Nature Structural & Molecular Biology: doi:10.1038/nsmb€¦ · 7,13563 (2016) ) and (c) DnaK...

Supplementary Figure 1

Pulldown analysis of Ssz1 with Zuo1 or Zuo1N and purification of the Ssz1–Zuo1N complex for crystallization.

(a) MBP-pulldown analysis of Ssz1 with MBP-Zuo1 or MBP-Zuo1N and of the coexpressed Ssz1-MBP-Zuo1N complex. (b) Uncropped SDS-PAGE of Fig. 1c showing the SEC fractions of the Ssz1-Zuo1N complex.

Nature Structural & Molecular Biology: doi:10.1038/nsmb.3349


Packing of the two molecules in the asymmetric unit.

Within the two molecules (Ssz1: gray and purple, Zuo1N: orange; cartoon representation) in the asymmetric unit, several regions are disordered. The disordered regions are depicted as dotted lines and the residues numbered. The N-, C-terminus and domains are indicated. ATP is represented as sticks and Mg

2+ is shown as a pink sphere.



Electron density map of typical regions in the Ssz1–Zuo1N complex and B factor plot for the Ssz1–Zuo1N structure.

(a) Stereoview of the Zuo1N EPVG motif (orange, yellow sticks). The 2Fo-Fc electron density map is contoured at 1 . (b) Stereoview

of the nucleotide bound to Ssz1. ATP (sticks), Mg2+

(green sphere) and water molecules (red spheres). The 2Fo-Fc electron density

map is contoured at 2 . (c) Ribbon representation of the Ssz1-Zuo1N complex with Ssz1 (left) and Zuo1N (right) colored according to

B-factors, scaled from 50 (blue) to 250 (red) Å2. ATP is represented as sticks. NBD and SBD are indicated.



Surface analyses of Hsp70 proteins (with bound ATP).

Comparison of electrostatic surface potentials of (a) Ssz1, (b) Ssb1 (Gumiero, A. et al. Interaction of the cotranslational Hsp70 Ssb with ribosomal proteins and rRNA depends on its lid domain. Nat. Commun. 7,13563 (2016)) and (c) DnaK (Kityk, R., Kopp, J., Sinning, I. & Mayer, M.P. Structure and dynamics of the ATP-bound open conformation of Hsp70 chaperones. Mol. Cell 48, 863-74 (2012)). The structures have been superimposed on the NBD and are shown side by side. The positively charged patch observed in Ssz1 is absent in canonical Hsp70s. (d) Close-up view of the positively charged patch in Ssz1. Surface charge spans from -4 kT/e (deep red) to +4kT/e (deep blue). (e) and (f) Conservation surface mapping of the Ssz1 patch shown in (d) as reported by ConSurf (Landau, M. et al. ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Res. 33, W299-302 (2005)), from variable (cyan) to conserved (deep red). Figures were produced using PDB2PQR (Dolinsky, T.J. et al. PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations. Nucleic Acids Res. 35, W522-5 (2007); Dolinsky, T.J., Nielsen, J.E., McCammon, J.A. & Baker, N.A. PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic Acids Res. 32, W665-7 (2004)) and APBS (Baker, N.A., Sept, D., Joseph, S., Holst, M.J. & McCammon, J.A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. U S A 98, 10037-41 (2001)) in PyMOL (Schrödinger, L.L.C. The PyMOL Molecular Graphics System, Version 1.8. (2015)).



Comparison of Ssz1–Zuo1N and DnaK to show the different domain arrangement.

Ribbon representation of (a) the Ssz1 inter-domain linker (dark blue), Ssz1 SBD (pale cyan), Zuo1N (orange), and (b)DnaK SBD

(green, 4B9Q (Kityk, R., Kopp, J., Sinning, I. & Mayer, M.P. Structure and dynamics of the ATP-bound open conformation of Hsp70

chaperones. Mol. Cell 48, 863-74 (2012)). Both structures have been superimposed on the NBD. NBD and SBD are indicated and the

NBDs are shown in surface representation (gray). SBD binds to different lobes of the NBD.



