RNA-mediated interaction between the peptide-binding and GTPase domains of the signal recognition particle

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1 R-medited interction etween the peptide-inding nd TPse domins of the signl recognition prticle Richrd J Spnggord 1, Fi Siu 1, ilong Ke 1 & Jennifer Doudn 1 4 The signl recognition prticle (SRP) trgets nscent proteins to cellulr memrnes for insertion or secretion y recognizing polypeptides contining n -terminl signl sequence s they emerge from the riosome. TP-dependent inding of SRP to its receptor protein leds to controlled relese of the nscent chin into memrne-spnning trnslocon pore. Here we show tht the ssocition of the SRP with its receptor triggers mrked conformtionl chnge in the complex, loclizing the SRP R nd the djcent signl peptide inding site t the SRP-receptor heterodimer interfce. The orienttion of the R suggests how peptide inding nd TP hydrolysis cn e coupled through direct structurl contct during cycles of SRP-directed protein trnsloction. The SRP includes universlly conserved TPse protein responsile for signl peptide recognition nd tightly ound R molecule, s well s dditionl protein components in rche nd eukryotes 1,2. In cteri, signl peptide inding y the SRP composed of the protein nd 4.5S R directs the trnslting riosome to the periplsmic memrne vi inding to the trnsloconssocited receptor TPse,. The phylogeneticlly conserved 4.5S R, which is essentil in vivo 3,4, sustntilly ccelertes the ssocition nd dissocition of nd, s well s the rte of TP hydrolysis in the complex 5,6. lthough this suggests direct interction etween the R nd the TPse domins of the - heterodimer, structurl dt re not yet ville for the intct SRP complex. crystl structure of the rchel SRP core reveled tht, together, the -terminl nd TPse domins of nd the -terminl M domin nd its ssocited SRP R form the opposing rms of U-shped ssemly in which the signl peptide inding site 7 is 60 Å wy from the TPse center (Fig. 1) 8. rystl structures of the - heterodimer showed how ound TP molecules re shred y oth proteins, suggesting mechnism for the oserved mutul TPse stimultion upon - ssocition 9,10. However, these structures did not provide insights into the function of 4.5S R in the SRP receptor complex ecuse the SRP core structure lcked the protein, wheres the heterodimer structures lcked the M domin nd the 4.5S R. In ddition, ecuse the 4.5S R M domin region of the SRP contins the universlly conserved signl peptide recognition site 11, these structures cnnot explin how peptide inding nd TP hydrolysis re coupled during TP-dependent cycles of peptide inding nd relese 12,13. To understnd the structurl sis for SRP-receptor interctions, we set out to determine the conformtionl rrngement of protein nd R components in the Escherichi coli SRP receptor complex. RESULTS onformtionl chnge of SRP upon receptor inding To determine the loction nd orienttion of the 4.5S R in the E. coli SRP complex, we used site-directed hydroxyl rdicl proing 14 to identify the reltive sptil reltionships of specific mino cid positions on nd with respect to the full-length 4.5S R. Using the crystl structures of the rchel SRP 8 nd the Thermus quticus complex 10 s guides, we egn y introducing single cysteine residues t vrious positions within tht reside within the M domin or long the - interfce (Fig. 1). Ech protein contining single cysteine ws then coupled with the cysteine-specific hydroxyl rdicl genertor romocetmidoenzyl-edt-fe (E-Fe). The E-Fe coupling efficiency for ech mutnt ws high, nd ech conjugted protein ws le to form sturle complex with the 4.5S R nd in the presence of the nonhydrolyzle TP nlog MPPP (Supplementry Figs. 1 nd 2 online). Ech E-Fe modified ws ssemled into n SRP complex, nd 4.5S R clevge within the complex ws nlyzed fter incution with peroxide nd scorte to produce free rdicls 15,16. SRP complexes contining E-Fe modified sites within the M domin (residues 344 nd 409, Fig. 1) led to 4.5S R clevge t sites close to the R tetrloop, consistent with previous crystl structures (Fig. 1, lnes 409 nd 344; for quntifiction of clevge nds see Supplementry Fig. 3 online) 8,17. These clevge sites occur in the presence or sence of MPPP, suggesting tht the core structure of the M domin does not chnge reltive to the ound 1 Deprtment of Moleculr nd ell iology, 2 Deprtment of hemistry nd 3 Howrd Hughes Medicl Institute, University of liforni, erkeley, liforni 94705, US. 4 Physicl iosciences Division, Lwrence erkeley tionl Lortory, erkeley, liforni 94720, US. orrespondence should e ddressed to J..D. (doudn@erkeley.edu). Received 21 Septemer; ccepted 25 Octoer; pulished online 20 ovemer 2005; doi: /nsm VOLUME 12 UMER 12 DEEMER 2005 TURE STRUTURL & MOLEULR IOLOY

