Structural evidence for consecutive -like modules in the spliceosomal ATPase Brr2 Lingdi Zhang, Tao Xu, Corina Maeder, Laura-Oana Bud, James Shanks, Jay Nix, Christine Guthrie, Jeffrey A. Pleiss, Rui Zhao
-S2 1851 -S1 1003 484 -S2 1909 -S1 1061 540 -S2 1957 -S1 1108 600 -S2 2012 -S1 1163 653 -S2 2071 -S1 1222 α1 α2 α3 α4 LIASHYGVSF FTIQSFVSSL SNTSTLK-NM LYVLSTAVEF ESVPLRKGDR ALLVKLS-KR NIASSFYINH ASMDVYNREL DEHTTQI-DL FRIFSMSEEF KYVSVRYEEK RELKQLL-EK HHH HHHHHHHHHH HH HH HHHHHHHHHH HHH HHHHHHH HH SRLYIDP LTGFIFHDVL SRMELSDIGA LHLICRTPDM ERLTVRKTD- SWVEEEAFRL α1 α2 α3 α5 α6 LPLRFPEHT- ---SSGS--V SFKVFLLLQA YFSRL---EL PV--DFQN-D LKDILEKVVP APIPIR--E- ---DIDD--P LAKVNVLLQS YFSQL--KFE GF--ALNS-D IVFIHQNAGR HHHHHHHH HHHHH H HH H HHHHHHHHHH RKELSYYPSD FSVEYDWFLS EVKTALCLKD WIEEKDEDEI CAKYGIAPGD LRRIVETAEW α4 α5 α6 α6 α7 α8 α9 LINVVVDILS ANGYLN-ATT ATT AMDLAQMLIQ GVWDVDNPLR QIPHF----N NKILEKCKEI LLRAMFEICL KRGWGHPTRM LLNLCKSATT KMWPTNCPLR QFKTC----P VEVIKRLEA- HHHHHHHHHH HH HHHHH HHHHHHHHHH HHH HHHHHHHH LSNAMNRIAE E--VGN-T-S VSGLTERIKH GVKEELLELV RIRHIGRVRA RKLYNAG--- α6 α7 α8 α9 α10 α11 α12 β1 β2 NVETVYDIMA LE-DEERDEI LTLTDSQLAQ VAAFVNNYPN VELTYSLNNS DSLISGVKQK STVPWGDYLQ LETPAEVGRA IRSE-KYGKQ VYDLLKRFPK MSVTCNAQPI TRSVMRFNIE HHHHHh H EEE EE HHHH HHHHH EEEEEEE EEEEEEEEE -IRNAEDIVR H--REKVASL I-----GRGI AERVVEGISV KS 686 α10 α11 α12 β2 β3 β4 β5 ITIQLTRDVE PENLQVTSEK YPFDKLESWW LVLGEVSKKE LYAIKKVTLN KET--QQYEL IIADWIWDMN VHGS------ -----LEPFL LMLEDTPGDS ILYYDVLFIT PDIVGHEFTL EEEEEEE EEE EEEE EEEEEEEEEE EEEE -S2 2129 -S1 1270 β6 β7 EFDTPTSG - KHNLTIWCVC DSYLDADKEL SFEINVK SFTYELKQHL PPNFFLTLIS ENWWHSEFEI PVSFN EEE EEEEEEEE E E Supplementary Fig. 1
Supplementary Figure 1. Sequence alignment of S2, S1, and domains 4 & 5. The alignment between S2 and is structure-based 1 and that between S1 and S2 is taken from Ponting 2. Secondary structures of S2 and derived from the crystal structures are labeled. Predicted secondary structures for S2 and S1 2 are also labeled as H (helix) and E (beta strands). Blue, green, and purple residues designate the N-terminal helical domain, the middle helical domain, and the C-terminal Fn3 domain in S2.
