Human CREB binding protein (bromodomain) in complex with acetylated lysine

Target ID:

CREBBPA

Aliases:

CBPCREBBPKAT3ARSTS

Deposition Date:

Thursday 30th September 2010

Families:

Bromodomain

Related Diseases:

CancerMetabolismChromatin and epigeneticsSignallingNeurobiology

Structure type:

protein-ligand

Download Coordinates:

3P1C

Authors:

Filippakopoulos, P., Picaud, S., Felletar, I., Fedorov, O., Muniz, J., Von Delft, F., Arrowsmith, C.H., Edwards, A.M., Weigelt, J., Bountra, C., Knapp, S.

Site:

Oxford

3P1C

tabs

Structure Details

CBP is a central protein in a variety of biological processes including cell growth and transformation, genomic stability, development, neuronal plasticity and memory formation as well as energy homeostasis. The fundamental role of CBP is reflected in the phenotype of the Cbp knockout: homozygous mutant mice die in utero with signs of defective blood vessel formation in the central nervous system, and developmental retardation as well as delays in both primitive and definitive hematopoiesis (Kalkhoven, 2004).

CBP acts as a general transcriptional coactivator and acetylates histones as well as non-histone proteins. Acetylation of non-histone proteins can have a positive or negative effect on transcriptional regulation by affecting protein-protein interaction, protein-DNA interaction, nuclear retention or protein half-life of these molecules (Kalkhoven, 2004).

Mutations in the CBP gene can lead to Rubinstein-Taybi syndrome (RTS), a rare human genetic disorder characterized by mental retardation and physical abnormalities; many patients with RTS either have breakpoints or microdeletions in chromosome 16p13.3 where the CBP gene is located but also heterozygous point mutations can lead to RTS (Rouaux et al., 2004). CBP plays also a role in the etiology of other human neurological disordes such as Amyotrophic lateral sclerosis, Alzheimer's disease and also in poly glutamine diseases, inflammatory diseases and several cancer forms (Matt, 2002; Hallam and Bourtchouladze, 2006).

CBP is a multidomain protein consisting of three cysteine-histidine-rich domains (CH1, CH2, CH3) serving as docking modules for numerous transcriptional regulators, the binding site for the CREB transcription factor (KIX domain), a bromodomain, two PHD type zinc finger motifs (LAP fingers, TTC fingers), an N-terminal nuclear receptor binding domain, a C-terminal glutamine-rich domain and a histone acetyltransferase (HAT) domain (Blobel, 2002). In order to enable the development of potent CBP inhibitor that target the bromodomain we solved co-crystal structures of the CBP bromodomains with acetyl lysine as well as complexes with three fragments that act as acetyl lysine competitive inhibitors. These molecules include N-Methyl-2-pyrrolidone (NMP), dimethylsulfoxide (DMSO) and a substituted hydroquinazolin.

Materials and Methods

Entry Clone Source: Synthetic

Entry Clone Accession: n/a

SGC Construct ID: CREBBPA-c003

GenBank GI number: gi|4758056

Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

Amplified construct sequence:
CATATGCACCATCATCATCATCATTCTTCT
GGTGTAGATCTGGGTACCGAGAACCTGTAC
TTCCAATCCATGCGCAAAAAAATTTTTAAA
CCGGAAGAACTGCGTCAGGCGCTGATGCCG
ACCCTGGAAGCGCTGTATCGCCAGGATCCG
GAAAGCCTGCCGTTTCGTCAGCCGGTGGAT
CCGCAGCTGCTGGGTATCCCGGATTATTTT
GATATTGTGAAAAACCCGATGGATCTGAGC
ACCATCAAACGCAAACTGGATACCGGCCAG
TATCAGGAACCGTGGCAGTATGTGGATGAT
GTTTGGCTGATGTTTAATAATGCGTGGCTG
TATAACCGTAAAACCAGCCGTGTGTATAAA
TTCTGTAGCAAACTGGCGGAAGTTTTTGAA
CAGGAAATTGATCCGGTGATGCAGAGCCTG
GGCTGACAGTAAAGGTGGATACGGATCCGA
A

Final protein sequence (tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfq*smRKKIFK
PEELRQALMPTLEALYRQDPESLPFRQPVD
PQLLGIPDYFDIVKNPMDLSTIKRKLDTGQ
YQEPWQYVDDVWLMFNNAWLYNRKTSRVYK
FCSKLAEVFEQEIDPVMQSLG
^ TEV cleave site

Tags and additions: MHHHHHHSSGVDLGTENLYFQ*SM, cleaved at the * with TEV protease.

Host: BL21(DE3)-R3: a phage resistant BL21(DE3) derivative .