2 Figure 1 Hydroxyl rdicl proing of the 4.5S R y in the E. coli SRP (residues ) complex. () Structure of the rchel SRP showing the nlogous loctions of the E. coli residues selected for mutgenesis (numers in prentheses represent the nlogous S. solftricus residue numers) 8.Residuesin contrsting colors represent those E- Fe modified positions tht produced loclized R clevge sites; residues in white re positions tht did not cleve the R (see text for detils). () Strnd scission of the full-length 4.5S R y in sturle SRP complex derivtized y E-Fe t positions 409, 344, 153 nd 17 ( positions 53 nd 234 re highlighted in Supplementry Fig. 4; clevge from these residues including position 289 is not shown; results re identicl to position 17). OH nd T1 indicte 4.5S R lkline hydrolysis nd prtil T1 Rse clevge lnes, respectively, nd lnes 1 nd 2 re 4.5S R lone in uffer or in the presence of cleving regents, respectively. nd + indicte the sence nd presence of MPPP, respectively. R nd +R indicte the sence nd presence of cleving regents, respectively. In nd c, successive s in the R sequence re indicted t left, positions of strnd scission re indicted y the letters nd rs nd re further nlyzed in Supplementry Figure 3, nd strred nds represent hyperctive nucleotides tht re cleved in the sence of E-Fe modified nd were not included in the nlysis. (c) Titrtion clevge experiment with (E153) E-Fe leled SRP with incresing concentrtions of in the presence nd sence of MPPP. First four gel lnes re s in. Experiments were done with incresing concentrtions s follows: 0 nm, 50 nm, 100 nm, 1 mm, 4 mm, 16 mm nd25mm. ll lnes re in the presence of the clevge regents. 4.5S R upon TP-dependent ssocition of with. To our surprise, however, SRP complexes contining E-Fe modified site in the domin (residue 153) cleved 4.5S R t the sme positions s oserved with the complex contining E- Fe t residue 344. Furthermore, 4.5S R clevges resulting from the E-Fe t residue 153 occurred only in the presence of MPPP (Fig. 1, lnes 153; for quntifiction of clevge nds see Supplementry Fig. 3). These clevges re sturle s function of SRP complex formtion, using either shorter construct (residues ) lcking the memrne-ssocition () domin 18 or longer construct used in previous study (residues ) 19 (Fig. 1c nd Supplementry Fig. 3, respectively). ecuse the shorter construct hs similr TPse ctivity to tht mesured for the full-length 20 nd shows more roust complex formtion with SRP s ssyed y nondenturing gel moility shift (Supplementry Fig. 3), (residues ) ws used for ll further experiments. The identicl sites of 4.5S R clevge produced y E-Fe modified positions 344 nd 153 in the SRP complex re not consistent with the rchel SRP crystl structure, in which the sites nlogous to the E. coli positions 344 nd 153 re 51 Å prt, well eyond the E-Fe proing rdius of 20 Å (see Methods for our internl clirtion of the E-Fe proing rdius). These clevge dt suggest tht in the SRP complex, the M domin nd the tetrloop of the 4.5S R re ner the domin, with residues 344 nd 153 close together in spce. The lck of 4.5S R clevge from E-Fe modified positions 17 nd 289 in (Fig. 1,, position 17 lnes; position 289, dt not shown) supports positioning of the M domin djcent to the domin in n orienttion tht protects the R from free rdicls produced from these sites. In ddition, the oservtion tht E-Fe modifiction t positions 53 nd 234 did not yield ny 4.5S R clevge sites implies tht the M domin nd R loclize to c - heterodimer interfce 0 nm ~60 Å R 3 Signl peptide inding cleft 25 µm 25 µm 50 nm ( ) ( ) +MPPP MPPP 5 3 side Loop 5 side ( R,+) 409 (+R,+) 409 (+R, ) 344 ( R,+) 344 (+R,+) 344 (+R, ) 153 ( R,+) 153 (+R,+) 153 (+R, ) 17 ( R,+) 17 (+R,+) 17 (+R, ) one side of the SRP complex (Supplementry Fig. 4 online). Previous cross-linking studies suggested interction etween the M domin nd the domin in the sence of 4.5S R 21, implying tht the linker etween the nd M domins hs the required length