ys2 ys2 ys2 ys2 ys2 ys2 10 20 30 40 50 60 ------LIASHYGVSFFTIQSFVSSLSNTSTLKNMLYVLSTAVEFESVPLRKGDRALLVK APLNLGMIAAYYYINYTTIELFSMSLNAKTKVRGLIEIISNAAEYENIPIRHHEDNLLRQ 70 80 90 100 110 120 LSKRLPLRFPEHTSSGSVSFKVFLLLQAYFSRLELPVDFQNDLKDILEKV---VPLINVV LAQKVPHKL-NNPKFNDPHVKTNLLLQAHLSRMQLSAELQSDTEEILSKAIMDVRLIQAC 130 140 150 160 170 180 VDILSANGYLN-ATTAMDLAQMLIQGVWDVDNPLRQIPHFNNKILEKCKEINVETVYDIM VDVLSSNGWLSPALAAMELAQMVTQAMWSKDSYLKQLPHFTSEHIKRCTDKGVESVFDIM 190 200 210 220 230 240 ALEDEERDEILTLTDSQLAQVAAFVNNYPNVELTYSLNNSDSLISGVKQKITIQLTRDVE EMEDEERNALLQLTDSQIADVARFCNRYPNIELSYEVVDKDSIRSGGPVVVLVQLEREEE 250 260 270 280 290 300 PENLQVTSEKYPFDKLESWWLVLGEVSKKELYAIKKVTLNKETQQYELEFDTPTSGKHNL VTG-PVIAPLFPQKREEGWWVVIGDAKSNSLISIKRLTLQQKAK-VKLDFVAPATGAHNY 310 320 330 TIWCVCDSYLDADKELSFEINVK--------- TLYFMSDAYMGCDQEYKFSVDVKEAETDSDSD Supplementary Figure 2. Sequence alignment between yeast S2 (ys2, residues 1851-2163) and human S2 (, residues 1811-2136) domains performed using program MULTALIN 3. Red and blue colors represent identical and similar residues, respectively.
a dsdna Supplementary Fig. 3 β-hairpin Domain 2 ssdna 2P6R Motif V β hairpin in Motif VI b 335-GVNLPARRVIVRSLYRFD---GYSKRIKVSEYKQMAGRAGR-372 Brr2 837-GVNLPAHTVIIKGTDVYSPEKGSWEQLSPQDVLQMLGRAGR-877 G GS S Q Q G G 877 eif4a LISTDLLARGID.... YIHRIGRGGRFG c WT brr2-3gs WT brr2-3gs 30 o C 18 o C MW (kda) 250 130 Brr2 Snu114 Prp8 d 250 130 WT brr2-3gs 37 o C 95 72 55 95 72 5 serial dilution
Supplementary Figure 3. (a) A prominent β-hairpin (red) between motifs V and VI in domain 2 of (PDB ID 2P6R 23 ) inserts into the dsdna and disrupts base-pairing i of the DNA duplex. Domains 1-4 are colored in dark blue, light blue, green, and purple, respectively. (b) Sequence alignment between and Brr2 in the putative β-hairpin region. Motifs V, VI, and the β-hairpin of are labeled, and identical residues between the two proteins are colored red. Motifs V and VI in DEAD-box protein eif4a (red designates residues conserved in many DEAD-box proteins) are also shown. Residues that have been changed to GSGSGS in Brr2 are underlined. (c) brr2-3gs mutant has impaired growth in all temperatures. In the right panel, Brr2 proteins in yeast cell extracts were pulled down using IgG resin and probed on a Western blot using anti-brr2, anti-prp8, and anti-snu114 antibodies. Note that Brr2 appears larger because it contains the Protein A tag. (d) SDS PAGE of purified Brr2 WT and Brr2-3GS mutant.
