Supplementary figures and tables
Diverse specificity of cellulosome attachment to the bacterial cell surface
Joana L.A. Brás1,2,a, Benedita Pinheiro1,3,a, Kate Cameron1,a, Fiona Cuskin4,a, Aldino Viegas3,5, Shabir
Najmudin1, Pedro Bule1, Virginia M.R. Pires1, Maria João Romão3, Edward A. Bayer6, Holly L. Spencer7,
Steven Smith7, Harry J. Gilbert4, Victor D. Alves1,*, Ana Luísa Carvalho3,* and Carlos M.G.A. Fontes1,2,*
1
Centro Interdisciplinar de Investigação em Sanidade Animal, Faculdade de Medicina Veterinária,
Universidade de Lisboa, 1300-477 Lisboa, Portugal; 2NZYTech Genes & Enzymes, Campus do Lumiar,
Estrada do Paço do Lumiar, Edifício E, r/c, 1649-038 Lisboa, Portugal;
3
UCIBIO-REQUIMTE,
Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516
Caparica, Portugal; 4Institute for Cell and Molecular Biosciences, Newcastle University, The Medical
School, Newcastle upon Tyne NE2 4HH, United Kingdom; 5Institute of Physical Biology, Heinrich Heine
University, Universitätsstr. 1, 40225 Düsseldorf, Germany; 6Department of Biomolecular Sciences, The
Weizmann Institute of Science, Rehovot, Israel; 7Department of Biomedical and Molecular Sciences,
Queen's University, Kingston, ON K7L 3N6, Canada.
Figure S1 - Detection of C. thermocellum type II Coh-Doc specificities and binding preferences as
evaluated by non-denaturing gel electrophoresis. A) XDoc module of ScaA was probed against the
Cohs of ScaF, ScaC, ScaH and ScaE. B) XDoc module of CipB was probed against the Cohs of ScaF,
ScaC, ScaH and ScaE. In panels A and B cohesins were used at a double molar concentration in
relation to dockerins. C) The method used to detect preferential partners for dockerin is illustrated. The
Doc is mixed with a double molar concentration of two potential Coh partners, and after a 30-min
incubation period the complex formed is visualized through non-denaturing gel electrophoresis. An
example of one such experiment is shown, where ScaA XDoc is mixed with ScaC2 Coh, forming
complex A (CA) or ScaF Coh, forming complex B (CB). When the XDoc module is mixed with the
two Cohs exclusively, complex A (CA) is formed, revealing that the ScaA XDoc module displays a
preference for binding to ScaC2. D) The method described in panel C) was used to identify preferred
Coh and Doc partners as listed.
Figure S2 - CtCohScaC2-XDocCipB and CtCohScaF-XDocScaA complex interfaces between the
dockerin and the X module (A and B) and between the dockerin and the cohesin (C, D, E and F). A),
C) and E) correspond to the CtCohScaC2-XDocCipB complex. B), D) and F) correspond to the
CtCohScaF-XDocScaA complex.
Figure S3 - Examples of the isothermal titration calorimetry (ITC) experiments performed using wild type CipB Xdoc, its mutant derivatives Phe124A and Leu147A and the wild-type cohesins ScaF A),
ScaE6 B) and ScaC2 C). The upper parts of each panel show the raw heats of binding, whereas the
lower parts are the integrated heats after correction for heat of dilution. The curve represents the best
fit to a single-site binding model. 1) Coh plus wild-type CipB XDoc. 2) Coh plus CipB XDoc
Phe124A. 3) Coh plus CipB XDoc Leu147A.
Figure S4 – Interaction of CipB XDoc and its seven mutant derivatives with ScaF, ScaC2 and ScaE6 at 45 ºC with complex formation probed by nondenaturing gel electrophoresis. The dockerins were loaded in lanes 1, 4 and 7. The cohesins were loaded in lanes 2 (ScaE6), 5 ( ScaF) and 8 (ScaC2).
Complexes were loaded in lanes 3 (Doc with ScaE6), 6 (Doc with ScaF) and 9 (Doc with ScaC2).
