Marc R. Fabian, Filipp Frank, Christopher Rouya, …...Marc R. Fabian, Filipp Frank, Christopher...
5
Supplementary Material for: Structural basis for the recruitment of the human CCR4–NOT deadenylase complex by Tristetraprolin Marc R. Fabian, Filipp Frank, Christopher Rouya, Nadeem Siddiqui, Wi S. Lai, Alexey Karetnikov, Perry J. Blackshear, Bhushan Nagar and Nahum Sonenberg Supplementary Figures 1-4 Nature Structural & Molecular Biology: doi:10.1038/nsmb.2572
Transcript of Marc R. Fabian, Filipp Frank, Christopher Rouya, …...Marc R. Fabian, Filipp Frank, Christopher...
Supplementary Material for:
Structural basis for the recruitment of the human CCR4–NOT deadenylase complex by Tristetraprolin
Marc R. Fabian, Filipp Frank, Christopher Rouya, Nadeem Siddiqui, Wi S. Lai, Alexey Karetnikov, Perry J. Blackshear, Bhushan Nagar and Nahum Sonenberg
Supplementary Figures 1-4
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2572
kDa95
72
55
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34
kDa95
72
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43
34
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43
Pull-down(GST alone)
Pull-down(1-551)
Pull-down(424-800)
Pull-down(727-1266)
Pull-down(1075-1575)
Pull-down(1325-1825)
GST-CNOT1 fragment:
Pull-down(1791-2371)
MBP-TTP:GST-CNOT1 fragment:
kDa
MBP-TTP:
Pull-down(800-1015)
+ ++ +
+ ++ +
+ ++ +
+ ++ +
+ ++ +
+ ++ +
+ ++ +
+ ++ +
Supplementary Figure 1. Human TTP directly binds a central domain of CNOT1. Recombinant glutathione-S-transferase (GST) or GST-tagged CNOT1 fragments was immobilized on glutathione-Sepharose beads and incubated with (MBP)-tagged full-length TTP. Precipitated proteins were separated by SDS-PAGE and visualized by Coomassie staining. Dashed lines separate each co-precipitation experiment. Magnification of CNOT1727–1266 binding to MBP-TTP (arrow) is shown in the grey box. MBP-TTP alone lanes equal 50% input.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2572
TTP -------------MDLTAIYESLLSLSPDVP----VPSDHGGTESSP--------GWGSS 35BRF1 MTTTLVSATIFDLSEVLCKGNKMLNYSAPSAGGCLLDRKAVGTPAGG--------GFPRR 52BRF2 MSTTLLSA--FYDVDFLCKTEKSLANLNLNN---MLDKKAVGTPVAAAPSSGFAPGFLRR 55 :. . :. * : . ** . *:
TTP GPWS--------LSPSDSSPSGVTSRLPGRS----------------------TSLVEGR 65BRF1 HSVT--------LPSSKFHQNQLLSSLKG------------------EP----APALSSR 82BRF2 HSASNLHALAHPAPSPGSCSPKFPGAANGSSCGSAAAGGPTSYGTLKEPSGGGGTALLNK 115 . : ... . . * . : .:
TTP SCGWVPPPPGFAPLAPRLGPELSPSPTSPTATSTTPSRYKTELCRTFSESGRCRYGAKCQ 125BRF1 DSRFRDRSFSEGGER------LLPTQKQPGGGQVNSSRYKTELCRPFEENGACKYGDKCQ 136BRF2 ENKFRDRSFSENGDRSQHLLHLQQQQKGGGGSQINSTRYKTELCRPFEESGTCKYGEKCQ 175 . : . . * . . . ..:********.*.*.* *:** ***
TTP FAHGLGELRQANRHPKYKTELCHKFYLQGRCPYGSRCHFIHNPSEDLAAPGHP------- 178BRF1 FAHGIHELRSLTRHPKYKTELCRTFHTIGFCPYGPRCHFIHNAEERR-ALAG--ARDLSA 193BRF2 FAHGFHELRSLTRHPKYKTELCRTFHTIGFCPYGPRCHFIHNADERRPAPSGGASGDLRA 235 ****: ***. .**********:.*: * ****.*******..* * .
TTP ----------------PVLRQSISFSGLPSGRRTSPPPPGLAGPSLSSSSFSPSS----- 217BRF1 D--------------RPRLQHSFSFAGFPS----AAATAAATGLLDSPTSITPPP----- 230BRF2 FGTRDALHLGFPREPRPKLHHSLSFSGFPSGHHQPPGGLESPLLLDSPTSRTPPPPSCSS 295 * *::*:**:*:** .. . *.:* :*..
TTP -------------------------------------------------------SPPPP 222BRF1 -------------------------------------ILS----ADDLLG------SPTL 243BRF2 ASSCSSSASSCSSASAASTPSGAPTCCASAAAAAAAALLYGTGGAEDLLAPGAPCAACSS 355 . .
TTP GDLPLSPSAFSAAPGTPLAR--------------------------------------RD 244BRF1 PDGTNNPFAFSSQELASLFA--------------------------------------PS 265BRF2 ASCANNAFAFG-PELSSLITPLAIQTHNFAAVAAAAYYRSQQQQQQQGLAPPAQPPAPPS 414 . . .. **. :.* .
