Download Protein overexpression and purification

Supplementary data
Supplementary methods
Plasmids
All genes were cloned into an overexpressing plasmid using a two-step strategy. bll2757
(fixK2), bll4943 (clpX), bll4944 (clpP1), bll5153 (clpA), bll5154 (clpS1) and, blr6518 (sspB)
were amplified by PCR from wild-type B. japonicum genomic DNA with primers listed in
Table S3. GFP proteins used in this study harbor two point mutations in the chromophore
(F64L S65T). All GFP derivatives were generated by PCR using the primers listed in Table S3.
PCR fragments were cloned first in the pGEM-T easy vector, digested with the respective
enzymes (Table S3) and ligated into a T7-based overexpressing plasmid. clpA was cloned into
pET16, clpS1, all gfp derivatives (GFP, GFPssrABj, GFPcA12, Fn9-GFP, GFP-Fc12, Fn9-GFPFc12)
and
clpP1
were
cloned
into
pET24.
As
annotated
in
Rhizobase
(http://genome.kazusa.or.jp/rhizobase/), ClpS1 from B. japonicum is 130 amino acids in length,
and contrary to all other rhizobia, it possesses a non-conserved N-terminal extension of 20
amino acids. Since we failed to overexpress the full-length protein in E. coli, we cloned and
overexpressed a shorter version lacking these first 20 amino acids. Curiously, ClpS 1 has a
second methionine in frame at position 21, which might be encoded by an alternative start
codon. All experiments were done with this shorter version of the predicted ClpS1. ClpP1 was
fused to a tobacco etch virus (TEV) recognition site and a His6-tag at its C-terminus. clpX and
sspBα were both cloned into pProEx-HTb with an N-terminal His6-tag followed by a TEV
recognition site. fixK2 was C-terminally fused to the Intein-Chitin Binding Domain tag in
pTXB1. All DNA sequences were confirmed by sequencing.
Protein overexpression and purification
All proteins were overexpressed in BL21 (DE3) cells from bacteriophage T7 promoterbased plasmids. ClpA, ClpS1 and all GFP derivatives were purified as previously described for
1
E. coli orthologs [1-2]. SspBα, ClpX and ClpP1 were purified over a Ni-NTA column (Qiagen)
and the His6-tag was cleaved off using the TEV protease. The tag-cleaved, TEV-free ClpX was
further purified on a Resource Q column (GE Healthcare) and eluted with 50 mM HEPES
KOH pH 7.5, 300 mM KCl, 10% glycerol, 2 mM EDTA. All proteins were stored in buffer S
(50 mM HEPES KOH pH 7.5, 300 mM KCl, 15% glycerol, 2 mM EDTA). ClpP 1 was also
further purified over a Q Sepharose column (GE Healthcare) and eluted with 50 mM Tris HCl
pH 7.5, 300 mM KCl, 10% glycerol, 2 mM EDTA and was subsequently loaded onto a
Superdex 200 10/30 HR column equilibrated with buffer S at pH 8 with 20% glycerol. FixK2
was purified with the IMPACT (intein-mediated purification with an affinity chitin-binding
tag) system (NEB). This purification procedure has the advantage to generate a protein without
additional vector-encoded amino acids, allowing us to generate a homogeneous protein sample
without additional amino acid extensions potentially masking a recognition site of the protease.
The purified tag-free FixK2 protein was stored in 50 mM HEPES KOH pH 7.2, 150 mM KCl,
0.1 mM EDTA. The correct molecular weight of each protein was confirmed by mass
spectrometry.
Enzymatic assays
ClpA and ClpX ATPase activity
ATPase activity was measured in a continuous spectrophotometric ATPase assay at 25 °C
as previously described [3]. All reactions were performed in buffer S supplemented with 1 mM
DTT, 20 mM MgCl2 and 5 mM ATP in a Cary UV-visible spectrophotometer (Varian).
ClpP peptidase assay
0.1 µM ClpP1 was incubated with 0.1 to 4 mM Suc-LY-AMC (Sigma Aldrich) at 25 °C in
buffer S with 20 mM MgCl2, 1 mM DTT and 11% DMSO. Substrate degradation was recorded
in a 96-well plate using a Victor3 fluorimeter (Perkin Elmer) with an excitation and emission
2
wavelength set at 380 nm and 460 nm, respectively. Initial slopes were recorded and analyzed
according to Michaelis-Menten theory.