10 20 30 40 50 60 MTEHETKDKAKKIREIYRYDEMSNKVLKVDKRFMNTSQNPQRDAEISQPKSMSGRISAKD 70 80 90 100 110 120 MGQGLCNNINKGLKENDVAVEKTGKSASLKKIQQHNTILNSSSDFRLHYYPKDPSNVETY 130 140 150 160 170 180 EQILQWVTEVLGNDIPHDLIIGTADIFIRQLKENEENEDGNIEERKEKIQHELGINIDSL 190 200 210 220 230 240 KFNELVKLMKNITDYETHPDNSNKQAVAILADDEKSDEEEVTEMSNNANVLGGEINDNED 250 260 270 280 290 300 DDEEYDYNDVEVNSKKKNKRALPNIENDIIKLSDSKTSNIESVPIYSIDEFFLQRKLRSE 310 320 330 340 350 360 LGYKDTSVIQDLSEKILNDIETLEHNPVALEQKLVDLLKFENISLAEFILKNRSTIFWGI 370 380 390 400 410 420 RLAKSTENEIPNLIEKMVAKGLNDLVEQYKFRETTHSKRELDSGDDQPQSSEAKRTKFSN 430 440 450 460 470 480 PAIPPVIDLEKIKFDESSKLMTVTKVSLPEGSFKRVKPQYDEIHIPAPSKPVIDYELKEI MKVEEL 490 500 510 520 530 540 T-SLPDWCQEAFPSSETTSLNPIQSKVFHAAFEGDSNMLICAPTGSGKTNIALLTVLKAL AESISSYAVGILKEEGIEELFPPQAEAVEKVFSG-KNLLLAMPTAAGKTLLAEMAMVREA motif I 550 560 570 580 590 600 SHHYNPKTKKLNLSAFKIVYIAPLKALVQEQVREFQRRLAFLGIKVAELTGDSRLSRKQI ------------IKGGKSLYVVPLRALAGEKYESF-KKWEKIGLRIGISTGDYESRDEHL 610 620 630 640 650 660 DETQVLVSTPEKWDITTRNSNNLAIVELVRLLIIDEIHLLHDD-RGPVLESIVARTFWAS GDCDIIVTTSEKADSLIRN--RASWIKAVSCLVVDEIHLLDSEKRGATLEILVTKMRRMN II 670 680 690 700 710 720 KYGQEYPRIIGLSATLPNYEDVGRFLRVPKEGLFYFDSSFRPCPLSQQFC--GIKERNSL KA----LRVIGLSATAPNVTEIAEWLDAD-----YYVSDWRPVPLVEGVLCEGTLELFDG III 730 740 750 760 770 780 KKLKAMNDACYEKVLESINEGNQIIVFVHSRKETSRTATWLKNKFAEENITHKLTKNDAG AFSTSRRVKFEELVEECVAENGGVLVFESTRRGAEKTAVKLSA------ITAKYVENEGL IV
790 800 810 820 830 840 SKQILKTEAANVLDPSLRKLIESGIGTHHAGLTRSDRSLSEDLFADGLLQVLVCTATLAW EKAILE-ENEGEMSRKLAECVRKGAAFHHAGLLNGQRRVVEDAFRRGNIKVVVATPTLAA 850 860 870 880 890 900 GVNLPAHTVIIKGTDVYSPEKGSWEQLSPQDVLQMLGRAGRPRYDTFGEGIIITDQSNVQ GVNLPARRVIVRSLYRFD---GYSKRIKVSEYKQMAGRAGRPGMDERGEAIIIVGKRDRE V VI 910 920 930 940 950 960 YYLSVLNQQLPIESQFVSKLVDNLNAEVVAGNIKCRNDAVNWLAYTYLYVRMLASPMLYK --IAVKRYIFGEPERITSKLGVETHLRFHSLSIICDGYAKTLEELEDFF----ADTFFFK 970 980 990 1000 1010 1020 VPDISSDGQLKKFRESLVHSALCILKEQELVLYDAENDVIEATDLGNIASSFYINHASMD QNEISLSYELERVVRQLENWGMVV-----------EAAHLAPTKLGSLVSRLYIDPLTGF 1030 1040 1050 1060 1070 1080 VYNRELDEHTTQIDLFRIFSMSEEFKYVSVRYEEKRELKQLLEKAPIPIREDIDDPLAKV IFHDVL----SRMELSDIGALHLICRTPDMERLTVRKTDSWVEEEAFRLRKELSYYPSDF 1090 1100 1110 1120 1130 1140 NVLLQSYFSQLKFEGFALNSDIVFIHQNAGRLLRAMFEICLKRGWGHPTRMLLNLCKSAT SVEYDWFLSEVK-TALCLKDWIEEKDED---------EICAK--YGIAPGDLRRIVETAE 1150 1160 1170 1180 1190 1200 TKMWPTNCPLRQFKTCPVEVIKRLEASTVPWGDYLQLETPAEVGRAIRSEKYGKQVYDLL WLSNAMNRIAEEVGNTSVSGLTERIKHGVK-EELLELVRIRHIGRVRARKLYNAGIRNA- 1210 1220 1230 1240 1250 1260 KRFPKMSVTCNAQPITRSVMRFNIEIIADWIWDMNVHGSLEPFLLMLEDTDGDSILYYDV -----EDIVRHREKVASLIGRGIAERVVEGISVKSLNPES 1270 1280 1290 1300 1310 1320 LFITPDIVGHEFTLSFTYELKQHNQNNLPPNFFLTLISENWWHSEFEIPVSFNGFKLPKK 1330 1340 1350 1360 1370 1380 FPPPTPLLENISISTSELGNDDFSEVFEFKTFNKIQSQVFESLYNSNDSVFVGSGKGTGK MKVEELAESISSYAVGILKEEGIEELFPP-QAEAVEKVF-SGKNLLLAMPTAAGK I 1390 1400 1410 1420 1430 1440 TAMAELALLNHWRQNKGRAVYINPSGEKIDFLLSDWNKRFSHLAGGKIINKLGNDPSLNL TLLAEMAMVREAIKG-GKSLYVVPLRALAGEKYESFKKW--EKIGLRIGISTGDYESRD- 1450 1460 1470 1480 1490 1500 KLLAKSHVLLATPVQFELLSRRWRQRKNIQSLELMIYDDAHEISQGVYGAVYETLISRMI EHLGDCDIIVTTSEKADSLIRN--RASWIKAVSCLVVDEIHLLDSEKRGATLEILVTKM- II