TTP PTPVCCPSCRRATPISVWGPLGGLVRTPSVQSLGSDPDEYASSGSSLGGSDSPVFEAGVF 304BRF1 MGLPGGGSP------TTFLFRPMSESPHMFDSPPSPQDSLSDQEGYLSS--SSSSHSGSD 317BRF2 ATLPAGAAAPPSPPFSFQLPRRLSDSP-VFDAPPSPPDSLSDRDSYLSGSLSSGSLSGSE 473 : : . .:: * *. :. . *.. *. :*
TTP APPQPVAAPRRLPIFNRISVSE- 326BRF1 SPT--LDNSRRLPIFSRLSISDD 338BRF2 SPS--LDPGRRLPIFSRLSISDD 494 :*. : ******.*:*:*:
Supplementary Figure 2. Alignment of human TTP, BRF-1 and BRF-2 amino acids. Tandem zinc finger RNA binding domains (yellow) and conserved C-terminal domain patches (black boxes) are marked accordingly.
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2572
Supplementary Figure 3. (A) Structural alignment of the MIF4G domain of eIF4GII (PDB ID 1HU3) (grey) with CNOT1800–999 (orange) using the CLICK structural alignment server. 113 Cα residues out of 196 and 180 total residues of eIF4GII and CNOT1, respectively, were matched and the alignment resulted in an RMSD of 2.29 Å. Isothermal titration calorimetry (ITC) trace for CNOT1800–999 titrated with (B) wild-type, (C) Phe319Ala and (D) phosphor-Ser323 TTP peptides. Upper panel shows the raw baseline corrected thermographs. Lower panel shows the integrated areas of the heat absorbed along with a fit from which the stoichiometry (N) and molar dissociation constant were calculated (Kd).
0.0 0.5 1.0 1.5 2.0 2.5 3.0-10
-8
-6
-4
-2
0
-0.8
-0.6
-0.4
-0.2
0.0
0 20 40 60 80 100 120
Time (min)
μC
al/s
eckC
al/m
ole
of in
ject
ant
Molar Ratio
TTP-WT
Time (min)
Molar Ratio
0 1.0 2.0 3.0 4.0 5.0
-1.7-1.6-1.5-1.4-1.3-1.2-1.1-1.0-0.9
-0.30
-0.25
-0.20
-0.15
-0.10
-0.05
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Time (min)
pS323-TTP
0 2 4 6 8 10
-1.4
-1.3
-1.2
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-1.0
-0.9
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-0.3
-0.2
-0.1
0.0
0 20 40 60 80 100 120
Molar Ratio
TTP-F319A
A
B C D
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2572
Hs CNOT1 836 -SKEIDDEANSYFQRIYNHPPHPTMSVDEVLEMLQRFKDSTIKREREVFNCMLRNLFEEYRFFPQY 900Xt CNOT1 835 -SKEIDDEANSYFQRIYNQPPHPTMSVDEVLEMLQRFKDSNIKREREVFNCMLRNLFEEYRFFPQY 899 Dm CNOT1 909 -SKEVEDEVNSYFQRIYNHQPNPTLSIDEVLDILQRFKESSNRREQEVFLCMLRNLFEEYRFFCQY 973 Ce CNOT1 922 FAEDIQEEANSYFEKIYS--VNNAMSVENLIDLLKRFRVSNDRRERLVLACVVKNLFEEYRFFHEY 985 Sc NOT1 593 INNDIEKEMQNYLQKMYS----GELAIKDVIELLRRLRDSDLPRDQEVFTCITHAVIAESTFFQDY 654
E893
Y900
Y609
Q606
Q602
Y653
E647
L605
Y851
Q848
N844
F847
α1α3
180°
human CNOT1 yeast NOT1human TTP
A
B
C
Supplementary Figure 4. (A) Van der Waals contacts are shown between TTP Phe319 and the relevant residues (outlined in yellow), which contribute to the surface of the pocket in CNOT1. TTP is shown in purple and CNOT1 in grey. Alignments of CNOT1 domain that binds TTP. (B) Alignment of human (Hs) CNOT1 domain that binds TTP with comparable regions from Xenopus tropicalis (Xt), Drosophila melanogaster (Dm), Caenorhabditis elegans (Ce) CNOT1 proteins and Saccharomyces cerevisiae (Sc) NOT1 protein. Human CNOT1 residues that interact with TTP are labeled with arrowheads. (Yellow) amino acids identical in 100% of proteins; (orange) conservative substitutions by related amino acids in TTP-interacting amino acids. (C) Structural alignment of the human CNOT1 MIF4G-like domain (residues 837–992) with yeast CNOT1 (PDB ID: 4B8B; residues 595–746). Cartoon representation of the human (grey) and yeast (green) structures and the TTP peptide (purple). 150 Cα atoms were matched by the CLICK structural alignment server 27 and the alignment resulted in an RMSD of 1.19 Å. (D) Close-up view of the TTP binding site. The residues in yeast CNOT1 corresponding to TTP-interacting residues in human CNOT1 are shown as sticks.
D
Nature Structural & Molecular Biology: doi:10.1038/nsmb.2572