Table S3 – Primary sequences of cohesins and dockerins involved in the formation of protein complexes
Protein complex
Cohesin
Dockerin
CtCohScaC2-XDocCipB MASAHIALELDKTKVKVGDVIVATVKAKNMTSMAGIQV MNNDSTDKTTVSGYISVDFDYPPESESKIKSGFNVKVAG
PDB
5k39
CtCohScaC2-XDocScaA
5g5d
CtCohScaF-XDocCipB
AcCohScaB3XDocScaA
AcCohScaB3XDocScaAN145G
AcCohScaB3XDocScaAN178G
Nd, not determined
NIKYDPEVLQAIDPATGKPFTKETLLVDPELLSNREYN
PLLTAVNDINSGIINYASCYVYWDSYRESGVSESTGII
GKVGFKVLKAANTTVKLEETRFTPNSIDGTLVIDWYGQ
QIVGYKVIQPDLEHHHHHH
MASAHIALELDKTKVKVGDVIVATVKAKNMTSMAGIQV
NIKYDPEVLQAIDPATGKPFTKETLLVDPELLSNREYN
PLLTAVNDINSGIINYASCYVYWDSYRESGVSESTGII
GKVGFKVLKAANTTVKLEETRFTPNSIDGTLVIDWYGQ
QIVGYKVIQPDLEHHHHHH
MASRADKASSIELKFDRNKGEVGDILIGTVRINNIKNF
AGFQVNIVYDPKVLMAVDPETGKEFTSSTFPPGRTVLK
NNAYGPIQIADNDPEKGILNFALAYSYIAGYKETGVAE
ESGIIAKIGFKILQKKSTAVKFQDTLSMPGAISGTQLF
DWDGEVITGYEVIQPDVLSLGDEPYEVEHHHHHH
MESYITMNFDKNTAEVGQIIKATVKINKITNFSGYQVN
IKYDPTVLQAVNPKTGVAYTNSSLPTSGELLVNEDYGP
IVQGVHKISEGILNLSRSYTALDVYRASESPEETGTVA
VVGFKALQKKATTVVFEHSVTMPNGIIGTTLFNWYGNR
ITSGYSVIQPGEINSE
MESYITMNFDKNTAEVGQIIKATVKINKITNFSGYQVN
IKYDPTVLQAVNPKTGVAYTNSSLPTSGELLVNEDYGP
IVQGVHKISEGILNLSRSYTALDVYRASESPEETGTVA
VVGFKALQKKATTVVFEHSVTMPNGIIGTTLFNWYGNR
ITSGYSVIQPGEINSE
MESYITMNFDKNTAEVGQIIKATVKINKITNFSGYQVN
IKYDPTVLQAVNPKTGVAYTNSSLPTSGELLVNEDYGP
IVQGVHKISEGILNLSRSYTALDVYRASESPEETGTVA
VVGFKALQKKATTVVFEHSVTMPNGIIGTTLFNWYGNR
ITSGYSVIQPGEINSE
TELSTKTDEKGYFEISGIPGDMREFTLEISKRNYLKRNV
TVNGTGKLVVSTEDNPLILWAGDVERKGVQDNAINMVDV
MEISKVFGTRAGDEEYVAELDLNMDGAINLFDIAIVIRH
FNALPSRY
MNKPVIEGYKVSGYILPDFSFDATVAPLVKAGFKVEIVG
TELYAVTDANGYFEITGVPANASGYTLKISRATYLDRVI
ANVVVTGDTSVSTSQAPIMMWVGDIVKDNSINLLDVAEV
IRCFNATKGSANYVEELDINRNGAINMQDIMIVHKHFGA
TSSDYDAQ
MNNDSTDKTTVSGYISVDFDYPPESESKIKSGFNVKVAG
TELSTKTDEKGYFEISGIPGDMREFTLEISKRNYLKRNV
TVNGTGKLVVSTEDNPLILWAGDVERKGVQDNAINMVDV
MEISKVFGTRAGDEEYVAELDLNMDGAINLFDIAIVIRH
FNALPSRY
MGSSHHHHHHSSGLVPRGSHMASGIVSEGTTVSGYINPDF
VTTSTTAPIVKAGFTVEIVGTTKSAVTDSNGYFEIKDVAAGTY
TVKITKANYLTREIANVSVTADKELSTSASPILMWAGDMAIG
GTQDGAINLEDILEICKAFNTSSTDAKYQVGLDLNRDGA
ISLEDVMIVAKHFNKVSSDY
MGSSHHHHHHSSGLVPRGSHMASGIVSEGTTVSGYINPDF
VTTSTTAPIVKAGFTVEIVGTTKSAVTDSNGYFEIKDVAAGTY
TVKITKANYLTREIANVSVTADKELSTSASPILMWAGDMAIG
GTQDGAINLEDILEICKAFGTSSTDAKYQVGLDLNRDGA
ISLEDVMIVAKHFNKVSSDY
MGSSHHHHHHSSGLVPRGSHMASGIVSEGTTVSGYINPDF
VTTSTTAPIVKAGFTVEIVGTTKSAVTDSNGYFEIKDVAAGTY
TVKITKANYLTREIANVSVTADKELSTSASPILMWAGDMAIG
GTQDGAINLEDILEICKAFNTSSTDAKYQVGLDLNRDGA
ISLEDVMIVAKHFGKVSSDY
5g5b
Nd
4u3s
4wi0
Table S5 – Primary sequence of recombinant cohesins and dockerins produced in the present study. Genes encoding cohesins were cloned into pET28 (NheIXhoI), and the corresponding recombinant protein contains a N-terminal His6 tag. XDockerin genes (encoding an X module fused to the dockerin) were cloned
into pET21a (NdeI -XhoI), and the recombinant protein contains an engineered C-terminal His6 tag.