Superposition of the SBDs of Ssz1 (with bound Zuo1N) and DnaK (ADP, substrate).

Ssz1-Zuo1N and DnaK are shown in ribbon representation, with Zuo1N in orange and Ssz1 SBD in pale cyan, and DnaK in green

(bound-substrate is in yellow; 1DKY (Zhu, X. et al. Structural analysis of substrate binding by the molecular chaperone DnaK. Science

272, 1606-14 (1996)). The Ssz1 specific insertion in 4– 5 would interfere with the lid closure observed in canonical Hsp70s.



Comparison of the substrate binding domains from Ssz1, DnaK (closed and open) and Ssb.

(a) Schematic representation of the twisted -sandwich, (b) ribbon representation of the structures of Ssz1 SBD –Zuo1N (pale cyan,

orange), DnaK-ADP SBD (Zhu, X. et al. Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272,

1606-14 (1996); green, sand) with bound substrate (yellow), DnaK-ATP SBD (Kityk, R., Kopp, J., Sinning, I. & Mayer, M.P. Structure

and dynamics of the ATP-bound open conformation of Hsp70 chaperones. Mol. Cell 48, 863-74 (2012); green, sand), and Ssb1-ATP

SBD (Gumiero, A. et al. Interaction of the cotranslational Hsp70 Ssb with ribosomal proteins and rRNA depends on its lid domain. Nat.

Commun. 7, 13563 (2016); dark red). (c) Corresponding residues in the putative substrate-binding pocket of Ssz1 SBD and the SBDs

of DnaK and Ssb1 are shown. The -strands are labeled, the residue are shown as sticks.



Binding assays of RAC to known Hsp70 substrates.

Fluorescence anisotropy binding assay of (a) EcDnaK, Ssz1 / Zuo1N or RAC with the NR peptide (NRLLLTG) (Schlecht, R., Erbse,

A.H., Bukau, B. & Mayer, M.P. Mechanics of Hsp70 chaperones enables differential interaction with client proteins. Nat. Struct. Mol. Biol. 18, 345-51 (2011); Gragerov, A. & Gottesman, M.E. Different peptide binding specificities of hsp70 family members. J Mol Biol 241, 133-5 (1994)) and (b) CtSsb, Ssz1 / Zuo1N or RAC with the CoxIV peptide

(Pfund, C. et al. The molecular chaperone Ssb from

Saccharomyces cerevisiae is a component of the ribosome-nascent chain complex. EMBO J. 17, 3981-3989 (1998)). (c) Binding constants (KD) values for peptide substrates binding with EcDnaK or CtSsb. Each value is the average of three technical replicates ± standard deviation.


1

Structural insights into a unique Hsp70/Hsp40 interaction in the Ribosome-

associated complex

Felix Alexander Weyer*, Andrea Gumiero *, Genís Valentín Gesé, Karine Lapouge

and Irmgard Sinning#.

Supplementary note 1-3


2

Supplementary Note 1

Multiple sequence alignment of Ssz1 family members and cytosolic Hsp70s.

Domain boundaries and secondary structure elements based on the crystal structure

of CtSsz1 and EcDnaK are depicted on the top and the bottom, respectively. Ct:

Chaetomium thermophilum, Sc: Saccharomyces cerevisiae, Sp:

Schizosaccharomyces pombe, Cg: Chaetomium globosum, Hs: Homo sapiens and

Ec: Escherichia coli. CtSsz1 domain boundaries are indicated above the secondary

structure elements colored as in Fig. 1.

nucleotide binding domain





nucleotide binding domain linker substrate binding domain

substrate binding domain

substrate binding domain

β1 β2 β3 β4 β5 β6

β7 β8 β9 β10

β11 β12 β13 β14

β15 β16

β17 β18

βL β1 β2

β3 β4 β5a

β6 β7

α1

α2 α3 α4 α5

α6 α7

α8 α9 α10

α11 α12 α13 α14 α15

α16 αL

α1

β19

β8

β1 β2 β3 β4 β5 β6

β7 β8 β9 β10

β11 β12 β13 β14

β15 β16

β18 β19

βL β1 β2

β3 β4

β6 β7

α1

α2 α3 α4 α5

α6 α7

α8 α9 α10

α11 α12 α13 α14 α15

α16

β5 β8α1

P I KHS A V I V K

P E G T L I V AAI A IL E V L V F L P

CtSsz1 1 M IG G A V N G R P Y G A V I NSNS I IA D D TIL DE K AESASKA...........APGERV TF S HTV.DDKAE E QI S V.DG YY QQ N F