Growth medium, induction protocol: Host cells transformed with the expression plasmids were plated out onto LB-agar plates containing 50µg/ml kanamycin. The next day several colonies were combined into 1 ml TB (Terrific Broth), 50µg/ml kanamycin, which was then grown overnight and stored as glycerol stocks at -80°C. The glycerol stock was used to inoculate a 10ml starter culture in TB + kanamycin (50µg/ml). This starter culture was grown overnight at 37°C and used to inoculate a 1 liter culture in the same medium. The culture was grown in baffled flasks at 37°C until the OD₆₀₀ reached ~3.5. After that the temperature was lowered to 18°C. Protein production was induced with 0.1mM IPTG and the recombinant bromodomain was incubation continued at 18°C overnight. The next day cells were harvested by centrifugation at 4000 rpm for 30 minutes. The cell pellet was stored at -80°C degrees.
Binding buffer:
Lysis buffer: 500mM NaCl, 50mM pH8.0 KH2PO4, 0.5mM TCEP, Benzonase 1ml/15 ml buffer, Protease inhibitor (1 ml/ml).
Affinity binding buffer: 10mM Imidazole, 500mM NaCl, 50mM pH8.0 KH2PO4, 0.5mM TCEP.
Extraction buffer, extraction method: The cell pellet (40g) from 4L cultue was re-suspended in one volume (40ml) of lysis buffer. The re-suspended cells were lysed by one passage through a Constant Systems cell breaker and subsequent sonication; the cell breaker was washed with 1x extraction buffer, bringing the total volume to 120ml. DNA was precipitation by addition of polyethyleneimine (PEI, pH 7.5) to a final concentration of 0.15% during an incubation time of 30 min on ice, followed by a centrifugation at 17,000 rpm (4°C); The supernatant was further cleared by filtration through a 0.2µm serum Acrodisc filter.

Column 1: Ni-affinity chromatography: HisTrap FF Crude, 5ml (GE Healthcare).

Column 1 Buffers:
Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 0.5mM TCEP

Column 1 Procedure: The supernatant was loaded by gravity flow on the DAE-52 column. The column was then washed with 2 x 50 ml binding buffer at gravity flow.

Enzymatic treatment and Tag removal: TEV protease (1:20 w/w), was added to the sample after gel filtration. The sample was incubated at 4°C overnight. The sample was then passed over a column of Ni-sepharose (0.5ml) to trap the cleaved tag and other Ni-binding proteins.

Column 2: Size exclusion chromatography HiLoad 16/60 Superdex 75

SEC-Buffers: 10 mM Hepes, pH 7.4, 500 mM NaCl, 5% glycerol, 0.5mM TCEP.

Column 2 Procedure: Fractions containing the expressed bromodomain were collected after his6-tag cleavage and were loaded on a SEC column at 1.0ml/min. Eluted fractions were >95% pure as judged by SDS-PAGE.

Mass spectrometry characterization (After Tag cleavage):
Expected MW: 14207 (cleaved protein)
Measured MW: 14207

Protein concentration: The protein was concentrated to 10mg/ml in SEC buffer using a centricon device with a 10kDa cut off.

Crystallisation:
Acetylated lysine complex (3P1C):The protein (10 mg/ml) in SEC buffer was mixed with an equal volume (100 nl) of reservoir solution (0.2M Potassium thiocyanate; 25% (w/v) PEG3350; 5% (v/v) ethylene glycol and equilibrated as a sitting drop at 4°C in the presence of 5mM of acetylated lysine.
N-Methyl-2-pyrrolidone (3P1D):The protein (10 mg/ml) in SEC buffer was mixed with an equal volume (100 nl) of reservoir solution (0.2M Potassium thiocyanate; 20% (w/v) PEG3350; 5% (v/v) ethylene glycol and equilibrated as a sitting drop at 4°C in the presence of 5mM of N-Methyl-2-pyrrolidone.
Dimethyl-sulfoxide (3P1E):The protein (10 mg/ml) in SEC buffer was mixed with an equal volume (100 nl) of reservoir solution (0.2M Potassium thiocyanate; 25% (w/v) PEG3350; 5% (v/v) ethylene glycol and equilibrated as a sitting drop at 4°C in the presence of 5mM of dimethyl sulfoxide.
3-methyl-3,4-dihydroquinazolin-2(1H)-one (3P1F):The protein (10 mg/ml) in SEC buffer was mixed with an equal volume (100 nl) of reservoir solution (0.15M Potassium thiocyanate; 20% (w/v) PEG3350; 10% (v/v) ethylene glycol and equilibrated as a sitting drop at 4°C in the presence of 5mM of 3-methyl-3,4-dihydroquinazolin-2(1H)-one.

Data Collection: Crystals were flash frozen in liquid nitrogen using the crystallization condition supplemented with 25% ethylene glycol. Diffraction data were collected at a RIGAKU FR-E+ SuperBright light source at a single wavelength of 1.54128 Å.

References

Blobel, G.A. (2002). CBP and p300: versatile coregulators with important roles in hematopoietic gene expression. Journal of leukocyte biology 71, 545-556.
Hallam, T.M., and Bourtchouladze, R. (2006). Rubinstein-Taybi syndrome: molecular findings and therapeutic approaches to improve cognitive dysfunction. Cell Mol Life Sci 63, 1725-1735.
Kalkhoven, E. (2004). CBP and p300: HATs for different occasions. Biochemical pharmacology 68, 1145-1155.
Matt, T. (2002). Transcriptional control of the inflammatory response: a role for the CREB-binding protein (CBP). Acta medica Austriaca 29, 77-79.
Rouaux, C., Loeffler, J.P., and Boutillier, A.L. (2004). Targeting CREB-binding protein (CBP) loss of function as a therapeutic strategy in neurological disorders. Biochemical pharmacology 68, 1157-1164.