Protein
Name
Organism
Primary Sequence
Cohesin
ScaF
Clostridium thermocellum DKASSIELKFDRNKGEVGDILIGTVRINNIKNFAGFQVNIVYDPKVLMAVDPETGKEFTSSTFPPGRTVLKNNA
XDockerin
ScaB6
Clostridium thermocellum
ScaC1
Clostridium thermocellum
ScaC2
Clostridium thermocellum
ScaH
Clostridium thermocellum
ScaE6
Clostridium thermocellum
ScaB3
Acetivibrio cellulolyticus
ScaA
Clostridium thermocellum
CipB
Clostridium thermocellum
ScaA
Acetivibrio cellulolyticus
YGPIQIADNDPEKGILNFALAYSYIAGYKETGVAEESGIIAKIGFKILQKKSTAVKFQDTLSMPGAISGTQLFD
WDGEVITGYEVIQPD
DSYVIMELDKTKVKVGDIITATIKIENMKNFAGYQLNIKYDPTMLEAIELETGSAIAKRTWPVTGGTVLQSDNY
GKTTAVANDVGAGIINFAEAYSNLTKYRETGVAEETGIIGKIGFRVLKAGSTAIRFEDTTAMPGAIEGTYMFDW
YGENIKGYSVVQPG
SRISMELDKTKANIGDIIIATIRIDNINNFSGYQLNIKYDPSYLQAVNPLTGEPIKKRTMPAVNGTVLLKGDQY
SITEVVENNVDEGILNFGKGYANLTEYRKSGKPETTGIIGKIGFKALKLGKTEIKFENTPVMPGAKEGTLLFDW
DAETITEYNVIQP
AHIALELDKTKVKVGDVIVATVKAKNMTSMAGIQVNIKYDPEVLQAIDPATGKPFTKETLLVDPELLSNREYNP
LLTAVNDINSGIINYASCYVYWDSYRESGVSESTGIIGKVGFKVLKAANTTVKLEETRFTPNSIDGTLVIDWYG
QQIVGYKVIQPD
AEANIQIVLDKNTAKKDEIITAKIILNNIPKIAGYQVNIKYDPNILQAVDLDTGKPLEDKQIPGGGDVLSNPDY
NVLPLAASDVKNGVINFAKAYVNVDEYKESNNPESSGVLALIGFKVLKEESTVISFADTPSMPNAVSGTYVYDW
DFNVLTNYSVGKGVKVN
YIKLEFDKNTASEGEIIRATVKVNNVKNLAGYQICIKYDPNVLQPVNPNTGAAYTTTTHLVDGELIVKQEYGST
SMAAHRLSNGILNFARTYLYVSDYKEDGKPEETGILGVIGFKVLKKEKTTVSFYADEALMPNSVSGTYLIDWNS
NKKTDYKVIQP
ESYITMNFDKNTAEVGQIIKATVKINKITNFSGYQVNIKYDPTVLQAVNPKTGVAYTNSSLPTSGELLVNEDYG
PIVQGVHKISEGILNLSRSYTALDVYRASESPEETGTVAVVGFKALQKKATTVVFEHSVTMPNGIIGTTLFNWY
GNRITSGYSVIQPGEINSE
MNKPVIEGYKVSGYILPDFSFDATVAPLVKAGFKVEIVGTELYAVTDANGYFEITGVPANASGYTLKISRATYL
DRVIANVVVTGDTSVSTSQAPIMMWVGDIVKDNSINLLDVAEVIRCFNATKGSANYVEELDINRNGAINMQDIM
IVHKHFGATSSDYDAQ
MNNDSTDKTTVSGYISVDFDYPPESESKIKSGFNVKVAGTELSTKTDEKGYFEISGIPGDMREFTLEISKRNYL
KRNVTVNGTGKLVVSTEDNPLILWAGDVERKGVQDNAINMVDVMEISKVFGTRAGDEEYVAELDLNMDGAINLF
DIAIVIRHFNALPSRY
GIVSEGTTVSGYINPDFVTTSTTAPIVKAGFTVEIVGTTKSAVTDSNGYFEIKDVAAGTYTVKITKANYLTREI
ANVSVTADKELSTSASPILMWAGDMAIGGTQDGAINLEDILEICKAFNTSSTDAKYQVGLDLNRDGAISLEDVM
IVAKHFNKVSSDY
Table S6 – Primers used to produce dockerin mutant derivatives obtained in the present study.
Dockerin derivative
XDocCipB M114A
XDocCipB M118A
XDocCipB S121A
XDocCipB F124A
XDocCipB L147A
XDocCipB F148A
XDocCipB I154A
Sequence (5’  3’)
GCAAGACAATGCTATTAATGCGGTGGATGTGATGGAAATATCC