ScSsz1 1 M IG G A V N G R P Y G A V I NTSS I IA D E SAL DE S.................SP.... TF S YINPKNDVD P AI S V.GE YH GQ LQ Q

SpSsz1 1 M IG G A V N G R P Y G A V I NQNS I LA E SIL E R QLKLTKTLPFSENFIMADSEEYKT SF S FNR.DGKTD E N QI S H.GDQ YH VQ G Q

CgSsz1 1 M IG G A V N G R P Y G A V I NSNS I IA D D TIL EE K SDTGAKPGP.........EPSERT TF S YLV.DDKAE E QI S V.DG YY QQ N F

HsHsp70L1 1 M IG G A V N G R P Y G A A V TS V VA D VV EE K A....................... HL C AC VYK.DGRAG DA VT A A S.EN IV LA Q S

EcDnaK 1 M IG G A V N G R P Y G A I I TTNS V L E D SII E K GK...................... DL C IMD.GTTPR E A TT A TQDG TLV QP R Q

CtSsz1 68 N N G LVR TV R L V V F I LT EI L RL PK AYF DI QDFKS.. .DP.THNHASAHPQEAG.DN V T K..DKAEEDAEPST VS ATRY R V G

ScSsz1 59 N N G LIR TI R I V F I LT EV L RL PK INF DF LPFDK..C.DV.SKCANGAPAVEVD.GK G V S.....RGEGKEEK VD VSRH N K L

SpSsz1 79 N N G LVR SV R L L I F I LT EA L RL AD TNF DL KSHDE.. .TL.HHCHYSANPVNVE.GQ G K TVQEDEESDPKEKI AH SVRH R T E

CgSsz1 70 N N G LVR TV R L I V F I LS EI I RL SE AYF DF QEFKS.. .DP.THNHASAHPQESG.ET S T K..DKAGEEEPAST IS ATRY R V G

HsHsp70L1 56 N N G IR TV K L A L Y I VN DV KM R IS MKV QI RSSSDPQ QKYIAESK..CLVIEKN.GK R E D......TGEETKF PE ARLIFS K E

EcDnaK 58 N N G AV TL K I V A V M I L KM T PQ FAI RL RRFQDEE QRDVSIMP..FKIIAADNGD WVE K..........GQK APPQ SAEV K K K

CtSsz1 141 A G V P F Q A I P A LA G EYL VT A ITI TE K L AA A L VL L E AV IVV G DA S KK S TN K A IA AA D E Q S A YD.ARPEATIS.DKI ADL SRS

ScSsz1 129 A G V P F Q A I P A LA G DYI V A LTV SE K L A I L IV E AL VVV G DA E SA KE TN E T KAS AK G Q QF N S H..AEQFPFEK.DVN ADF IRS

SpSsz1 154 A G V P F Q A I P A LA G DFL VN MSV TD R L AA A L VL L D VI VVV ES E TK GC VY A K ES NE G P Q H A LM.YSEEVLI..DKT ANF ATRS

CgSsz1 143 A G V P F Q A I P A LA G EYL VT A ITV ND K L AA A L VL L E AA AVI G DS S KK S TN K A IA AA D E Q S I YD.ARPEAKVQ.DKI ADL TRS

HsHsp70L1 127 A G V P F Q A I P A LA G L AN V ITV E K L AA A VL L E AL ILV G T HSV SD D FD G K N GE RA GFN R H S YG.IGQDSPTG.KSN FKL TSL S