to ccommodte this conformtionl rerrngement. Orienttion of R t the SRP-receptor heterodimer interfce To determine the position nd orienttion of 4.5S R with respect to the - heterodimer interfce, single cysteine residues were introduced into long the interfce surfce (Fig. 2). fter E-Fe conjugtion nd testing to confirm full modifiction nd ility to ind SRP s done with the proteins just discussed, ech mutnt protein ws ssemled into n SRP complex nd 4.5S R clevge ws nlyzed. otly, E-Fe modifiction of position 359 in, locted ner position 153 in the heterodimer structure (Fig. 2), results in R clevges ner the R tetrloop (nucleotides 55 57), where position 153 lso cleves (nucleotides 54 56) (Fig. 2). s oserved with position 153, E-Fe induced clevges from position 359 re sturle nd occur only in the presence of MPPP, indicting tht the R is only cleved upon TP-dependent formtion of the SRP complex. Further proing using contining E-Fe t positions 244, 433 or 392 under conditions in which the SRP complex is fully formed (Supplementry Fig. 2) produced, respectively, 4.5S R clevge sites successively frther wy from the tetrloop (Fig. 2c; for quntifiction of the clevge nds see Supplementry Fig. 5 online). In contrst, E-Fe modifiction of t positions 212 nd 322 on the ck side of the - interfce (Supplementry Fig. 4) produced no specific clevge ptterns in the 4.5S R, consistent with the clevge dt from E-Fe modified positions 53 nd 234 of (Fig. 2c, position 212 lnes; position 322 dt not shown). Mpping the clevge sites from ech E-Fe modified protein 3 side Loop 5 side TURE STRUTURL & MOLEULR IOLOY VOLUME 12 UMER 12 DEEMER

3 c TPse center Heterodimer interfce onto the 4.5S R tertiry structure defines specific orienttion for the ound R in which the highly conserved R tetrloop is djcent to the TPse domin; the other end of the R hirpin uts the domin (Fig. 2c, right pnel). otly, ll four E-Fe modified positions nd the 153 position tht elicited loclized 4.5S R clevge re locted on one side (the front) of the - heterodimer (Fig. 2). This suggests tht the R loclizes exclusively t or ner the front of the - heterodimer interfce, consistent with the sence of specific R clevge sites produced y E-Fe modifictions on the ck side of the interfce (Supplementry Fig. 4). Model of the SRP receptor ternry complex Using distnce constrints derived from the E-Fe induced clevge dt nd clirted using ville high-resolution crystllogrphic structures, moleculr model of the E. coli SRP complex ws constructed nd refined using torsion-ngle moleculr dynmics 22 (Fig. 3,; Supplementry Fig. 6 online; see Methods). In this model, nd shre the sme R-inding site, consistent with the oserved lck of chnges in the footprint of on the 4.5S R upon inding to 23. Furthermore, ll of the R-protein structurl contcts occur within the most conserved portion of the 4.5S R, consistent with the oservtion tht the 244 ( ) 244 (+) 433 ( ) 433 (+) 392 ( ) 392 (+) 359 ( ) 359 (+) 212 ( ) 212 (+) 3 side Loop 5 side nm 16 µm 16 µm Ftsy (359-E) 50 nm (359-E) ( ) ( ) +MPPP MPPP 3 positions 392 nd 359 clevge sites positions 244 nd 433 clevge sites 5 3 side Loop 5 side Figure 2 Hydroxyl rdicl proing of the 4.5S R y (residues ) in the E. coli SRP complex. () Structure of the T. quticus nd heterodimer showing the nlogous loctions of the E. coli residues selected for mutgenesis 10 (numers in prentheses represent the nlogous T. quticus residue numers). Residues in contrsting colors represent those E-Fe positions tht produced loclized R clevge sites; residues in white re positions tht did not cleve the R (see text for detils). () Titrtion clevge experiment with SRP nd incresing concentrtions of (359) E-Fe in the presence nd sence of MPPP. Lnes 1 nd 2 re s in Figure 1 ( OH nd T1 re omitted). Experiments were done with incresing (359) E-Fe concentrtions s follows: 0 nm, 50 nm, 100 nm, 1 mm, 4 mm nd16mm. In nd c, ll lnes re in the presence of the clevge regents, the clevge sites re indicted y letters nd rs nd strred nds represent hyper-rective nucleotides tht re cleved in the sence of E-Fe modified nd were not included in the nlysis. (c) Left, strnd scission of the full-length 4.5S R in sturted SRP complex y derivtized with E-Fe t positions 244, 433, 392, 359 nd 212 (Supplementry Fig. 4), respectively (for position 322 (see Supplementry Fig. 4), dt not shown; result