3
Table S1. Compilation of genes coding for ClpA, ClpX, ClpP, ClpS, and SspB orthologs in rhizobiaa.
Rhizobiaa
ClpA
ClpX
ClpP
ClpS
SspB
B. japonicum USDA110
bll5153
bll4943
blr0611
bll2636
blr6518
bll4944
bll5154
mlr0748
mll0664
mlr8472
mll2736
SMc01903
SMc02110
SMc02720
SMc02694
Mesorhizobium loti
mll0663
mlr8474
MAFF303099
Sinorhizobium meliloti 1021
SMc02109
SMc01904
mlr1474
SMc01450
SMc03841
Rhizobium etli CFN42
RHE_CH01907
RHE_CH01588
RHE_CH01414
RHE_CH01906
RHE_CH01587
RHE_CH03030
RHE_CH02800
RHE_CH03069
Rhizobium leguminosarum
RL2213
RL1689
bv. viciae 3841
RL1170
RL2212
RL1688
RL3476
RL3257
RL3515
Bradyrhizobium sp. BTAi1
BBta_4772
BBta_4571
BBta_4573
BBta_2472
BBta_6105
BBta_4773
Bradyrhizobium sp. ORS278
Rhizobium sp. NGR234
Azorhizobium caulinodans
BRADO4545
NGR_c13260
AZC_1595
BRADO4197
NGR_c10350
AZC_1609
BRADO0526
BRADO2155
BRADO4198
BRADO4546
NGR_c10340
NGR_c13250
NGR_c23470
NGR_c23260
AZC_1608
AZC_1594
BRADO5583
NGR_c20590
AZC_4485
ORS571
a
Mutualistic symbiotic rhizobia whose genome sequences have been deposited in the Rhizobase
(http://genome.kazusa.or.jp/rhizobase/) as of September 2012, and contain at least an annotated FixK-coding gene.
Blast searches were done using blastp tool in the Rhizobase
(http://genome.microbedb.jp/blast/blast_search/rhizobase/genes) and E. coli K12 orthologs as query sequences
(http://www.ecogene.org/3.0/). In the case of SspB, C. crescentus CC_2102 (Q9A6J2; [4]) was used as query.
4
Table S2. Expression data of clp genes at transcriptomic and proteomic levels.
Sequence
identity
with E. coli
proteinsb
Sequence identity
with C. crescentus
proteinsc
Oxic
blr0611 ClpP2
58 %
66%
308 ± 41
bll2636
ClpS2
43 %
39%
507 ± 99
959 ± 206
248 ± 72
bll4943
ClpX
73 %
84%
5208 ± 770
6838 ± 1022
5564 ± 507
4180 ± 981
64
bll4944
ClpP1
65 %
73%
4841 ± 595
5441 ± 1196
4573 ± 812
1551 ± 345
42
bll5153
ClpA
58 %
73%
4821 ± 526
9567 ± 2351
4628 ± 620
4706 ± 1238
64
bll5154
ClpS1
57 %
68%
3349 ± 852
5700 ± 2899
3553 ± 571
3082 ± 1052
2
28 %
44%
1551 ± 167
1319 ± 574
1491 ± 267
853 ± 214
2
Genea Protein
blr6518 SspBα
Transcriptomicd
Microoxic
a
201 ±
89
Anoxic
244 ± 41
Proteomice
Symbiosis
231 ± 104
335 ±
65
Symbiosis
-
Gene nomenclature according to Kaneko et al. [5].