EcDnaK 126 A G V P F Q A I P A LA G DYL VT A ITV ND R A A L V I E AA IAV G DT E EP E AY A Q TKD GRI G E KR N T YGLDK....GTGNRT YDL GTF

CtSsz1 219 L D G L EF R L K LS V VL GMY I AT I K D N R LAK R E R N A T ASRS.... T VH YEYH IALDKV DHFSK L KNPGAK P E P S LEA ST A RST

ScSsz1 206 L D G L EF R L K LS A VI GIF I AT V A K N R LAK K K S A A AVRN.... T AH LSLG DNLDTE EYF S Q KYQ..ANP K A S ANSSIT T NAT

SpSsz1 231 L D G L EF R L K LS V VV GL I AS V A K D N R LAK R E R T A S SVKG.... MT VH ENLG EQLTDV NFF K E KNG..I P K A S AQC IT V NGT

CgSsz1 221 L D G L EF R L K LS V IV GMY V AT M A K D N R LAK R E R N A A ASRG.... T VH YEFA VALDKV DHF K L KNSGAK P E A S LES ST A LGA

HsHsp70L1 205 L D G L EF R L K LS L VM GIY V T A A R D N R MMK E H S A S EVNS.... R S NT DNIG AHFTET QYL S Q SFK..H V G A A TNSA VA S TLG

EcDnaK 202 L D G L EF R L K LS I II F V AT I V K D M R K E Q S EIDEVDGEKT E NG THLG EDFDSR NYL E K DQG..I L NDPLA Q EAA KA IE SAQ T

CtSsz1 295 R D V GG P S VESL D G DF TIN LRYE IA V V AV A L V IM S RI FP FS I .... L AS T RT FEGFNRL ES KK G DPL DE S T NT AANFRYI E

ScSsz1 280 R D V GG P T IDSL D G DY SIN MRYE VA V V VI A L I LL S KL L P IS A .... F HA L NK FAQFSSF DS AK E DPL DA T V FT TTN EYTL E

SpSsz1 305 R D V GG P S VDSL D G DF SIN LRYD AA V AV A M I IL S KL M FP AA A .... I HS L SATLNRMADL TE EK N EPF SE A A NT TSL ESI E

CgSsz1 297 R D V GG P S VESL D G DF TIN LRYE IA V V AV A L V IL S RI L FP FS S .... I AS T RT FEGINRL ES KK G DVL DE S T HT ASN SNI Q

HsHsp70L1 279 R D V GG P N LDSL E G DF NVS ARFE L L I LL I VL S KL I FP CF Y .... Q DC L CSP FNKCIEA RG DQNGFTAD NK C S RI QQL KDL A

EcDnaK 280 R D V GG P L I P VT AK E LV L L AL A L I IL T V V FG DVN PY TADATG KHMNIK L S ED VNRSIEP KV QD G SVS DD V Q RM M QKK AEF K

CtSsz1 371 P E G T I L L A AALQA LI E I V V IGV F I STR LAPST.DPSA N S Q R S QEF TED EQSTHAA TTMPH TNA VSVSESGEEK VP IAPE A

ScSsz1 356 P E G T I L A AALQA LI D L I L IGL F V SVE LGPQN.KNASNN N A S R SDY ADE AEALQPV VNTPH KKP I....GAKGE HP LLAE S

SpSsz1 381 P E G T I L L A VQA LI D I V L IGI F I QTI RSSSSVTPLQ D S T I SG S GHF AAD AASTDAQ VDVPH TAP .....NEGEN VT FDIE A

CgSsz1 373 P E G T I L A A AALQA LI E I V V IGV F I TTR LSPST.EVTA N S E R S QDY AED EQSTHAA TTVPH SNA ISVNEAGEEV VP VPPE A