1510 1520 1530 1540 1550 1560 FIATQLEKKIRFVCLSNCLANARDFGEWAGMTKSNIYNFSPSERIEPL--EINIQSFKDV ---RRMNKALRVIGLSATAPNVTEIAEWLD-ADYYVSDWRPVPLVEGVLCEGTLELFDGA III 1570 1580 1590 1600 1610 1620 EHISFNFSMLQMAFEASAAAAGNRNSSSVFLPSRKDCMEVASAFMKFSKAIEWDMLNVEE FSTSRRVKFEELVEECVAENGGVLVFESTRRGAEKTAVKLSAITAKY---VENEGL---E IV 1630 1640 1650 1660 1670 1680 EQIVPYIEKLTDGHLRAPLKHGVGILYKGMASNDERIVKRLYEYGAVSVLLISKDCSAFA KAILEENEGEMSRKLAECVRKGAAFHHAGLLNGQRRVVEDAFRRGNIKVVVATPTLAAGV 1690 1700 1710 1720 1730 1740 -CKTDEVIILGTNLYDGAEHKYMPYTINELLEMVGLASGNDSMAGKVLILTSHNMKAYYK NLPARRVIVRSLYRFDGYSKRIKVSEYKQMAGRAG-RPGMDERGEAIIIVGKRDREIAVK V VI 1750 1760 1770 1780 1790 1800 KFLI-EPLPTESYLQYIIHDTLNNE--IANSIIQSKQDCVDWFTYSYFYRRIHVNPSYY- RYIFGEPERITSKLGVETHLRFHSLSIICDGYAKTLEELEDFFADTFFFKQNEISLSYEL 1810 1820 1830 1840 1850 1860 --GVRDTSPHGISVFLSNLVETCLNDLVESSFIEIDDTEAEVTAEVNGGDDEATEIISTL ERVVRQLENWGMVVEAAHLAPTKLGSLV--SRLYIDPLTGFIFHDVLSRM-ELSDIGALH 1870 1880 1890 1900 1910 1920 SNGLIASHYGVSFFTIQSFVSSLSNTSTLKNMLYVLSTAVEFESVPLRKGDRALLVKLSK LICRTPDMERLTVRKTDSWVEEEAFRLRKELSYYPSDFSVEYDWF-LSEVKTALCLK--- 1930 1940 1950 1960 1970 1980 RLPLRFPEHTSSGSVSFKVFLLLQAYFSRLELPVDFQNDLKDILEKVVPLINVVVDILSA ----DWIEEKDEDEICAKYGIA--------------PGDLRRIVETAEWLSNAMNRIAEE 1990 2000 2010 2020 2030 2040 NGYLNATTAMDLAQMLIQGVWDVDNPLRQIPHFNNKILEKCKEINVETVYDIMALEDEER VGN---TSVSGLTERIKHGVKEELLELVRIRHIGRVRARKLYNAGIRNAEDIVRHREKVA 2050 2060 2070 2080 2090 2100 DEILTLTDSQLAQVAAFVNNYPNVELTYSLNNSDSLISGVKQKITIQLTRDVEPENLQVT SLIGRGIAERVVEGISVKSLNPES------------------------------------ 2110 2120 2130 2140 2150 2160 SEKYPFDKLESWWLVLGEVSKKELYAIKKVTLNKETQQYELEFDTPTSGKHNLTIWCVCD ------------------------------------------------------------ 2170 SYLDADKELSFEINVK ----------------
Supplementary Figure 4. Sequence alignment between and Archaeoglobus fulgidus performed using program MULTALIN 3. Red and blue indicate identical and similar residues, respectively. Major helicase motifs of are underlined. Notice that the second putative helicase domain of Brr2 is much more deviated from the canonical helicase motifs. Also notice that there is very low sequence similarity in regions outside of the helicase domain (i.e., the S1 and S2 regions) and the sequence alignment in these regions is not very accurate. A much more accurate alignment based on the structure of S2 and is presented in Supplementary Fig. 1. References: 1. Holm, L. & Sander, C. Dali: a network tool for protein structure comparison. Trends Biochem Sci 20, 478-80 (1995). 2. Ponting, C.P. Proteins of the endoplasmic-reticulum-associated degradation pathway: domain detection and function prediction. Biochem J 351 Pt 2, 527-35 (2000). 3. Corpet, F. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16, 10881-90 (1988).