is identicl to position 212). Positions of strnd scission re further nlyzed in Supplementry Figure 5. Right,the tertiry structure of the stem-loop end of the E. coli 4.5S R (nucleotides 29 76, ref. 17), where the colored nucleotides highlight the clevge sites. hirpin end of 4.5S R is necessry nd sufficient for SRP function in vivo 17. The proximity of the M domin to the nd domins is lso consistent with dt from fluorescence nd cross-linking studies showing high-ffinity interction etween isolted M nd domins nd domin shifts in upon inding to the 4.5S R nd to 24.Inddition, wek electron density for the linker region etween the M nd domins in previous crystl structure of the intct T. quticus protein suggested linker flexiility 25, nd cross-linking showed tht even in the sence of the 4.5S R, the M domin cn swing into the - interfce 21. SRP conformtionl chnge requires receptor inding The tethered hydroxyl rdicl proing dt nd resulting model show tht the 4.5S R ridges the - interfce, ut does locliztion of the R to this region occur efore or fter SRP complex formtion? One possiility is tht oth TP nd re necessry for the oserved conformtionl rerrngement in. lterntively, TP inding to lone 5 might e sufficient to induce the oserved conformtionl rerrngement, thus prepositioning the SRP for inding. To test these possiilities, contining E-Fe modified position 153 ws ound to 4.5S R with incresing concentrtions of MPPP in the presence nd sence of (Fig. 4). otly, in the sence of no loclized clevge of the 4.5S R with the intensities oserved in the previous experiments (Fig. 1)ws oserved (Fig. 4, lnes). However, s efore, upon ddition of the 1118 VOLUME 12 UMER 12 DEEMER 2005 TURE STRUTURL & MOLEULR IOLOY

4 Figure 3 Moleculr model of the E. coli SRP complex. () Model of the SRP complex ws constructed using the hydroxyl rdicl proing dt s sptil constrints to position the 4.5S R nd M domin in the complex s shown. nd re depicted in the spce-filling mode;, green;, lue; M domin, drk lue; signl peptide inding site, dshed circle; 4.5S R, gold. Those E-Fe modified positions tht cleved the 4.5S R re highlighted in contrsting colors, nd their corresponding clevge sites re indicted on the R in the corresponding colors (numers in prentheses represent the nlogous T. quticus residue numers). White residues represent positions tht filed to cleve the R. () Loction of the two E-Fe moieties t positions 153 nd 344, where the cleved TPse center Heterodimer interfce nucleotides re highlighted in red. The clevge pttern generted from these positions suggests plcement of the E-Fe in the R mjor groove leding to strnd scission in the two djcent minor grooves (in this cse one of the minor grooves is the R loop) 36. The sme nlysis ws done for the positions in Supplementry Figure 6. previously oserved R clevge sites reppered (Fig. 4, + lnes; see Supplementry Fig. 7 online for quntifiction of clevge nds). These results show tht the ssocition of SRP with its receptor cuses the mrked conformtionl rerrngement in the SRP, ringing the 4.5S R to the - interfce nd the ssocited M domin proximl to the TPse domins of nd. 4.5S R tetrloop ffects SRP receptor TPse ctivity The proximity of the 4.5S R tetrloop nd the nd TPse domins in the SRP model rised the possiility tht the evolutionrily conserved tetrloop nucleotides directly influence TPse ctivity of the SRP complex. Hydrolysis of TP y oth nd is known to e stimulted once the SRP complex is formed 26. In ddition, the sequence of the 4.5S R tetrloop ws previously shown to ffect formtion of the SRP complex 23. To test for possile effect of the tetrloop on TPse 5 onserved 4.5S R tetrloop Signl peptide inding site M domin onserved 4.5S R tetrloop M domin ctivity in the SRP complex, 4.5S R mutnt ws prepred in which the tetrloop ws mutted from to UU, chnge tht cuses lethl phenotype in vivo (F. Siu nd J.. Doudn, unpulished dt) 23. Under our rection conditions, this mutnt 4.5S R ssemles into stle SRP complex s detected y nondenturing gel moility shift (Fig. 4), in contrst to results from previous study 23. This difference in the ehvior of the complex tht we oserved could rise from n effectively higher concentrtion of MPPP in our experiments, owing to the use of chromtogrphiclly purified MPPP smple (see Methods). otly, the UU mutnt 4.5S R considerly reduced the rte of TP hydrolysis