HsHsp70L1 355

EcDnaK 360 P E G T V A A AAVQ VL V LGI L EPRK.........D N D V I GG TG.........DVKD LLLDVTPLS ETMGG....VMTT IAKN T

CtSsz1 450 P V ARRT L A V V V E EE VH D. PKEGGD L K V GSTHINVIKPEPKAKEDGETK.EKTEDADDDGDFDDDDEEEEEE EEEKREKV W

ScSsz1 431 P V K L I V E DE F Q KLT K.QAK..GDFL G Y GDHHIEEKTLEPIPKE.........ENA......EEDDESEWSD PEVVREKL Y

SpSsz1 456 P L ARKT V A A I I E EE VE I. PKE.GA F P Y AERSVKVTKVEPEPID........EEEA......FSDDE....E PEEIKERI A

CgSsz1 452 P V ARRT V A V V V E EE VH S. P.NGGD L K V GNTHIKVTQPEPKAKENGESKAAKVEDADDSD..EEDSDFSEDE EEEKREKV W

HsHsp70L1 422 P L ARRQ L A V L L E EE HT Q. PGSISS C E Y SD......................GKNS..............AK ........ .

EcDnaK 418

CtSsz1 528 G S LA A V V VEV NV D V VTARE R TL KI..G T E A RG K....KGAK TI NT LT I VGGK G.V G SA..................... .

ScSsz1 493 G T LM L I VEI NI D L VTARD K L TL..G K E G K.......NANG IF NK GA R LKT. NAV GE ....................... .

SpSsz1 516 G T IA I L V IEL QI E L ASAR R S IP..K L T T PD S....PNAK VL DA GK T PKDGK TNV G TA...................... .

CgSsz1 528 G S LA A V V VEV TV D V VTARE R TL KI..G T E G KG K....KGGK SI NS LS I VGGK G.V G TSA.................... .

HsHsp70L1 456 G T A V L L I A TM D L VT D SI ..... KF Q V QD DKKENGLRD L VL KR GS H CT QET. KCEAI EIAS................... .

EcDnaK 447 G L L I IEV I D L VSAKD K TI RAADNKS GQFN DG NPAPRGMPQ TFD DA GI H KNS. KEQ I KASSGLNEDEIQKMVRDAEANAE A

βL2,3

β5b

PT QVFST EDNQSA T H LQGE...................................................R

VEL ........NS PPDE IPIG E G IGK NLL EDSLMIECSARDI VKG DE.....SGASR TV FPSGT

β17


3


Multiple sequence alignment of Zuo1 family members. Domain boundaries and

secondary structure elements based on the crystal structure of CtZuo1N, the

predicted structure of the charged region and the crystal structure of the Zuo1 helical

domain1,2 are depicted. Ct: Chaetomium thermophilum, Sc: Saccharomyces

cerevisiae, Sp: Schizosaccharomyces pombe, Cg: Chaetomium globosum, and Hs:

Homo sapiens.

Zuo1-N

J-domain

Zuotin homology domain

Charged region

Helical domain

Charged region

J-domain

β1 β2 α1

α2 α3 α4

α1

Zuotin homology domain

CtZuo1 1 M P EPVG F R S L L TL E R L LAHAR H RTF E DRI A A E NATV.VS P PEGWAA KDFKAIGKLTQEGSSM T PH VR K D Q QAK KKI QDS.

ScZuo1 1 M P EPVG F R S L TL E N T R V L HA R HTW E ERI A V DE .......FS TSDITV V ..SSATK.. PFVR P KF Q Q TL N F E EKN KTV SNV

SpZuo1 1 M P EPVG F R S L T E S S R V LAHAR H RTF E ERL V V EE SAGDSIQ FP TKQAI.. A FVPHGKI.. PLIK A PL QR G D E KNK Q.. VK.

CgZuo1 1

HsMpp11 1

CtZuo1 79 WK D Y GL R A QI AH V HPDK A G F D D SE EDPMLL D KD TQ H VL SK WR T KR RK LKH K A A DD F KC S ....I E AR A K Y ED K A R... D N

ScZuo1 70 WK D Y GL R A QI AH V HPDK A G F D D TE D LL D RD T AM SK FR T K RK VK A L D F K P ELLFD LA ED TH A A L A L ES I Q Y QS GS.. DQ G I

SpZuo1 74 WK D Y GL R A QI AH V HPDK A G F E E EDPAML D KE QQ H VL SK YK KK K LKH K I DD F KC ES....ED E RA P A Y DTE L K A S N... N S