Sequence identity of B. japonicum USDA110 Clp proteins and E. coli K12 orthologs (http://www.ecogene.org/3.0/): ClpX
(P0A6H1), ClpP (P0A6G7), ClpA (P0ABH9), ClpS (P0A8Q6), SspB (P0AFZ3). Blast searches were performed by using
blastp (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi?PAGE=Proteins).
c Sequence identity of B. japonicum USDA110 Clp proteins and C. crescentus CB15 orthologs (http://cmr.jcvi.org/cgibin/CMR/GenomePage.cgi?org=gcc): ClpX (P0CAU2), ClpP (P0CAU1), ClpA (Q9A5H9), ClpS (Q9A5I0), SspBα (Q9A6J2).
d Average and standard deviation of normalized signal intensities detected in the transcriptomic experiments of cells grown
under free-living conditions (oxically, microoxically, anoxically) and in soybean nodules (symbiosis) [6-7].
e Number of spectra for the corresponding protein detected in bacteroids isolated from soybean nodules in a proteomic
approach [8].
b
5
Table S3. List of proteins used in this study and primers used for amplification of their genes. The
protein names refer to the protein overexpressed and not the final protein. Restriction enzyme sites are
underlined in the primer sequence, additional sequences (tags or amino acids) are in bold. For: forward
primer; Rev: reverse primer; TEV: tobacco etch virus recognition sequence; CBD: Chitin Binding
Domain; cA12: 12 C-terminal amino acids of ClpA; Fn9: 9 N-terminal amino acids of FixK2; Fc12: 12 Cterminal amino acids of FixK2.
Protein
Plasmid
backbone
Restriction
sites
Primers (5’-3’)
ClpA
pET16a
NcoI, NdeI
For: CTGAGCCCATGGCGACTTTTTCTCAAAGC
Rev: CGGGTTCATATGTCAAGCCTTGACCAG
ClpS1ΔN20
pET24a
NdeI, NotI
For: GCGCATATGAGCAACGACGAGAACCG
Rev: CCGCGGCCGCCTTATTTCTTTTCCATCAC
His6-TEV-ClpX
pProEx-HTbb
NdeI, BamHI
For: GCATGCCATATGAGTAAGGTCGGCACGA
Rev: ACATCGGGATCCCTAGTTCAGGCGCTGGCGTTCTC
ClpP1-TEV-His6
pET24a
For: GATAATCATATGCGCGATCCGGTTGAAACCTAC
NdeI, BamHI Rev: ACACGGGATCCCTAGTGGTGGTGGTGGTGGTGAGACTGGAAGTA
CAGGTTTTCCTGGGTCTTCGCCGCGGCCGGCTCT
His6-TEV-SspBα
pProEx-HTbb
NdeI, EcoRI
For: GCATGCCATATGGCGACCGATCATATCCGATAC
Rev: GCGAATTCTGGCCATGAGCACATCCATTCC
NdeI, SpeI
For: GCGCATATGCTGACCCAGACAC
Rev: ACTAGTGCATCTCCCGTGATGCAGGCGTCGAGATTGTGCAGGC
For: GCGGCTAGCATGAGTAAAGGAGAAGAAC
Rev: CGCGAATTCTCATTTGTATAGTTCATCCATGCCATGTG
FixK2-Intein-CBD pTXB1c
GFP
pET24a
NheI, EcoRI
GFPssrABj
pET24a
For: GCGGCTAGCATGAGTAAAGGAGAAGAAC
NheI, EcoRI Rev: CGAATTCTCACGCGGCCTGCGCCACCGGCGCAAAGTTATCGTTT
GCTGCTTTGTATAGTTCATCCATGCCATG
GFP-cA12
pET24a
For: GCGGCTAGCATGAGTAAAGGAGAAGAAC
NheI, HindIII Rev: CGAAGCTTTCAAGCCTTGACCAGCGGCCCCTTGGAGGTCGGCCC
CTTTTTGTATAGTTCATCCATGCCATG
Fn9-GFP
pET24a
For: GCGCTAGCATGCTGACCCAGACACTCAAGACCCAGAGTAAAGGA
NheI, EcoRI GAAGAACTTTTCACTGGGGTTG
Rev: CGCGAATTCTCATTTGTATAGTTCATCCATGCCATGTG
GFP-Fc12
pET24a
For: GCGGCTAGCATGAGTAAAGGAGAAGAAC
NheI, EcoRI Rev: GCGAATTCTCAGGCGTCGAGATTGTGCAGGCGCTGGCGGTTGC
GCAGTTTGTATAGTTCATCCATGCCATGTG
Fn9-GFP-Fc12
pET24a
NheI, EcoRI
For: GCGCTAGCATGCTGACCCAGACACTCAAGACCCAGAGTAAAGGA
GAAGAACTTTTCACTGGGGTTG
Rev: GCGAATTCTCAGGCGTCGAGATTGTGCAGGCGCTGGCGGTTGC
GCAGTTTGTATAGTTCATCCATGCCATGTG
a
Novagen
Invitrogen
c
New England Biolabs
b
6
A.