GGATATTTCCATCACATCCACCGCATTAATAGCATTGTCTTGC
GCTATTAATATGGTGGATGTGGCGGAAATATCCAAAG
CTTTGGATATTTCCGCCACATCCACCATATTAATAGC
GTGGATGTGATGGAAATAGCCAAAGTTTTTGGCAC
GTGCCAAAAACTTTGGCTATTTCCATCACATCCAC
GGAAATATCCAAAGTTGCTGGCACAAGAGCCGGAGATG
CATCTCCGGCTCTTGTGCCAGCAACTTTGGATATTTCC
GGACGGAGCAATCAATGCATTTGATATAGCTATAGTTATCAGGC
GCCTGATAACTATAGCTATATCAAATGCATTGATTGCTCCGTCC
GGACGGAGCAATCAATTTAGCTGATATAGCTATAGTTATCAGGC
GCCTGATAACTATAGCTATATCAGCTAAATTGATTGCTCCGTCC
GATATAGCTATAGTTGACAGGCATTTTAACGCATTACC
GGTAATGCGTTAAAATGCCTGTCAACTATAGCTATATC
Direction
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Table S7 – Data collection and refinement statistics of C. thermocellum coh-doc complexes.
Coh-XDoc complex
Space Group
Unit cell parameters
a, b, c (Å)
α, β, γ (°)
Matthews parameter (Å3/Da)
Data collection statistics
X-ray source
Wavelength (Å)
No. of unique reflections
Resolution limits (Å)
Completeness (%)
Redundancy
Average I/σ(I)
Rmerge (%)
Rpim (%)
Half-dataset correlation CC(1/2)
Refinement statistics
Resolution limits (Å)
R-work
R-free
No. protein residues in the asymmetric unit
No. water molecules in the asymmetric unit
No. atoms in the asymmetric unit
rmsd bond length (Å)
rmsd bond angles (°)
Average temperature factor (Å2)
main chain
side chain
Calcium 1
Calcium 2
solvent molecules
outliers (%)
Ramachandran plot
favored (%)
PDB ID codes
CtCohScaF-XDocCipB
P212121
CtCohScaC2-XDocCipB
C121
CtCohScaC2-XDocScaA
I222
43.4, 63.7, 141.2
90.0, 90.0, 90.0
2.6
116.7, 78.6, 35.8
90.0, 95.8, 90.0
2.2
52.4, 125.8, 130.5
90.0, 90.0, 90.0
3.1
ESRF, ID29
0.9762
63360
47.31 – 1.5 (1.53-1.50)
99.5 (99.6)
4.4 (4.5)
18.8 (2.3)
3.7 (64.8)
2.0 (33.8)
0.999 (0.736)
ESRF, ID14-4
0.9735
21909
39.31 – 1.98 (2.09 – 1.98)
97.8 (98.2)
3.6 (3.7)
13.10 (5.5)
9.4 (17.3)
5.8 (10.4)
not determined
ESRF, ID29
0.9537
8989
45.28 – 3.00 (3.18 – 3.00)
100 (100)
7.0 (7.2)
5.9 (1.5)
18.9 (118)
8.2 (50.1)
0.991 (0.737)
47.3 - 1.5
0.190
0.203
333
201
2855
0.006
1.243
18.8 – 1.98
0.187
0.247
320
322
2819
0.009
1.154
48.3 – 3.0
0.255
0.303
316
0
2420
0.007
1.20
22.2
24.2
14.8
19.5
34.9
0
98.5
5m0y
17.8
18.7
17.0
14.6
36.8
0
97.0
5k39
48.0
48.4
64.4
62.3
0
95
5g5d
Rmerge = [Σ |I-<I>|]/Σ <I>, where I is the observed intensity, and <I> is the statistically weighted average intensity of multiple observations.