within the SRP complex to nerly the level of on its own (Fig. 4c). These dt support the proximity of the 4.5S R tetrloop nd the TPse ctive sites within the SRP complex, s proposed in our structurl model. Furthermore, the influence of the R tetrloop nucleotides on TP hydrolysis is intriguing ecuse the Fe Fe 1 mm 1 mm 0 nm MPPP 0 nm MPPP ( ) + ( ) 64 WT 4.5S R UU 4.5S R SRP- SRP SRP- SRP c Kos (min 1) S R 4.5S R [] (µm) Figure 4 (residues ) is required for the oserved SRP conformtionl rerrngement, nd the 4.5S R tetrloop ffects the heterodimer TPse ctivity. () Strnd scission of the full-length 4.5S R ound y derivtized y E-Fe t position 153 in the sence nd presence of with n incresing mount of MPPP (incresing MPPP concentrtions s follows: 0 nm, 10 nm, 100 nm, 500 nm, 100 mm, 500 mm nd1mm).el lels re s in Figure 1, nd ll protein lnes re in the presence of cleving regents. rrows indicte those lnes used in the quntifiction nlysis (see Supplementry Fig. 7). () el moility shift ssy of SRP contining the wild-type (WT) 4.5S R nd the UU tetrloop R with under similr rection conditions to those reported erlier (see Methods) 23. WT 4.5S R lnes re s follows: lne 1, WT 4.5S R lone; lne 2, WT 4.5S R nm ; lne 3, WT 4.5S R nm nm. UU 4.5S R lnes re the sme s the lnes for the WT 4.5S R gel except the UU tetrloop 4.5S R ws used. (c) TPse ctivity of the SRP complex. The oserved rte of the stimulted TP hydrolysis y the SRP complex ws determined with smll fixed mount of nd vrying concentrtions of (residues ) in the presence ( ) nd sence () of the 4.5S R nd in the presence of the UU tetrloop 4.5S R (&). The mximl rte constnts were s follows: 13.8 ± 0.95 min 1 in the presence of the 4.5S R, 1.11 ± 0.16 min 1 in the sence of the 4.5S R nd 1.24 ± 0.13 min 1 in the presence of the UU tetrloop 4.5S R. K 1/2 vlues were s follows: 5.91 ± 1.26 mm in the presence of the 4.5S R, 5.85 ± 2.62 mm in the sence of the 4.5S R nd 6.17 ± 2.04 mm inthe presence of the UU tetrloop 4.5S R. TURE STRUTURL & MOLEULR IOLOY VOLUME 12 UMER 12 DEEMER

5 loop is djcent to the signl peptide inding site within the ssocited M domin of 7. Together, these oservtions imply n R-medited connection etween the signl peptide inding nd regultory centers of the SRP complex. DISUSSIO The proposed conformtionl chnge in the SRP upon inding to its receptor explins severl oservtions out SRP function tht hve een puzzling in light of previous structurl nd iochemicl dt. First, our model sed on the tethered-proe clevge dt depicts n extended R interction surfce creted y oth the nd proteins, ridging the heterodimer interfce. otly, lone cnnot ind to 4.5S R, showing tht oth nd MPPP re necessry to crete stle R-recognition site (Supplementry Fig. 2) 26. The R conformtion induced upon M domin inding 17 my e prerequisite to 4.5S R interction, nlogous to SRP19 priming of the 7S R to ind the homolog SRP54 in the mmmlin SRP 27,28. The presence of the 4.5S R t the protein-protein interfce of the SRP complex, where it could influence protein structure or dynmics directly, my explin its 200-fold ctlytic effect on SRP- ssocition nd dissocition 6. Second, the loction of the M domin in the complex would position the ound nscent signl peptide directly djcent to the TPse domins of nd, coordinting productive peptide inding with TP hydrolysis y the SRP complex. The pprecile conformtionl rerrngement of nd 4.5S R orienttion discovered in this work is therefore plusile mechnism for direct communiction etween the signl peptide recognition site nd TPse domins of the SRP complex. Finlly, our results suggest function for the evolutionrily conserved tetrloop of the 4.5S R s structurl nd functionl link etween the signl peptide inding nd regultory centers of the SRP receptor complex. The plcement nd sequence of the 4.5S R tetrloop re thus of crucil importnce to SRP ctivity, helping to explin their high level of evolutionry conservtion 4. The, nd 4.5S R interctions clrified here involve SRP components conserved in higher orgnisms, implying tht the conformtionl rerrngement we descrie my lso occur in rchel nd eukryotic SRPs. In support of this possiility, previous cross-linking studies with the riosome-ound mmmlin SRP showed conformtionl rerrngements in SRP54 upon receptor inding to the complex 29. This suggests tht the oserved conformtionl rerrngement in the E. coli SRP could