Zn finger
ClpXEc
ClpXCc
ClpXBj
MTDKRKDGSGKLLYCSFCGKSQHEVRKLIAGPSVYICDECVDLCNDIIREEIKEVAPHRE 60
MTKAASGDTKSTLYCSFCGKSQHEVRKLIAGPTVFICDECVELCMDIIREEHK-IAFVKS 59
MSKVGTSDSKNTLYCSFCGKSQHEVRKLIAGPTVFICDECVELCMDIIREENK-SSLVKS 59
*:. ...: . ********************:*:******:** ****** * : :.
ClpXEc
ClpXCc
ClpXBj
RSALPTPHEIRNHLDDYVIGQEQAKKVLAVAVYNHYKRLRNGDTSNGVELGKSNILLIGP 120
KDGVPTPREICEVLDDYVIGQGHAKKVLAVAVHNHYKRLNHASKNNDVELAKSNILLVGP 119
RDGIPTPKEICKVLDDYVIGQSHAKKVLSVAVHNHYKRLNHQTKHNDVELAKSNILLIGP 119
:..:***:** : ******** :*****:***:******.: . *.***.******:**
D2 domain
Walker A
ClpXEc
ClpXCc
ClpXBj
D2 domain
TGSGKTLLAETLARLLDVPFTMADATTLTEAGYVGEDVENIIQKLLQKCDYDVQKAQRGI 180
TGTGKTLLAQTLARIIDVPFTMADATTLTEAGYVGEDVENIVLKLLQAADYNVERAQRGI 179
TGSGKTLLAQTLARILDVPFTMADATTLTEAGYVGEDVENIILKLLQAADYNVERAQRGI 179
**:******:****::*************************: **** .**:*::*****
Walker B
D2 domain
ClpXEc
ClpXCc
ClpXBj
VYIDEIDKISRKSDNPSITRDVSGEGVQQALLKLIEGTVAAVPPQGGRKHPQQEFLQVDT 240
VYIDEIDKISRKSDNPSITRDVSGEGVQQALLKIMEGTVASVPPQGGRKHPQQEFLQVDT 239
VYIDEIDKISRKSDNPSITRDVSGEGVQQALLKIMEGTVASVPPQGGRKHPQQEFLQVDT 239
*********************************::*****:*******************
ClpXEc
ClpXCc
ClpXBj
SKILFICGGAFAGLDKVISHRVETGSGIGFGATVKAKSDKASEGELLAQVEPEDLIKFGL 300
TNILFICGGAFAGLEKIISARGAAKS-IGFGAKVTDPEERRT-GEILRNVEPDDLQRFGL 297
TNILFICGGAFAGLEKIISARGRSTS-IGFGAQVLAPEDRRT-GEIFRHVEPEDLLKYGL 297
::************:*:** * : * ***** *
.:: : **:: :***:** ::**
ClpXEc
ClpXCc
ClpXBj
IPEFIGRLPVVATLNELSEEALIQILKEPKNALTKQYQALFNLEGVDLEFRDEALDAIAK 360
IPEFIGRLPVVATLEDLDEAALVKILTEPKNAFVKQYQRLFEMENIGLTFTEDALHQVAK 357
IPEFVGRLPVVATLEDLDETSLKKILTEPKNALVKQYQRLFEMENIELTFADEALGAVAR 357
****:*********::*.* :* :**.*****:.**** **::*.: * * ::** :*:
ClpXEc
ClpXCc
ClpXBj
KAMARKTGARGLRSIVEAALLDTMYDLPSMEDVEKVVIDESVIDGQSKPLLIYGKPEAQQ 420
KAIARKTGARGLRSIMEGILLETMFELPTYEGVEEVVVNAEVVEGRAQPLLIYAEKKGGA 417
KAIERKTGARGLRSILEAILLETMFDLPGLEGVEEVVISREVVEGTARPLYIYADRSDRA 417
**: ***********:*. **:**::** *.**:**:. .*::* ::** **.. .
ClpXEc
ClpXCc
ClpXBj
ASGE-- 424
ASA--- 420
VENASA 423
..
