T h r 1 1 4 , L y s 1 1 8 , A r g 1 4 0 , A s p 1 8 9 , L y s 2 4 8 , A s p 2 5 0 , & G l y 2 7 5 . 2 + E . c o l i M a k e a h o m o l o g y m o d e l o f t h e N G - d o m a i n o f t h e S R P , F f hE . c o l iD o c k G T P - M g t o F f h , a n d i t s r e c e p t o r , F t s Y2 + E . c o l iD o c k t h e t w o p r o t e i n sE v a l u a t e t h e d o c k e d c o m p l e xC o m p u t e t h e e l e c t r o s t a t i c p o t e n t i a l u s i n g t h e P o i s s o n - B o l z m a n n e q u a t i o nC a l c u l a t e p K a s o f F f h a n d F t s Y : a r e t h e r e r e s i d u e s w h e r e t h e p K a s a r e s h i f t e d ?W h a t r o l e s d o t h e s e r e s i d u e s p l a y i n G T P - M g b i n d i n g o r p r o t e i n - p r o t e i n a s s o c i a t i o n ?2 +E v a l u a t e c o n f o r m a t i o n a l c h a n g e s F i g u r e 4 : R e s u l t s o f U H B D e l e c t r o s t a t i c s c a l c u l a t i o n s u s i n g C H A R M m f o r c e f i e l d p a r a m e t e r s . T h e p r o t e i n s a r e s h o w n i n t h e s a m e o r i e n t a t i o n a s t h e y w e r e p o s i t i o n e d i n t h e c o m p l e x . L e f t : F f h , R i g h t : F t s Y . C o n t o u r l e v e l s f o r b o t h p r o t e i n s : - 0 . 1 t o 0 . 1 . P o s i t i v e l y c h a r g e d r e g i o n s a r e s h o w n i n b l u e a n d n e g a t i v e l y c h a r g e d r e g i o n s a r e s h o w n i n r e d . E . c o l i E . c o l i * W e h a v e m a d e a h o m o l o g y m o d e l o f F f h* W e h a v e d o c k e d G T P - M g t o b o t h F f h a n d F t s Y* W e a r e e v a l u a t i n g a p r o p o s e d m o d e l o f t h e F f h - F t s Y c o m p l e x* T h e e l e c t r o s t a t i c s c a l c u l a t i o n s i n d i c a t e t h a t t h e p o s i t i v e l y c h a r g e d F f h a n d n e g a t i v e l y c h a r g e d F t s Y a r e s o m e w h a t c o m p l e m e n t a r y i n t h e o r i e n t a t i o n f r o m t h e p r o p o s e d m o d e l* S D A ( s i m u l a t e d d i f f u s i o n a l a s s o c i a t i o n ) c a l c u l a t i o n s a r e c u r r e n t l y u n d e r w a y t o e v a l u a t e o t h e r p o s s i b l e a s s o c i a t i o n m o d e s f o r t h e 2 p r o t e i n s* T h e M d o m a i n o f F f h a n d t h e 4 . 5 S R N A a r e c u r r e n t l y b e i n g m o d e l l e d i n t o t h e c o m p l e x