CgZuo1 77 WK D Y GL R A QI AH V HPDK A G F D E SE EDPMML E KD SQ H VL SK YK T KR RK LRH K A A DD F KC G ....I P QR A K Y EE K A R... D N

HsMpp11 65 WK D Y GL R A QI AH V HPDK A G F D E Q E PML D KD NQ H VL H YK T K K LKH R A D Y C E ....L LE F KTL P A G V QR A AM K EPIKEG ND T

CtZuo1 152 I KA E L D R S D D P VF R S P G V FY FW Q I PVK RQFD V A I P K K NYYELW E EA F K PV STR E E N Y A L EE P T KQLA G GK N IQ GF NEN E A

ScZuo1 148 I KA E L D R S D D P VF R S P G V FY FW Q K QYD A V P K FYEAW E EA F K PI SSK E E F T T SN A C FV P K ..GTDYD GP A KT SL NKD K Q A H

SpZuo1 147 I KA E L D R S D D P VF R S P G V FY FW Q I PVR RQFD V A V P K TFFELW E EA F K PV STR E D N Y Y S EN E ES..TT E TP S KQ SL TIE A N

CgZuo1 150 I KA E L D R S D D P VF R S P G V FY FW Q V PIK RQFD V A V P K K NYY LW EA F K PV TK D E N Y T L EE E T KQLA G K SQ KS VH TF DDKA E N

HsMpp11 141 I KA E L D R S D D P VF R S P G V FY FW T M PVK R F V K K NFFEVF E W V SS D D S Y Y S A N PTF NSV S S.EA D TP RNS NKKN KL DMN FE I

CtZuo1 232 FDSWR F LD RD R E N R K R D RIK F K N S EY EDVPDD EN K M RK KK DNA LR LVE A R E A KR ERE N Q H ANA K AE K E QAG E R Q AN A NK L

ScZuo1 226 FDSWR F LD RD R E N R K R D RIK F K T EF EDVPDD N K I RK KK DNA L LVE A K E RK ERER SS H Y KAA D TA VK R VSE P M E EKKE ER W

SpZuo1 225 FDSWR F LD RD R E N R K R D RIK F K N S EY DIPDD ES K KK K DNA LR LVD A K A RK ERE K G N FQ RSE Q N AR N T LAS P L EQEK A AA W

CgZuo1 230 FDSWR F LD RD R E N R K R D RIK F K N S EY EDVPDD EN K M RK KK DNA LR MLD R E A KR ERE N Q H TNA K AE K DCSAG E R Q AN A NK F

HsMpp11 220 FDSWR F LD RD R E N R K R D RIK F K N Y EE E E R I K R E IR LVD A K E A K K E S EK KA C E W Q RAT AQ KE MN T N YSC P K E EK K EAE KA A

CtZuo1 312 E A KK K K A K K F A R AE KKEA R E E KA A A A EA K KRVLR SV E NY E EA A ID AE KA EARL E.... RK A E K ERE .A A N V N G A V G SA .T

ScZuo1 306 E A KK K K A K K F A R AE K EA K E E KA A A A EA K KRAIR SA E Y D D ID GA AE A.KA A A.... KA S A N SAK DK A A K N N AD G A K.AT.T

SpZuo1 305 E A KK K K A K K F A R A KKE R E KA A A A E R KKVVK L D NY DV A VD GA EA AAAQK E ER... AE AAA S .AA .N DK K Q D NA F SAT PS EH

CgZuo1 310 E A KK K K A K K F A K AE KKEA K E E KA M A A EA K KRVLR SV D NY E DA A ID AE KA DARL E.... AK A E K DRE .T A N V N G A V G SP .T

HsMpp11 300 E A KK K K A K K F R E R A E V L DI K R LR S N D EA V M EAK KEQ AKEKQ Q LEAARL KEKE EE RQQAL .A E Q K I E QK N C TW H S N ER .K .

