C-terminal Binding Protein
(CtBP)
Marco Nardini
Dept. of Physics-INFM, University of Genova, Italy
Consorzio Mario Negri Sud, Chieti, Italy
Stefania Spanò, Alberto Luini, Daniela Corda
ESRF synchrotron, Grenoble, France
ID14-2 beamline
EMBL-DESY synchrotron, Hamburg, Germany
BW7A beamline
CtBP: protein of 430 a.a. residues
•
localization: Cellular nucleus
•
function:
Co-repression activity
nucleosoma
cromatina
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NATURE | VOL 422 | 17 APRIL 2003 | www.nature.com/nature
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C-terminal Binding Protein
• transcriptional regulator (co-repressor)
• homodimer
• PXDLS recognition motif in DNA-binding proteins and
other regulatory proteins
• regulated by NAD+/NADH
REPORTS
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www.sciencemag.org SCIENCE VOL 295 8 MARCH 2002
NAD+/NADH
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Nicotinamide Adenine Dinucleotide
C-terminal Binding Protein
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Questions
• Dimerization
• NAD+/NADH binding site
• Peptide recognition site (PxDLS)
repressor – co-repressor interaction site
Crystallization
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• vapour diffusion technique
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• reservoir solution:
1.8 – 2.1 M ammonium formate,
100 mM Hepes buffer, pH 7.5
• T=296 K, crystals after one week
• space group P6422
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X-ray diffraction experiments
CtBP native
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CtBP + peptide
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X-ray diffraction data
• native data set: 2.3 Å resolution
(EMBL-DESY, Hamburg, Germany)
- Multiple wavelength Anomalous Dispersion (MAD: SeMet)
- Rfactor/Rfree (%) = 22.2/27.2
• t-CtPB-peptide complex: 3.1 Å resolution
(ESRF, Grenoble, France)
- peptide = PIDLSKK (PXDLS)
- Rfactor/Rfree (%) = 23.1/31.5
MAD experiment
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MAD data collection statistics
Absorption peak Inflection point
Remote
Wavelength (Å)
0.9778
0.9783
0.9321
Resolution (Å)
40.0-3.2
40.0-3.2
40.0-3.2
Mosaicity (°)
0.68
0.68
0.68
Completeness (%)
99.9 (100)
99.9 (100)
99.9 (100)
Rmerge (%)
5.4 (53.0)
5.7 (54.1)
5.9 (64.0)
Total reflections
84,641
84,384
85,714
Unique reflections
11,504
11,511
11,551
Redundancy
7.4
7.3
7.4
Average I/σ(I)
32.5 (4.4)
28.7 (4.5)
28.2 (3.8)
MAD experiment
lambda 1
label Wavelength # 1
rawmadfile ./bars_r_p6222_ano_solve.sca ! datafile
wavelength 0.9321
fprimv_mad -2.0861
! f' value at this wavelength
fprprv_mad 3.4733
lambda 2
rawmadfile ./bars_p_p6222_ano_solve.sca
wavelength 0.97780
fprimv_mad -5.8404
fprprv_mad 3.8334
lambda 3
rawmadfile ./bars_i_p6222_ano_solve.sca
wavelength 0.97826
fprimv_mad -6.1161
fprprv_mad 3.8367
nres 358
nanomalous 8
! approx number of residues in the protein molecule
! number of anomalously scattering atoms per protein
t-CtBP Sequence
His-tag
1
MGHHHHHHMSGVRPPIMNGPMHPRPLVALLDGRDCT
VEMPILKDVATVAFCDAQSTQEIHEKVLNEAVGALMY
HTITLTREDLEKFKALRIIVRIGSGFDNIDIKSAGDLGIAV
CNVPAASVEETADSTLCHILNLYRRTTWLHQALREGTR
VQSVEQIREVASGAARIRGETLGIIGLGRVGQAVALRAK
AFGFNVLFYDPYLSDGIERALGLQRVSTLQDLLFHSDCV
TLHCGLNEHNHHLINDFTVKQMRQGAFLVNTARGGLV
DEKALAQALKEGRIRGAALDVHESEPFSFSQGPLKDAP
NLICTPHAAWYSEQASIEMREEAAREIRRAITGRIPDSLK
NCVNKDHLTAATH
350
Se-Met sites
0.594432
0.295841
0.774250
0.660929
0.140777
0.242292
0.252017
0.636193
0.472125
0.784566
0.812143
0.615367
0.768459
0.293221
0.258739
0.949095
0.922143
0.440777
0.458711
0.630251
0.574630
0.450519
0.241912
0.398332
0.923300
0.939996
0.901555
0.842179
0.924001
0.835246
0.881202
0.894022
0.937302
0.929420
0.943545
0.973473
16.50
14.70
11.84
10.97
5.25
5.15
5.13
4.73
4.36
4.01
3.67
3.66
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3
4
5
6
7
8
9
10
11
12
Se-Met sites
t-CtBP Sequence
His-tag
1
15
MGHHHHHHMSGVRPPIMNGPMHPRPLVALLDGRDCT
VEMPILKDVATVAFCDAQSTQEIHEKVLNEAVGALMY
HTITLTREDLEKFKALRIIVRIGSGFDNIDIKSAGDLGIAV
CNVPAASVEETADSTLCHILNLYRRTTWLHQALREGTR
VQSVEQIREVASGAARIRGETLGIIGLGRVGQAVALRAK
AFGFNVLFYDPYLSDGIERALGLQRVSTLQDLLFHSDCV
TLHCGLNEHNHHLINDFTVKQMRQGAFLVNTARGGLV
DEKALAQALKEGRIRGAALDVHESEPFSFSQGPLKDAP
NLICTPHAAWYSEQASIEMREEAAREIRRAITGRIPDSLK
NCVNKDHLTAATH
345
350
initial map
final map
solvent
flattening
3D-Structure
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Topology
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NAD-binding fingerprint motif
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Dimerization
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Peptide binding site
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Structural homologues
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Domain closure
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Conclusions
•
PXDLS
CtBP:NAD+/NADH:peptide
NAD(H)
• NAD+/NADH binding:
closed, dimeric form
NAD(H)
PXDLS
• favourable position of the
N-terminal PXDLS recognition
site
Nardini, M. et al., (2003) EMBO J. 22, 3122-3130
Future Plans
• CtBP full-length structure
• HDAC (1, 2) complex (co-repression activity)
• ADP-ribosylation
• Small Angle Scattering of full-length CtBP
CtBP full-length crystals !!