E . c o l i2 +

A l i g n m e n t u s e d f o r E . c o l i F f h m o d e l o f N G - d o m a i n ‘ALLEADV’ E. coli Ffh KFFH 5 LT DRLSRTLRNI SGRGRLTEDN VKDTLREVRM ALLEADVALP A. ambivalens SRP54 1J8M 3 LL DNLRDTVRKF LTGSSSYDKA VEDFIKELQK SLISADVNVK T. aquaticus Ffh 1JPJ 1 MFQQ----LS ARLQEAIGRL RGRGRITEED LKATLREIRR ALMDADVNLE T. aquaticus Ffh 1NG1 1 MFQQ----LS ARLQEAIGRL RGRGRITEED LKATLREIRR ALMDADVNLE E. coli FtsY 1FTS 201 RSLL----KT KENLGSGFIS LFRG--KKID DDLFEELEEQ -LLIADVGVE E. coli Ffh KFFH 47 VVR-FINRVK EKAVGHEVNK SLTPGQEFVK IVRNELVAAM GEEN-QTLNL A. ambivalens SRP54 1J8M 45 LVFSLTNKIK ERLKNEKPPT YIERREWFIK IVYDELSNLF GGDK-EPKVI T. aquaticus Ffh 1JPJ 47 VARDFVERVR EEALGKQVLE SLTPAEVILA TVYEALKEAL GGE--ARLPV T. aquaticus Ffh 1NG1 47 VARDFVERVR EEALGKQVLE SLTPAEVILA TVYEALKEAL GGEA--RLPV E. coli FtsY 1FTS 244 TTRKIITNLT EGASRKQLR- ---DAEALYG LLKEEMGE-- ILAKVDEPLN |--P-loop-| |--------G1--------| |---G2--- E. coli Ffh KFFH 95 AAQPP-AVVL MAGLQGAGKT TSVGKLGKFL REKHKKKVLV VSADVYRPAA A. ambivalens SRP54 1J8M 94 PDKIP-YVIM LVGVQGTGKT TTAGKLAYFY KKK-GFKVGL VGADVYRPAA T. aquaticus Ffh 1JPJ 95 LKDR--NLWF LVGLQGSGKT TTAAKLALYY KGK-GRRPLL VAADTQRPAA T. aquaticus Ffh 1NG1 95 LK-DR-NLWF LVGLQGSGKT TTAAKLALYY KGK-GRRPLL VAADTQRPAA E. coli FtsY 1FTS 288 VEGKAPFVIL MVGVNGVGKT TTIGKLARQF EQQ-GKSVML AAGDTFRAAA ---| |--G3-- E. coli Ffh KFFH 144 IKQLETLAEQ VGVDFFPSDV GQK-PVDIVN AALK-EAKLK F-YDVLLVDT A. ambivalens SRP54 1J8M 142 LEQLQQLGQQ IGVPVYGEPG EKD-VVGIAK RGVE-KFLSE K-MEIIIVDT T. aquaticus Ffh 1JPJ 142 REQLRLLGEK VGVPVLEVMD GES--PESIR RRVEEKA-RL EARDLILVDT T. aquaticus Ffh 1NG1 142 REQLRLLGEK VGVPVLEVMD GES--PESIR RRVEE-KARL EARDLILVDT E. coli FtsY 1FTS 337 VEQLQVWGQR NNIPVIAQH- TGADSASVIF DAIQ-AAKAR N-IDVLIADT -----| E. coli Ffh KFFH 191 AG--RLHVDE AMMDEI-KQV HASIN----- -PVETLFVVD AMTGQ---DA A. ambivalens SRP54 1J8M 189 AGRHGYGEEA ALLEEM-KNI YEAIK----- -PDEVTLVID ASIGQ---KA T. aquaticus Ffh 1JPJ 189 AGRLQID--- EPLMGELARL KEVLG----- -PDEVLLVLD AMTGQ---EA T. aquaticus Ffh 1NG1 189 AGRLQID--- EPLMGELARL KEVLG----- -PDEVLLVLD AMTGQ---EA E. coli FtsY 1FTS 384 AGRLQNK--- SHLMEELKKI VRVMKKLDVE APHEVMLTID ASTGQNAVSQ |--------G4--------| |closing E. coli Ffh KFFH 229 ANTAKAFNEA LPLTGVVLTK VDGDARGGAA LSIRHIT-GK PIKFLGVGEK A. ambivalens SRP54 1J8M 229 YDLASKFNQA SKIGTIIITK MDGTAKGGGA LSAVAAT-GA TIKFIGTGEK T. aquaticus Ffh 1JPJ 227 LSVARAFDEK VGVTGLVLTK LDGD-ARGGA ALSARHVTGK PIYFAGVSEK T. aquaticus Ffh 1NG1 227 LSVARAFDEK VGVTGLVLTK LDGD-ARGGA ALSARHVTGK PIYFAGVSEK E. coli FtsY 1FTS 431 AKLFHE---A VGLTGITLTK LDGTAKGGV- IFSVADQFGI PIRYIGVGER loop| E. coli Ffh KFFH 278 TEALEPFHPD RIASR--ILG MGD 298 A. ambivalens SRP54 1J8M 278 IDELEVFNPR RFVAR--L-H HHH 297 T. aquaticus Ffh 1JPJ 276 PEGLEPFYPE RLAGR--ILG M-- 294 T. aquaticus Ffh 1NG1 276 PEGLEPFYPE RLAGRILG-M --- 294 E. coli FtsY 1FTS 477 IEDLRPFKAD DFIEAL--FA R-- 495