D2 domain
ClpP loop
7
B.
ClpS binding region
ClpAEc
ClpACc
ClpABj
--MLNQELELSLNMAFARAREHRHEFMTVEHLLLALLSNPSAREALEACSVDLVALRQEL 58
MPSFSRPLEESLHRAVAYANQRKHEYATLEHLLLSLTDDEDAAGVMRACDVDLAALKKSL 60
MPTFSQSLEQSLHRALAIANERHHQYATLEHLLLSLIDDSDAAAVMRACSVDLDKLRTSL 60
:.: ** **: *.* *.:::*:: *:*****:* .: .* .:.**.*** *: .*
ClpAEc
ClpACc
ClpABj
EAFIEQTTPVLPASEEERDTQPTLSFQRVLQRAVFHVQSSGRNEVTGANVLVAIFSEQES 118
SNYLDVELTSLVVDDEE-DAKPTAGFQRVIQRAVIHVQSSGREEVTGANVLVAIFSERES 119
VNYLETEFENLVTDGAD-DAKPTAGFQRVIQRAVIHVQSSGREEVTGANVLIAIFAERES 119
:::
* .. : *::** .****:****:*******:********:***:*:**
ClpAEc
ClpACc
ClpABj
QAAYLLRKHEVSRLDVVNFISHGTRK----DEPTQSSDPGSQPNSEEQAG-------GEE 167
HAAYFLQEQDMTRYDAVNFIAHGIAKKAGASEPKSVKGASTGSNATEEDGDKSTTKTGGE 179
HAAYFLQEQDMTRYDAVNYISHGIAKRPGVSEARPVRGVDEETETKGSD----DAKKKGE 175
:***:*::::::* *.**:*:** *
.*.
. . .:: .