tke plce on the riosome. omprison of our structurl model to tht of the mmmlin SRP ound to the 80S riosome 30, determined y single-prticle cryo-em, revels some similrities. In the cryo-em structure, modeling of SRP54 on the riosome with ound signl peptide suggested tht the domin of SRP54 is oriented in etween the nd M domins nd djcent to the M domin ssocited SRP R 30,31. From this nlysis it ws predicted tht the signl peptide inding site my e positioned ner the TPse domin of SRP54, similr to our model of the E. coli SRP complex 32.Differencesintheplcementoftheconservedtetrloop reltive to the SRP54 domin in the two models my e due to the sence of the mmmlin SRP receptor in the cryo-em structure nd to the presence of dditionl R nd protein components in the mmmlin SRP. The conformtionl rerrngement we detect in the SRP receptor my occur on the riosome upon receptor inding or could e structurl chnge relevnt to the postriosome SRP complex. It is tempting to propose tht the oserved conformtionl chnge in the E. coli SRP upon receptor inding cts s moleculr switch to signl the riosome tht the SRP receptor is ound. Indeed, this conformtionl switch could lso influencetheffinityofthesrp complexfortheriosomlexit site, thus providing mechnistic precursor to shuttling the riosome to the trnslocon upon productive signl peptide nd receptor inding. METHODS Protein nd R expression nd purifiction. loning of the wild-type nd UU tetrloop 4.5S Rs ws done s descried in ref. 33. In these studies we used two different -terminlly truncted versions of (one construct consists of residues nd the other construct consists of residues ) nd -terminlly truncted version of (residues 1 432), which respectively form the heterodimer complex nd ind R with wild-type ffinities (dt not shown). loning of nd (residues ) contining n -terminl His 6 tg ws done s descried in ref. 33. protein-expression plsmid encoding (residues ) ws generously provided y Shu-ou Shn (liforni Institute of Technology, Psden, liforni, US). Site-directed mutgenesis of nd (residues ) ws crried out with the Quikhnge site-directed mutgenesis kit (Strtgene). The only endogenous cysteine in the SRP complex, t position 406 of, ws mutted to serine to produce cysteine-free protein 33. Incorportion of single cysteines into nd ws verified y sequencing of ll constructs. (residues ) ws expressed nd purified s descried in ref. 5. The ntive nd single-cysteine nd (residues ) proteins were expressed nd purified s follows. The nd expression vectors were trnsformed into E. coli L21 (DE3) cells expressing plyss. Freshly trnsformed cells were grown in 750 ml L medium supplemented with 100 mg ml 1 mpicillin nd 34 mg ml 1 chlormphenicol t 37 1 until the sornce t 600 nm reched 0.4. Protein expression ws induced with 0.5 mm IPT, nd the cells were grown t 28 1 for n dditionl 3.5 h. ells were hrvested y centrifugtion (7,000 r.p.m. in eckmn J-10 rotor) nd resuspended in cell lysis uffer (50 mm Tris (ph 7.5), 300 mm l, 10 mm Mgl 2, 10% (v/v) glycerol, 5 mm -mercptoethnol (-ME), 1% Triton X-100 (v/v)). Lysozyme (40 mg ml 1 finl concentrtion) ws dded to the cell resuspension, which ws then frozen t ell resuspensions were thwed t 37 1 for 10 min, nd 20 mm MgSO 4, 2 mm l 2, 100 units of Rse-free Dse, 50 mgml 1 PMSF nd 10 mg ml 1 leupeptin were dded nd incuted for 30 min t 4 1, then for 20 min t ell resuspensions were centrifuged t 10,000g for 1 h, nd the cell lystes were removed. rude cell lystes were pplied to 5-ml ed volume contining Superflow i 2+ resin (Qigen) equilirted with the following uffer: 50 mm Tris (ph 7.5), 300 mm l, 10% (v/v) glycerol, 5 mm -ME, 5 mm imidzole). The nickel nitrilotricetic cid slurry ws wshed with five column volumes of the uffer just descried nd five column volumes of the sme uffer supplemented with 10 mm imidzole. The proteins were eluted y dding five column volumes of the sme uffer supplemented with 400 mm imidzole nd nlyzed y SDS-PE. To remove the histidine tg, 1 mg of recominnt His 6 -tgged tocco etch virus (TEV) protese ws dded to the nickel-purified proteins nd incuted t 4 1 for 48 h in TEV clevge uffer (50 mm Tris, ph 7.5, 300 mm l, 10% (v/v) glycerol, 5 mm -ME). The TEV rections were pplied to 5-ml ed volume contining Superflow i 2+ resin (Qigen) equilirted with uffer (50 mm Tris (ph 7.5), 300 mm l, 10% (v/v) glycerol, 5 mm -ME, 