Rpim = [Σ (1/(n-1)) Σ |I-<I>|]/Σ <I>, a redundancy-independent version of Rmerge
Rwork = Σ ||Fcalc|− |Fobs||/Σ |Fobs|× 100, where Fcalc and Fobs are the calculated and observed structure factor amplitudes, respectively (Rfree is calculated for a randomly chosen 5%
of the reflections).
Geometry values from Molprobity.
Values in parentheses are for the highest resolution shell.
Table S8 – Data collection and refinement statistics of A. cellulolyticus coh-doc complexes.
Coh-XDoc complex
Space Group
Unit cell parameters
a, b, c (Å)
α, β, γ (°)
Matthews parameter (Å3/Da)
Data collection statistics
X-ray source
Wavelength (Å)
No. of unique reflections
Resolution limits (Å)
Completeness (%)
Redundancy
Average I/σ(I)
Rmerge (%)
Rpim (%)
Half-dataset correlation CC(1/2)
Refinement statistics
Resolution limits (Å)
R-work
R-free
No. protein residues in the asymmetric unit
No. water molecules in the asymmetric unit
No. atoms in the asymmetric unit
rmsd bond length (Å)
rmsd bond angles (°)
Average temperature factor (Å2)
main chain
side chain
Calcium 1
Calcium 2
solvent molecules
outliers (%)
Ramachandran plot
favored (%)
PDB ID codes
AcCohScaB3XDocScaA_N145G
P6522
AcCohScaB3XDocScaA_N178G
P212121
72.3, 72.3, 231.5
90.0, 90.0, 120.0
2.3
38.5, 88.3, 92.0
90.0, 90.0, 90.0
2.3
Diamond, IO3
0.976
45113
62.63 – 1.64 (1.68 – 1.64)
100 (100)
26.7 (29.5)
13.5 (3.2)
11.3 (70.2)
2.7 (13.0)
0.998 (0.856)
ESRF, ID29
0.976
29281
92.02 – 1.93 (1.98 – 1.93)
90.8 (92.3)
3.3 (3.1)
5.0 (2.0)
13.6 (49.7)
8.0 (30.4)
0.987 (0.561)
62.6 – 1.64
0.171
0.206
323
341
2843
0.009
1.371
63.7 – 1.93
0.197
0.241
324
219
2680
0.006
1.097
25.5
28.3
16.2
19.7
31.9
0
98.5
4u3s
23.3
24.3
19.6
19.9
28.9
0
98.5
4wi0
Rmerge = [Σ |I-<I>|]/Σ <I>, where I is the observed intensity, and <I> is the statistically weighted average intensity of multiple observations.
Rpim = [Σ (1/(n-1)) Σ |I-<I>|]/Σ <I>, a redundancy-independent version of Rmerge.
Rwork = Σ ||Fcalc|− |Fobs||/Σ |Fobs|× 100, where Fcalc and Fobs are the calculated and observed structure factor amplitudes, respectively (Rfree is calculated for a randomly chosen 5%
of the reflections).
Geometry values from Molprobity.
Values in parentheses are for the highest resolution shell.