*
ClpAEc
ClpACc
ClpABj
RMENFTTNLNQLARVGGIDPLIGREKELERAIQVLCRRRKNNPLLVGESGVGKTAIAEGL 227
ALEAYCVDLNEKARQGKVDPLIGRANEVERAIQILCRRTKNNPLLVGDPGVGKTAIAEGL 239
ALETYCVNLNKKARDGKIDPVIGRNSEINRAIQVLCRRQKNNPLFVGEAGVGKTAIAEGL 235
:* : .:**: ** * :**:*** .*::****:**** *****:**:.***********
ClpAEc
ClpACc
ClpABj
AWRIVQGDVPEVMADCTIYSLDIGSLLAGTKYRGDFEKRFKALLKQLEQDTNSILFIDEI 287
ARKIVTHQVPEVLEGATIYSLDMGALLAGTRYRGDFEERLKQVVKELENHPNAVLFIDEI 299
AKRIVDSEVPEVLAAATVFSLDMGTLLAGTRYRGDFEERLKQVLKELEAHPNAILFIDEI 295
* :** :****: .*::***:*:*****:******:*:* ::*:** ..*::******
ClpAEc
ClpACc
ClpABj
HTIIGAGAASGGQVDAANLIKPLLSSGKIRVIGSTTYQEFSNIFEKDRALARRFQKIDIT 347
HTVIGAGATSGGAMDASNLLKPALASGTLRCMGSTTYKEFRQHFEKDRALVRRFQKIDVN 359
HTVIGAGATSGGAMDASNLLKPALASGTIRCMGSTTYKEYRQHFEKDRALVRRFQKIDIN 355
**:*****:*** :**:**:** *:**.:* :*****:*: : *******.*******:.
ClpAEc
ClpACc
ClpABj
EPSIEETVQIINGLKPKYEAHHDVRYTAKAVRAAVELAVKYINDRHLPDKAIDVIDEAGA 407
EPTVEDTIKILKGLKTYYEDFHKLKYTADALKVAVELSAKYITDRKLPDKAIDVIDEAGA 419
EPTVEDAIAILKGLKPYFEDYHRLKYTNEAIEAAVQLSSRYIHDRKLPDKAIDVIDESGA 415
**::*::: *::***. :* .* ::** .*:..**:*: :** **:***********:**
ClpAEc
ClpACc
ClpABj
RARLMPVSKRKKTVNVADIESVVARIARIPEKSVSQSDRDTLKNLGDRLKMLVFGQDKAI 467
SQMLLPESRRKKTIGVKEIESVVAKIARIPPKSVSKSDTEALKELESDLKRAVFGQDEAL 479
AQMLVAENKRKKTIGIKEIETTIASMARIPPKSVSKDDAEVLKHLEQTLKRTVFGQDKAI 475
*:. .:****:.: :**:.:* :**** ****:.* :.**.* . ** *****:*:
ClpAEc
ClpACc
ClpABj
EALTEAIKMARAGLGHEHKPVGSFLFAGPTGVGKTEVTVQLSKALGIELLRFDMSEYMER 527
SQLAAAMKLARAGLREPNKPIGSYLFSGPTGVGKTEAAKQLAQTLGIEMLRFDMSEYMER 539
ESLAASIKLARAGLREPEKPIGCYLFSGPTGVGKTEVAKQLAASLGVELLRFDMSEYMER 535
. *: ::*:***** . .**:*.:**:*********.: **: :**:*:***********
ClpS binding region
N-domain
Linker
D1 domain
Walker A
D1 domain
Walker B
D1 domain
D1 domain
D1 domain
D2 domain
D2 domain
Walker A
8
D2 domain
Walker B
ClpAEc
ClpACc
ClpABj
HTVSRLIGAPPGYVGFDQGGLLTDAVIKHPHAVLLLDEIEKAHPDVFNILLQVMDNGTLT 587
HTVSRLIGAPPGYVGFDQGGQLTDAVDQHPHAVVLLDEIEKAHGDVYNILLQVMDNGTLT 599
HTVSRLIGAPPGYVGFDQGGLLTDGVDQHPHCVVLLDEIEKAHPDLYNVLLQIMDHGRLT 595
******************** ***.* :***.*:********* *::*:***:**:* **
ClpAEc
ClpACc
ClpABj
DNNGRKADFRNVVLVMTTNAGVRETERKSIGLIHQDNSTDAMEEIKKIFTPEFRNRLDNI 647
DSNGKKVDFRNVVLIMTTNAGASDAQRNSIGFGRSKVEGEEEAALKRLFTPEFRNRLDAV 659
DHNGKQVNFRNVILIMTTNAGASDLAKQAFGFTRSKREGDDHEAINRQFAPEFRNRLDAI 655
* **::.:****:*:******. : ::::*: :.. . :
::: *:******** :
ClpAEc
ClpACc
ClpABj
IWFDHLSTDVIHQVVDKFIVELQVQLDQKGVSLEVSQEARNWLAEKGYDRAMGARPMARV 707
VAFKPLSADIIRQVVQKFVMQLEAQLADRNITIELSDDAADWLAKNGFDELYGARPLARV 719
VSFSHLSVEVIGTVVEKFVLQLEAQLGDRDVTIELSEPAKAWLVQHGYDEQMGARPMARV 715
: *. **.::* **:**:::*:.** ::.:::*:*: * **.::*:*. ****:***
ClpAEc
ClpACc
ClpABj
IQDNLKKPLANELLFGSLVDGGQVTVALD-------KEKNELT-YGFQSAQK-------- 751
IQEHIKKPLADDILFGRLVRGGHVKVVLK-------DSKIDFE-IDSSTAPKA------- 764
IQEHIKKPLADEVLFGKLKSGGHVRVVLVKDEADETKEKIGFEFVEGPVTPKQEKLPGAR 775
**:::*****:::*** * **:* *.*
..* :
: *
ClpAEc
ClpACc
ClpABj
-------HKAEAAH------------- 758
----GKTDETEPAMAE----------- 776
KRPPGKPKPGGPGGSKGPTSKGPLVKA 802
.
..
D2 domain
ClpP loop
Autodegradation tag
9
C.