P r o t e i n O r g a n i s m P e r c e n t I d e n t i t y . p d b S t r u c t u r e R e f . Ffh Escherichia coli 100 M domain in 1DUL with 4.5S RNA, (this homology model KFFH work) Ffh Thermus aquaticus 46 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) SRP54 Acidianus ambivalens 35 1J8M, apo, NG-domain (4) (Desulfurolobus ambivalens) (archaebacteria) FtsY Escherichia coli 33 1FTS, apo, NG domain (1) T a b l e 1 : S w is s - M o d e l ( h t t p : / / w w w . e x p a s y . c h / s w i s s m o d / S W I S S - M O D E L . h t m l ) P r e d i c t P r o t e i n m a x i m u m h o m o l o g y a l i g n m e n t s u m m a r y f o r E . c o l i F f h a n d b a c t e r i a l / a r c h a e b a c t e r i a l s i g n a l r e c o g n i t i o n p a r t i c l e p r o t e i n s a n d r e c e p t o r s f o r w h i c h c r y s t a l s t r u c t u r e s a r e k n o w n a n d w e r e u s e d t o m a k e t h e h o m o l o g y m o d e l K F F H ( r e d ) . C o l o r e d i n b l u e a r e t h e s t r u c t u r e s u s e d t o m o d e l / i n s e r t t h e M - d o m a i n a n d 4 . 5 S R N A t o K F F H .

K e l l y M . E l k i n s , I r m g a r d S i n n i n g , a n d R e b e c c a C . W a d e1 * 2 1G T P - b i n d i n g a n d A s s o c i a t i o n o f t h e i g n a l R e c o g n i t i o n P a r t i c l e , F f h , a n d i t s R e c e p t o r , F t s YE s c h e r i c h i a c o l i SA b s t r a c t

C o n c l u s i o n s & O u t l o o k

B Z H

1 . M o n t o y a , G . , S v e n s s o n , C . , L u i r i n k , J . , & S i n n i n g , I . ( 1 9 9 7 ) . C r y s t a l s t r u c t u r e o f t h e N G d o m a i n f r o m t h e s i g n a l r e c o g n i t i o n p a r t i c l e r e c e p t o r F t s Y . , , 3 6 5 - 3 6 8 . 2 . F r e y m a n n , D . M . , K e e n a n , R . J . , S t r o u d , R . M . , & W a l t e r , P . ( 1 9 9 9 ) . F u n c t i o n a l c h a n g e s i n t h e s t r u c t u r e o f t h e S R P G T P a s e o n t h e b i n d i n g o f G D P a n d M g G D P . . , , 7 9 3 - 8 0 1 . 3 . P a d m a n a b h a n , S . , & F r e y m a n n , D . M . ( 2 0 0 1 ) . T h e c o n f o r m a t i o n o f b o u n d G M P P N P s u g g e s t s a m e c h a n i s m f o r g a t i n g t h e a c t i v e s i t e o f t h e S R P G T P a s e . , , 8 5 9 - 8 6 7 . 4 . M o n t o y a , G . , t e K a a t , K . , M o l l , R . , S c h a e f e r , G . , & S i n n i n g , I . ( 2 0 0 0 ) . T h e c r y s t a l s t r u c t u r e o f t h e c o n s e r v e d G T P a s e o f S R P 5 4 f r o m t h e a r c h e o n a n d i t s c o m p a r i s o n w i t h r e l a t e d s t r u c t u r e s s u g g e s t s a m o d e l f o r t h e S R P : S R P r e c e p t o r c o m p l e x . , , 5 1 5 - 5 2 5 . 5 . K e e n a n , R . J . , F r e y m a n n , D . M . , W a l t e r , P . , & S t r o u d , R . M . ( 1 9 9 8 ) . C r y s t a l s t r u c t u r e o f t h e s i g n a l s e q u e n c e b i n d i n g s u b u n i t o f t h e s i g n a l r e c o g n i t i o n p a r t i c l e . , , 1 8 1 - 1 9 1 . 6 . B a t e y , R . T . , R a m b o , R . P . , L u c a s t , L . , R h a , B . , & D o u d n a , J . A . ( 2 0 0 0 ) . C r y s t a l s t r u c t u r e o f t h e r i b o n u c l e o p r o t e i n c o r e o f t h e s i g n a l r e c o g n i t i o n p a r t i c l e . , , 1 2 3 2 - 1 2 3 9 .7 . D e m c h u k , E . & W a d e , R . C . ( 1 9 9 6 ) . I m p r o v i n g t h e c o n t i n u u m d i e l e c t r i c a p p r o a c h t o c a l c u l a t i n g p K a s o f i o n i z a b l e g r o u p s i n p r o t e i n s . . , , 1 7 3 7 3 - 1 7 3 8 7 .8 . M a d u r a , J . D . , D a v i s , M . E . , G i l s o n , M . K . , W a d e , R . C . , L u t y , B . A . , & M c C a m m o n , J . A . ( 1 9 9 4 ) . B i o l o g i c a l a p p l i c a t i o n s o f e l e c t r o s t a t i c c a l c u l a t i o n s a n d b r o w n i a n d y n a m i c s s i m u l a t i o n s . I n : “ “ , L i p k o w i t z , K . B . , & B o y d , D . ( E d s . ) , V C H P u b l i s h e r s , I n c . , N e w Y o r k . 9 . G a b d o u l l i n e , R . R . & W a d e , R . C . ( 1 9 9 7 ) . S i m u l a t i o n o f t h e d i f f u s i o n a l a s s o c i a t i o n o f b a r n a s e a n d b a r s t a r . . , , 1 9 1 7 - 1 9 2 9 .1 0 . G a b d o u l l i n e , R . R . & W a d e , R . C . ( 2 0 0 1 ) . P r o t e i n - p r o t e i n a s s o c i a t i o n : i n v e s t i g a t i o n o f f a c t o r s i n f l u e n c i n g a s s o c i a t i o n r a t e s b y b r o w n i a n d y n a m i c s s i m u l a t i o n s . . , , 1 1 3 9 - 1 1 5 5 .