5 mm imidzole) to remove the cleved histidine tg nd TEV protese. leved smples were pplied to Mono-S column (Phrmci) equilirted in uffer (50 mm Tris (ph 8.0), 10% (v/v) glycerol, 5 mm DTT, 0.5 mm EDT). ws eluted using 17 column volume liner grdient to 100% uffer (50 mm Tris (ph 8.0), 1 M l, 10% (v/v) glycerol, 5 mm DTT, 0.5 mm EDT). leved smples were dilyzed into the following uffer overnight t 4 1: 50 mm MES (ph 6.0), 100 mm l, 10 mm Mgl 2, 10% (v/v) glycerol. They were then pplied to Mono-S column equilirted in uffer (50 mm MES (ph 6.0), 10 mm Mgl 2, 10% (v/v) glycerol) nd eluted using 16 column volume liner grdient to 100% uffer D (50 mm MES (ph 6.0), 1Ml,10mMMgl 2, 10% (v/v) glycerol). Mono-S frctions were nlyzed y SDS-PE, comined nd concentrted to finl concentrtion of mm. Protein concentrtions were estimted y io-rd protein ssys. ll 1120 VOLUME 12 UMER 12 DEEMER 2005 TURE STRUTURL & MOLEULR IOLOY

6 purified proteins were dilyzed overnight t 4 1 into storge uffer (50 mm Tris (ph 7.5), 300 mm l, 20% (v/v) glycerol, 1 mm DTT) nd stored t E-Fe modifiction of single-cysteine proteins. onjugtion of the singlecysteine nd proteins with the nucleic cid cleving regent E- Fe ws done initilly y incuting 1 ml of mm protein solution with 1 mm DTT for 5 min t room temperture efore 3-h dilysis into degssed modifiction uffer (50 mm Tris (ph 8.0), 300 mm l, 10% (v/v) glycerol) until ll of the DTT hd een removed. Ech 1 ml of protein solution ws divided into two 500-ml smples, of which one ws mixed with 2 ml of 250 mm E-Fe solution (in DMSO) nd the other ws mixed with 2 ml DMSO. Ech protein smple ws incuted t room temperture for 6 h in the drk. Once the conjugtion rection ws complete, 49 ml of ech rection ws removed nd rected with 1 ml of 1.5 mm fluorescein nd incuted t room temperture for 20 min in the drk. Ech fluorescein rection ws filtered to remove ny unrected fluorescein using Micro io-spin 6 columns (io-rd), nd 10 ml of these rections were pplied to 10% SDS-PE gel. The extent of E-Fe conjugtion ws nlyzed on 310-nm UV tle (Supplementry Fig. 1) nd y electrospry ioniztion mss spectrometry. The residul E- Fe conjugtion rections were quenched y removing ny excess E-Fe y dilysis overnight into storge uffer (50 mm HEPES (ph 7.5), 150 mm potssium cette, 1.5 mm mgnesium cette). E-Fe modified proteins were stored t SRP complex formtion. The complex etween E-Fe modified, nd 4.5S R ws oserved using gel moility shift ssys (Supplementry Fig. 2). 4.5S R (300 nm) ws incuted with E-Fe or ntive (1 mm) in uffer contining 50 mm HEPES (ph 7.5), 150 mm potssium cette, 1.5 mm mgnesium cette nd 1 mm MPPP for 5 min t room temperture to form the SRP. R ws heted to 90 1 for 1 min nd cooled on ice for 1 min efore use. E-Fe or ntive (4 mm) ws incuted with the SRP t room temperture in the drk for 30 min (30 ml finl rection volume). fter the incution, 10 ml of n 80% (v/v) glycerol solution ws dded to the inding rections nd loded onto 6% nondenturing gel. omplexes were visulized y SYR old stining (Moleculr Proes) on 310-nm UV tle. For the gel moility shift ssy of SRP contining the wildtype 4.5S R nd the UU tetrloop R (Fig. 4), 1 nm wild-type or UU tetrloop 4.5S R in the presence of MPPP (200 mm, Mono-Q purified) ws used with 0.9 nm nd 1.1 nm (residues ) in 20-ml finl rection volume. el moility shift ssys of this kind were lso conducted under the kinetic ssy conditions, nd SRP complexes were oserved with oth Rs (dt not shown). ffinity cleving ssys. SRP complexes were preformed s descried erlier except tht the inding rections were spiked with g- 32 P5 end leled 4.5S R. fter incution s descried erlier, clevge of the R ws initited y the ddition of hydrogen peroxide nd sodium scorte (finl concentrtion of 0.01% (v/v) nd 5 mm, respectively) followed y 2-min incution t room temperture. Distilled wter ws dded to ech smple, nd two successive phenol extrctions nd ethnol precipittion of the R were then performed. leved 4.5S R ws resuspended in formmide loding dye nd nlyzed on 12% denturing gel. Dt were otined from the gels using storge phosphor utordiogrphy nd STORM PhosphorImger (Moleculr Dynmics). Quntifiction of clevge nds. E-Fe clevge sites were nlyzed nd quntified using procedures descried efore 34. riefly, individul nd intensities were quntitted y PhosphorImger nlysis, wherein trces were drwn down the clevge