Axial loop
ClpPEc
ClpPCc
ClpP1Bj
ClpP2Bj
MSYSGERDNFAPHMALVPMVIEQTSRGERSFDIYSRLLKERVIFLTGQVEDHMANLIVAQ
M-----YDPVSTAMNLVPMVVEQTSRGERAFDIFSRLLKERIIFLTGPVEDGMASLICAQ
M-----RDPVETYMNLVPMVVEQTNRGERAYDIFSRLLKERIIFLTGPVEDGMSTLVVAQ
M-----RD----MLQLVPMVVEQSARGERSFDIYSRLLRERIIFLNGEVNDAMSGLVCAQ
*
*
: *****:**: ****::**:****:**:***.* *:* *: *: **
ClpPEc
ClpPCc
ClpP1Bj
ClpP2Bj
Δ Δ
Δ
●
MLFLEAENPEKDIYLYINSPGGVITAGMSIYDTMQFIKPDVSTICMGQAASMGAFLLTAG
LLFLESENPKKEIAMYINSPGGVVTAGLAIYDTMQYIKSPVSTVCMGMAASMGSLLLAAG
LLFLEAENPKKEISMYINSPGGVVTSGLAIYDTMQFIRPPVSTLCTGQAASMGSLLLAAG
LLFLEAENPNRPINLYINSYGGVVTSGLAMYDTMQFIKAPVHTLCMGTARSMGSFLLMAG
:****:***:: * :**** ***:*:*:::*****:*:. * *:* * * ***::** **
ClpPEc
ClpPCc
ClpP1Bj
ClpP2Bj
ClpPEc
ClpPCc
ClpP1Bj
ClpP2Bj
●
Handle region
AKGKRFCLPNSRVMIHQPLGGYQGQATDIEIHAREILKVKGRMNELMALHTGQSLEQIER
AAGQRISLPNARIMVHQPSGGFRGQASDIERHAEDIIKTKRRLNEIYVKHCGRTYEEVER
EKDMRFSLPNARIMVHQPSGGFQGQATDIMLHAQEILNLKKRLNEIYVKHTGQTYKTIED
ERGHRAALPNASLHVHQPLGGFQGQASDILIHANEMQETKRRITRLYAQHCGRTEAEVER
. * .***: : :*** **::***:** **.:: : * *:..: . * *::
:*
●
DTERDRFLSAPEAVEYGLVDSILTHRN--------- 207
TLDRDHFMSADEAKAWGLVDHVYDSRDAAEAGAE-- 209
ALERDKFLTANDAKEFGLVDRVIDKRAEEPAAAKTQ 211
TLDRDHFMTAQQGVEWGLIDRVFAERDAT------- 200
:**:*::* :. :**:* :
*
60
55
55
51
120
115
115
111
180
175
175
171
D.
ClpSEc
ClpSCc
ClpS1Bj
ClpS2Bj
M----GKTND---------WLDFDQLAEEKVRDALKPPSMYKVILVNDDYTPMEFVIDVL
MICPPGENKSMAERKQGGQGNGVGSSVVTEVKPKTQKPSLYRVLILNDDYTPMEFVVYVL
--MSNDENRS-------GSPTGPNTSVITKVKPKTKRPNLYRVLILNDDYTPMEFVVHVL
----------MND---------AVTKPKTRTKTKVERPKLHKVILINDDYTPREFVTMIL
..:
: *.:::*:::****** *** :*
ClpSEc
ClpSCc
ClpS1Bj
ClpS2Bj
QKFFSYDVERATQLMLAVHYQGKAICGVFTAEVAETKVAMVNKYARENEHPLLCTLEKAERFFNKSREDATRIMLHVHQNGVGVCGVYTYEVAETKVAQVIDSARRHQHPLQCTMEKDEKFFQKDVEAATKIMLHVHHHGIGECGVFTYEIAETKVTQVMDFARKHQHPLQCVMEKKKAEFRMTEDQAYKVMITAHKLGACVVAVFTRDVAETKATRATDAGRAKGYPLLFTTEPEE
: *
: * ::*: .* *
.*:* ::****.: . . .* : :** . *
47
60
51
41
ClpA binding region
10
106
119
110
101
E.
Substrate binding
SspBEc
SspBαCc
SspBαBj
M-------DLSQLTPRRPYLLRAFYEWLLDNQLTP---------HLVVDVTLPGVQVPME 44
MSQTEPPEDLMQYEAMAQDALRGVVKAALKKAAAPGGLPEPHHLYITFKTKAAGVSGPQD 60
MAT-----DHIRYDVLARDALRGVLRKVLTDAASHG-LPGEHHFFITFVSKAEGVKLSSR 54
*
* :
**.. . * . :
.:.. . **. .