N a t u r eN a t u r e S t r u c t . B i o lS t r u c t u r e ( C a m b . )A c i d i a n u s a m b i v a l e n s S t r u c t u r eC e l lS c i e n c eJ . P h y s . C h e mR e v i e w s i n C o m p u t a t i o n a l C h e m i s t r y , V o l u m e VB i o p h y s . J J . M o l . B i o l

3 8 5 69 89 42 8 71 0 07 2 3 0 6

2 +

T h e s i g n a l r e c o g n i t i o n p a r t i c l e ( S R P ) i s a u n i v e r s a l l y c o n s e r v e d s y s t e m f o r p r o t e i n t r a f f i c k i n g . M a n y S R P s a r e G T P - b i n d i n g p r o t e i n s . T h e i r c r u c i a l r o l e i n e n s u r i n g t h a t p r o t e i n s a r e n o t m i s p l a c e d i n t o t h e w r o n g c e l l u l a r l o c a t i o n m a k e s t h e m p o t e n t i a l t a r g e t s f o r d r u g d e s i g n . T h e a i m o f o u r w o r k i s t o d e r i v e s t r u c t u r a l m o d e l s o f t h e i n t e r a c t i o n s o f t h e S R P a n d i t s r e c e p t o r . T h i s i s b e i n g d o n e f o r t h e S R P s y s t e m f r o m , w h i c h i s m u c h s i m p l e r t h a n t h a t f o u n d i n h u m a n s a n d t h u s p r o v i d e s a g o o d m o d e l s y s t e m . T h e S R P i s a r i b o n u c l e o p r o t e i n c o m p l e x c o m p o s e d o f a 4 8 k D a G T P a s e p r o t e i n a n d a 4 . 5 S R N A . W h i l e t h e c r y s t a l s t r u c t u r e o f t h e S R P r e c e p t o r F t s Y , a l s o a G T P a s e , h a s b e e n s o l v e d , t h e s t r u c t u r e o f t h e S R P p r o t e i n i t s e l f h a s n o t . C o n s e q u e n t l y , w e h a v e u s e d c o m p a r a t i v e m o d e l l i n g t e c h n i q u e s t o b u i l d a m o d e l o f t h e S R P p r o t e i n , F f h , o n t h e b a s i s o f S R P s t r u c t u r e s f r o m o t h e r o r g a n i s m s a n d t o d o c k G T P a n d M g i n t o t h e i r h y p o t h e s i z e d s i t e s o n b o t h F f h a n d F t s Y . T h e s e m o d e l l e d s t r u c t u r e s a r e b e i n g u s e d t o b u i l d a m o d e l o f t h e a c t i v e S R P : S R P r e c e p t o r c o m p l e x . T h i s m o d e l s h o u l d p r o v e u s e f u l i n u n d e r s t a n d i n g h o w t h e c o m p o n e n t s o f t h e S R P i n t e r a c t t o d i r e c t p r o t e i n t r a f f i c w i t h i n a n d o u t o f t h e c e l l .E s c h e r i c h i a c o l i E . c o l i E . c o l iE . c o l i E . c o l i 2 + R e s u l t s