lnes nd their corresponding ckground lnes (see Supplementry Figs. 3, 5 nd 7). SRP complex model uilding nd refinement. SWISS-MODEL ( swissmodel.expsy.org//swiss-model.html) ws used to generte the structurl model of the E. coli - heterodimer using the T. quticus structure s templte (PD entry: 1RJ9) 10. The model, which contins residues of nd of, ws further refined using torsion-ngle moleculr dynmics with S 22 to remove minor steric clshes. Inspection of the E. coli M domin ound to the 4.5S R structure (PD entry: 1DUL) 17 revels tht the proing distnces etween the tom of the E-Fe leled ln409 nd the 2 -hydroxyl of the cleved R ses re in the rnge of 17.5 ± 7.5 Å. This oservtion ws used s moleculr ruler to clirte the rest of the distnce constrints etween the 4.5S R nd E- Fe modified residues on nd proteins. n OE signl ws ssigned etween the tom of the leled residue nd the 2 -hydroxyl of the R se for ech of the clevge signls with the verge distnce, d minus nd d plus vlues set to 17.5 Å, 7.5Ånd 7.5 Å, respectively, using the r 6 verging option. The initil structurl model ws generted using restrined rigid ody refinement 35. The domin heterodimer nd the M domin R complex were treted s two rigid odies, whose position nd orienttion were systemticlly vried until ll OE constrints were stisfied. The model ws further refined y introducing dditionl OE constrints etween residues 393 nd 400 of the M domin nd residues 17 nd 289 of the domin. The resulting structurl model ws refined using restrined torsion-ngle moleculr dynmics to resolve minor steric clshes etween vrious prts of the model. The - heterodimer nd M domin R complexes were refined in different strting orienttions nd sptil rrngements, which produced similr structurl models fter refinement. The M domin finger loop from the T. quticus crystl structure (PD entry: 2FFH) 25 ws used to model the missing counterprt in the E. coli crystl structure (Leu338 sp370). Similrly, residues ly293 lu318 in the Sulfolous solftricus SRP crystl structure (PD entry: 1QZW) 8 were used to model prt of the missing linker (ly296 Ser321) connecting the M domin nd the domins of the E. coli. The two structures were ligned, nd the linker in the S. solftricus structure ws then moved to pck ginst the M domin in the model using simple swinging motion. n dditionl 7-residue linker ws engineered to model the rest of the linker (Lys322 ly338) lcking in the E. coli model. TPse ctivity ssys. The TPse ctivity of the SRP complex ws mesured in severl turnover rections s descried in ref. 5. riefly, ll TPse rections were crried out t 25 1 in the following uffer: 50 mm HEPES (ph 7.5), 150 mm potssium cette, 1.5 mm mgnesium cette, 0.01% (v/v) ikkol nd 2 mm DTT. The oserved rte of the stimulted TP hydrolysis y the SRP complex ws determined with smll fixed mount of nd vrying concentrtions of (residues ) in the presence nd sence of the 4.5S R (1 mm nd5mm with nd without R present, respectively) nd in the presence of the UU tetrloop 4.5S R (5 mm ). two-fold excess of R reltive to ws used in this nlysis (2 mm wild-type 4.5S R nd 10 mm UU tetrloop 4.5S R). (residues ) concentrtions used were s follows: 1, 2, 5, 10, 20 nd 30 mm. The rections were initited y dding 100-mM finlconcentrtionof fst protein liquid chromtogrphy purified TP doped with gel-purified [g- 32 P]TP, nd ech rection ws monitored nd kinetic rtes were estimted s descried in ref. 5. ote: Supplementry informtion is ville on the ture Structurl & Moleculr iology wesite. KOWLEDMETS We thnk I.J. McRe for helpful discussions on the E-Fe modifiction sites of nd nd K. Krstein, W. ilert,.s. Frser,.H. hmiel, J.W. Hershey nd H.F. oller for review of the mnuscript. The work ws supported y grnt M22778 from the US tionl Institutes of Helth. OMPETI ITERESTS STTEMET The uthors declre tht they hve no competing finncil interests. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/ 1. Keenn, R.J., Freymnn, D.M., Stroud, R.M. & Wlter, P. The signl recognition prticle. nnu. Rev. iochem. 70, (2001). 2. Doudn, J.. & tey, R.T. Structurl insights into the signl recognition prticle. nnu. Rev. iochem. 73, (2004). 3. rown, S. & Fournier, M.J. The 4.5S R gene of Escherichi coli is essentil for cell growth. J. Mol. iol. 178, (1984). TURE STRUTURL & MOLEULR IOLOY VOLUME 12 UMER 12 DEEMER

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