SspBEc
SspBαCc
SspBαBj
----YARDGQIVLNIAPRAVGNLELANDEVRFNA--RFGGIPRQVSVPLAAVLAIYAREN 98
LLSKYPDEMTIVLQHQ-----YWDLAPGETFFSVTLKFGGQPKRLSVPYAALTRFYDPSV 115
LLAQYPEEMTIILQHQ-----FWDLTVLEDRFEVGLSFGGIPERLVVPFSAIKSFLDPSV 109
*. : *:*:
:*: * *..
*** *.:: ** :*: :
.
Substrate binding
Substrate binding
SspBEc
SspBαCc
SspBαBj
Linker
ClpX binding
GAGTMFEPEAAYDEDTSIMNDEEASADNETVMSVIDGDKPDHDDDTHPDDEPPQPPRGGR 158
QFALQFSAP----EIIEDEPE-----PDP----E----PEDKANQGASGDE-------GP 151
KFGLQFDTS----DVAEVAPETLPAAPAPSALSV----PTPATDKAETAEEPTPPSQGGA 161
. *..
: .
:
:. . :*
*
ClpX binding
SspBEc
SspBαCc
SspBαBj
PALRVVK---- 165
KIVSLDQFRKK 162
EVVRLDRFRKK 172
: : :
Fig. S1. Alignment of Clp proteins from E. coli, C. crescentus and B. japonicum. Alignment was
performed
with
T-Coffee
using
the
Clustalw-pairwise
method
(http://www.igs.cnrs-
mrs.fr/Tcoffee/tcoffee_cgi/index.cgi?stage1=1&daction=TCOFFEE::Advanced; [9]). The main features
known for E. coli proteins are annotated above the sequences: the Walker A and Walker B motifs are
underlined [10] and the ClpP loop is marked as bold in ClpX and ClpA sequences [11]. The vertical bar
represents the propetide cleavage in ClpP [12]. Note that the site of the propeptide cleavage is a
prediction for C. crescentus. Rond circle: catalytic triad of the ClpP protein [13-14]. Empty triangle:
hydrophobic patch of ClpP making contact with the ClpP loop of the chaperone ClpA or ClpX [14].
Boxed amino acids: key residues involved in ClpA/ClpS interaction [15]. Other amino acids important
for ClpS interaction are marked with grey shade in ClpA alignment [16]. Amino acids in bold and
marked with an arrow in ClpS alignment represent the residues involved in N-end residue contact [1718]. Star represents identical amino acids, colon conserved substitutions and dot semi-conserved
substitutions. Ec: E. coli K12; Cc: C. crescentus CB15; Bj: B. japonicum USDA110.
11
Fig. S2. B. japonicum ClpP1 is N-terminally processed. The molecular mass detected by mass
spectrometric analysis of purified protein corresponds to the mature protein with the nine N-terminal
amino acids being cleaved off plus a C-terminal tag (ENLYFQ), which remains after cleavage with
TEV protease. The sample was desalted and analyzed by ESI-MS. The m/z data were then deconvoluted
into MS-data using the MaxEnt1 software.
12
Fig. S3. ClpA is autodegraded in the absence of substrate by recognition of its C-terminal region. ClpA
autodegradation by ClpAP1 followed on Blue Coomassie-stained SDS-PAGE in the absence (A, upper
panel) or presence (A, bottom panel) of ClpS1. (B) Degradation of GFP protein fused with the 12 Cterminal amino acids of ClpA (GFP-cA12) in the absence or presence of ClpS1 was followed by
fluorescence. (C) Alignment of the C-terminal twelve residues of ClpA from B. japonicum (Bj),
Neisseria gonorroehae (Ns), E. coli (Ec), Myxococcus xanthus (Mx), and Helicobacter pylori (Hp).
Alignment was performed with T-Coffee using the Clustalw-pairwise method [9].
Fig. S4. Comparison of degradation rates of Fn9-GFP-Fc12 with that of FixK2 by ClpAP1. The
degradation was followed by Blue Coomassie-stained SDS-PAGE and quantified with AlphaImager.
Results are the means ± standard deviations (error bars) of at least 3 independent experiments.
13
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[12]
[13]
[14]
[15]
[16]
[17]
[18]
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14