F i g u r e 1 : H o m o l o g y m o d e l o f t h e N G - d o m a i n o f t h e s i g n a l r e c o g n i t i o n p a r t i c l e F f h ( r e d t r a c e ) s u p e r i m p o s e d o n t h e t e m p l a t e s ( b l u e t r a c e s ) .E s c h e r i c h i a c o l i T a b l e 2 : S e q u e n c e a l i g n m e n t a n d t e m p l a t e x - r a y c r y s t a l s t r u c t u r e s u s e d t o p r o d u c e F f h h o m o l o g y m o d e l . E . c o l i

F i g u r e 2 : G T P - M g i s d o c k e d i n t o t h e F f h m o d e l . R e s i d u e s w h i c h a r e w i t h i n l i g a t i n g d i s t a n c ea r e s h o w n , i n c l u d i n g : G l n 1 0 8 , G l y 1 0 9 , A l a 1 1 0 , G l y 1 1 1 , L y s 1 1 2 , T h r 1 1 3 , T h r 1 1 4 , L y s 1 1 8 , A r g 1 4 0 , A s p 1 8 9 , L y s 2 4 8 , A s p 2 5 0 , & G l y 2 7 5 . 2 + E . c o l i M a k e a h o m o l o g y m o d e l o f t h e N G - d o m a i n o f t h e S R P , F f hE . c o l iD o c k G T P - M g t o F f h , a n d i t s r e c e p t o r , F t s Y2 + E . c o l iD o c k t h e t w o p r o t e i n sE v a l u a t e t h e d o c k e d c o m p l e xC o m p u t e t h e e l e c t r o s t a t i c p o t e n t i a l u s i n g t h e P o i s s o n - B o l z m a n n e q u a t i o nC a l c u l a t e p K a s o f F f h a n d F t s Y : a r e t h e r e r e s i d u e s w h e r e t h e p K a s a r e s h i f t e d ?W h a t r o l e s d o t h e s e r e s i d u e s p l a y i n G T P - M g b i n d i n g o r p r o t e i n - p r o t e i n a s s o c i a t i o n ?2 +E v a l u a t e c o n f o r m a t i o n a l c h a n g e s F i g u r e 4 : R e s u l t s o f U H B D e l e c t r o s t a t i c s c a l c u l a t i o n s u s i n g C H A R M m f o r c e f i e l d p a r a m e t e r s . T h e p r o t e i n s a r e s h o w n i n t h e s a m e o r i e n t a t i o n a s t h e y w e r e p o s i t i o n e d i n t h e c o m p l e x . L e f t : F f h , R i g h t : F t s Y . C o n t o u r l e v e l s f o r b o t h p r o t e i n s : - 0 . 1 t o 0 . 1 . P o s i t i v e l y c h a r g e d r e g i o n s a r e s h o w n i n b l u e a n d n e g a t i v e l y c h a r g e d r e g i o n s a r e s h o w n i n r e d . E . c o l i E . c o l i * W e h a v e m a d e a h o m o l o g y m o d e l o f F f h* W e h a v e d o c k e d G T P - M g t o b o t h F f h a n d F t s Y* W e a r e e v a l u a t i n g a p r o p o s e d m o d e l o f t h e F f h - F t s Y c o m p l e x* T h e e l e c t r o s t a t i c s c a l c u l a t i o n s i n d i c a t e t h a t t h e p o s i t i v e l y c h a r g e d F f h a n d n e g a t i v e l y c h a r g e d F t s Y a r e s o m e w h a t c o m p l e m e n t a r y i n t h e o r i e n t a t i o n f r o m t h e p r o p o s e d m o d e l* S D A ( s i m u l a t e d d i f f u s i o n a l a s s o c i a t i o n ) c a l c u l a t i o n s a r e c u r r e n t l y u n d e r w a y t o e v a l u a t e o t h e r p o s s i b l e a s s o c i a t i o n m o d e s f o r t h e 2 p r o t e i n s* T h e M d o m a i n o f F f h a n d t h e 4 . 5 S R N A a r e c u r r e n t l y b e i n g m o d e l l e d i n t o t h e c o m p l e x

E . c o l i2 +

Rebecca Wade, European Media LaboratoryIrmi Sinning, University of Heidelberg

Timm EssigkeRazif GabdoullineTing Wang

J. William Fulbright Foreign Scholarship Board and the German Fulbright KommissionKlaus Tschira Stiftung (KTS)