CtZuo1 386 V L D DLV KI EI LA LN A I V V V G L I AV N IT DAD AA GK GLTV ..DE KN WKEE SRL GA K KEGD KALVA.................

ScZuo1 379 V LI L EL A I A A V A V G L L ...EQ G VDS NDE VST DK KAN.. ..AG KE LKES KTI DS K PSSL SYFV..................

SpZuo1 380 V L D DVI KL EL LA IN A V V A I G M V SV K MS GEG GQ AD AEKA GAAS QA FDKF KMF ER S SSAD VFFAQ.................

CgZuo1 384

HsMpp11 377

CtZuo1 ................................................................................

ScZuo1 ................................................................................

SpZuo1 ................................................................................

CgZuo1 ................................................................................

HsMpp11 427 EEAEARMRQASKNTEKSTGGGGNGSKNWSEDDLQLLIKAVNLFPAGTNSRWEVIANYMNIHSSSGVKRTAKDVIGKAKSL

CtZuo1 ................................................................................

ScZuo1 ................................................................................

SpZuo1 ................................................................................

CgZuo1 ................................................................................

HsMpp11 507 QKLDPHQKDDINKKAFDKFKKEHGVVPQADNATPSERFEGPYTDFTPWTTEEQKLLEQALKTYPVNTPERWEKIAEAVPG

CtZuo1 ...................................

ScZuo1 ...................................

SpZuo1 ...................................

CgZuo1 ...................................

HsMpp11 587 RTKKDCMKRYKELVEMVKAKKAAQEQVLNASRAKK

M P EPVG F R S L L L E N T R L LAHAR H RTF E DRI A A EE EATV.VP P A PEGWES N FIPKGKI.. SFTQ T PY AR K D Q QER KKV ND.

M P EPVG F R S L L SA T S V A VK R S L L EE ....... L ADGR...G AITHALT.. ASTLCQ RW E F RN NA A .FQE EDKKE S.. SE.

V M E E L RL L L LT V A .. E K CD ELAS QC NET SCT....KE GK ........................ALEKQIEEINEQIRKEK

V L D ELI KI EI LA LN A I V V V G L A AV G QA DAD AA GK GLKV ..DE KG WKAE ERL GA K QEGE KALSA.................


4


Characterization of the Ssz1-Zuo1 interaction. ITC titrations are shown for SUMO-

Ssz1 with MBP-Zuo1 (a) and for SUMO-Ssz1 with MBP-Zuo1N (b). (c) Kd values and

thermodynamic parameters for relevant ITC measurements. The Ct variants of Ssz1

and Zuo1 were used. Data are the mean of two biological replicates ± standard

deviation.

Supplementary Note References 1. Zhang, Y. et al. Structural basis for interaction of a cotranslational chaperone

with the eukaryotic ribosome. Nat. Struct. Mol. Biol. 21, 1042-6 (2014). 2. Leidig, C. et al. Structural characterization of a eukaryotic chaperone--the

ribosome-associated complex. Nat. Struct. Mol. Bio.l 20, 23-8 (2013).

a

0.0 0.5 1.0 1.5 2.0 2.5 3.0-50.00

-40.00

-30.00

-20.00

-10.00

0.00-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0 20 40 60 80 100 120 140 160 180Time (min) b

0.0 0.5 1.0 1.5 2.0-12.00

-10.00

-8.00

-6.00

-4.00

-2.00

0.00-0.35-0.30-0.25-0.20-0.15-0.10-0.050.000.050.10

0 20 40 60 80 100 120 140Time (min)

Molar Ratio (Zuo1/Ssz1) Molar Ratio (Zuo1N/Ssz1)

Kd (nM) N H (kcal/mol) T S (kcal/mol) G (kcal/mol)

Ssz1 / Zuo1 121 ± 54 1.62 -33.73 ± 41.25 -22.15 -11.58

Ssz1 / Zuo1N 118 ± 104 0.64 -29.34 ± 19.66 -17.89 -11.45


Date post:	31-Jul-2020
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Nature Structural & Molecular Biology: doi:10.1038/nsmb€¦ · 7,13563 (2016) ) and (c) DnaK...

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